- "<.‘.:“- ..

¢ , w.
m.‘~u- " . '7
." - - “f‘O-rxht-‘
m".

--
r.-
“s...“
‘0 a .. -
V n.

. «~. .‘
.:::T:w~
» «.9

a
:{li

33%. I.»
:x 1:5»

ltxi
3.11?
' i

C: fig ‘5:
v‘ 1' '~ ': 1‘
“£32,! Hik'j?!

'h‘" ;

".1558?
. ?-'1!}jf‘" . I
"’1‘: .

' Jr}

'4‘“.
‘u,“3

, w -.
3.2

n.“

N'.‘ *

‘ 5
‘ ‘ ‘1’.
fl. 1
' ' ‘-
’£‘S‘a;", -

«;3;r

"EI

WEE-'4‘
TSP-'7‘ '
3’ka-

....
at.

}

2v
~35?“

v.

‘

hi " {“1
}

fifty. 21‘}:

0

u.
an

-w~

‘13

 

“SIS

or 3 U0

This is to certify that the

dissertation entitled
THE ROLE OF TUMOR SUPPRESSOR GENE p53
IN MALIGNANT TRANSFORMATION AND THE
ROLE OF THE hREVl GENE IN DAMAGE-
INDUCED MUTAGENESIS OF HUMAN

FI BROBLAST CELLS
presented by

Xi-De Wang

has been accepted towards fulfillment
of the requirements for

Ph.D. degree“, Microbiology and

 

 

Molecular Genetics

    

Major professor

Date June 14, 2002

MS U i: an Affirmative Action/Equal Opportunity Institution 0— 12771

 

 

 

LIBRARY
Michigan State
University

 

 

 

PLACE IN RETURN BOX to remove this checkout from your record.
To AVOID FINES return on or before date due.
MAY BE RECALLED with earlier due date if requested.

 

DATE DUE DATE DUE DATE DUE

 

 

 

 

 

 

 

 

 

 

 

 

 

 

6/01 cJCIRC/DaleDuopSS—p. 1 5

 

 

THE ROLE OF TUMOR SUPPRESSOR GENE p53 IN MALIGNANT
TRANSFORMATION AND THE ROLE OF THE hREV1 GENE IN DAMAGE-
INDUCED MUTAGENESIS OF HUMAN FIBROBLAST CELLS
By

Xi-De Wang

A DISSERTATION

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Microbiology and Molecular Genetics

2002

DNA

ACUI

 

grow
mute
This
func4
and

freqL
cons

ager

UIIra'
’Dett
SIabI
DErc.
addit

IGSI I

ABSTRACT
THE ROLE OF TUMOR SUPPRESSOR GENE p53 IN MALIGNANT
TRANSFORMATION AND THE ROLE OF THE hREV1 GENE IN DAMAGE-
INDUCED MUTAGENESIS OF HUMAN FIBROBLAST CELLS
By

Xi-De Wang

Alterations in oncogenes, tumor suppressor genes, or genes involved in
DNA repair have been demonstrated to play causal roles in carcinogenesis.
Activation of oncogenes or inactivation of tumor suppressor genes promotes cell
growth, whereas a decreased rate of DNA repair or aberrant repair increases the
mutation rate of the genome, including oncogenes and tumor suppressor genes.
This dissertation concerns two aspects of carcinogenesis, i.e., the role of loss of
function of p53 in the malignant transformation of human fibroblasts in culture
and the role of hREV1 in human cell mutagenesis. The p53 gene is the most
frequently altered tumor suppressor gene in human cancers, and hREV1 is
considered to be an essential gene for mutagenesis induced by DNA damaging
agents.

McCormick and colleagues found that carcinogens such as y-radiation,
ultraviolet (UV) radiation, benzo[a]pyrene diol epoxide (BPDE), or
methylnitrosourea, can induce focus formation in the immortal, chromosomally-
stable, non-tumorigenic human fibroblast cell strain MSU-1.1, and that a high
percentage of the focus-derived cell strains have lost the function of p53. In
addition, only the cell strains that lost p53 could form tumors in athymic mice. To

test whether loss of p53 plays a causal role in allowing cells to form foci in

 

 

the

daIT
hurt
hurt
this

cell

dete
and
Item
exos
reSIl
dam
Virtu
HPF,
”OUT

the k

culture and malignant tumors in mice, I transfected a wild-type p53 gene into a
focus-derived malignant cell strain that had lost p53 function, and determined
the effects of exogenous p53 expression on the cells' transformed phenotypes.
The results indicate Clearly that p53 suppresses focus formation, anchorage-
independence, and the ability to form tumors, strongly suggesting that loss of
p53 predisposes carcinogen-treated MSU-1.1 cells to transformation.

It has been determined that in Saccharomyces cerevisiae, the function of
the REV1 gene is required for mutagenesis induced by a variety of DNA
damaging agents. To determine whether this is also the case for mutagenesis in
human cells, Christopher Lawrence of the University of Rochester Cloned a
human gene homologous to the yeast REV1 gene, and l determined whether
this hREV1 gene is required for human cell mutagenesis. To do so, I generated
cell strains that express hREV1 antisense RNA at high levels and used them to
determine the effect of antisense RNA expression on the mutagenic effect of UV
and of BPDE. The results showed that expression of antisense RNA greatly
reduced the frequency of UV- or BPDE-induced mutants. I then expressed
exogenous hRev1 protein in cells that express antisense RNA and found that it
restored their capability of generating mutations in response to such DNA
damage. These data strongly support the hypothesis that hREV1 is required for
virtually all damage-induced mutations. Amplification and sequencing of the
HPRT cDNA from a series of independent BPDE-induced mutants derived from
normal parental cells and from cells expressing exogenous hRev1 revealed that

the kinds of base substitutions induced were virtually identical.

To my parents and the whole family for their love, encouragement and support
To my wife, Lin-Fang Zeng, for the love she dedicates to our little family

To my teachers and friends who care about us

 

 

 

 

(23'
,..l
rese
fact

aIIOI.

 

EXEC

field

from

Feli;

eDVI
WOU
frien
Ban
Kap
Meg
War
'abo

Critic

ACKNOWLEDGEMENTS

I sincerely thank my mentor, Dr. Veronica M. Maher and Co-Director of the
Carcinogenesis Laboratory, Dr. J. Justin McCormick, for providing me such a
great opportunity to learn and to do research in the exciting field of cancer
research. Their guidance, support, and encouragement are the most pivotal
factors in my pursuance of the Ph.D. degree. Studying under their direction
allowed me an opportunity to learn the ways of great scientists to design and
execute the research, to manage the laboratory, and to strive to succeed in the
field that they have chosen.

I would also like to indicate my appreciation for the advice and support
from the other members of my graduate committee, Drs. Walter J. Esselman,
Felipe Kierszenbaum, and John L. Wang.

My dissertation research could not have been done without the excellent
environment of the Carcinogenesis Laboratory and of Michigan State University. I
would like to thank the present and former members of the laboratory for their
friendship and assistance. In particular, I would like to acknowledge Michele
Battle, Katherine Bergdolt, Clarissa Dallas, Jackie Dao, Bethany Heinlen, Evan
Kaplan, Susanne Kleff, Suzanne Kohler, Ziqiang Li, Hongyan Liang, Glenn
McGregor, Terry McManus, Sandra O’Reilly, Beatrice Tung, Lijuan Wang, Yun
Wang and Hong Zhang. I also thank Dr. Katheryn Meek and the staff in her
laboratory for their advice and friendship. I also appreciate Dr. Winnie Chiang for

critically reading part of the literature review and many professors, fellow

 

 

 

 

gav
Cat

DEI

graduate students, undergraduate students and supporting staff at Michigan
State University who taught me or helped me.

I am indebted to the members of my whole family as well. My wonderful
wife, Lin-Fang Zeng, who enjoys the happiness and shares the difficulties of life
with me, is my strongest support. I cherish with her the past, and the present,
and I look forward to the future. Members of my family, including my parents, my
brothers and sisters and their respective families, and members of my wife’s
family, including her parents and her brother, Yibin, and sister-in-Iaw, Bei, all
gave me their full support. I also appreciate the cooperation of my babies,
Catherine Angie and William Jianing. Without the love and/or support of all these

persons, I would not be where I am now.

vi

 

LIST OF T
LIST OF F
LIST OF A

INTRODUC

 

CHAPTER

Literature R

I Carc
A. C
B. E

1. C
a.
b.

_’\J
:7

U
--4..
HPWPVTHZJ-{OO

(D an FTP)

TABLE OF CONTENTS

page
LIST OF TABLES ................................................................................................ xii
LIST OF FIGURES ............................................................................................. xiii
LIST OF ABBREVIATIONS ................................................................................ xv
INTRODUCTION .................................................................................................. 1
CHAPTER 1
Literature Review .................................................................................................. 5
l. Carcinogenesis is a multistep process ....................................................... 5
A. Characteristics Of cancer ...................................................................... 5
B. Evidence for the multistep nature of carcinogenesis ............................. 5
1. Colorectal tumorigenesis ...................................................................... 6
a. Mutations found in colorectal tumors ............................................... 6
b. Stages of the occurrence of mutations .......................................... 10
2. Malignant transformation studies of the MSU-l cell lineage ............... 12
3. Transformation of normal cells with defined steps .............................. 13
C. Cancer genes ..................................................................................... 14
1. Oncogenes ......................................................................................... 14
2. Tumor suppressor genes .................................................................... 15
3. Repair genes ...................................................................................... 16
D. The p53 gene ...................................................................................... 19
1. From a tumor antigen to an oncogene, and from an oncogene to a
tumor suppressor gene ....................................................................... 19
2. Functional domains of p53 .................................................................. 20
3. Activation of p53 ................................................................................. 21
4. Tumor suppression functions of p53 ................................................... 23
a. Growth arrest ................................................................................. 23
b. Apoptosis ....................................................................................... 24
c. Inhibition of angiogenesis .............................................................. 26
d. Senescence ................................................................................... 27
e. Role in DNA repair and homologous recombination ...................... 28
II. The RADG pathway for dealing with DNA damage ................................... 31
A. Cellular responses to DNA damage .................................................... 31
1. Sources of DNA damage .................................................................... 31
2. DNA repair and damage tolerance ..................................................... 32
a. Three major pathways in dealing with DNA damage ..................... 32
b. Excision repair ............................................................................... 33

vii

C. Homologous recombination ........................................................... 34

B. Damage tolerance: RADG pathway in Saccharomyces cerevisiae ..... 34
1. Principal genes involved ..................................................................... 34
a. RADG and RAD18 .......................................................................... 34
b. RAD5 ............................................................................................. 36
c. RAD30 and PCNA ......................................................................... 36
d. Ub, MMSZ and UBC13 .................................................................. 39
e. Genes for mutagenesis: REV3, REV7 and REV1 .......................... 41
f. Damage avoidance and mutagenesis subpathways ...................... 42
2. Damage avoidance mechanisms ........................................................ 43
a. Recombination-dependent mechanism In the error-free damage
tolerance pathway .......................................................................... 43
b. Coordination of two Ubiquitin-conjugating enzymes Rad6 and
Mms2/ch13 ................................................................................. 43
3. Mutagenic mechanisms involving REVS ............................................. 45
a. Molecular Cloning of yeast REV3, REV7 and REV1 genes ............ 45
b. Rev3 and Rev7 proteins form Pol zeta .......................................... 46
c. Studies on Rev1 ............................................................................ 46
1) The Rev1 protein has a deoxycytidyl transferase activity ........... 47
2) Rev1 as a dCMP transferase in abasic site bypassing ............... 47
3) Evidence for a second function of Rev1 ..................................... 49
C. Damage tolerance RAD6 pathway in humans .................................... 51
1. Damage avoidance mechanisms ........................................................ 51
a. hHR6A, hHRGB and hRAD18 ........................................................ 51
b. hMMSZ and UBC13 ....................................................................... 52
2. Translesion synthesis mechanisms .................................................... 53
a. hREV3 ........................................................................................... 53
1) Cloning of hREV3 gene .............................................................. 53
2) The level of hRev3 protein in human cells might be low ............. 54
3) Importance of the function of hREV3 in mammals ..................... 55
4) The role of hRE V3 in mutagenesis ............................................. 56
b. hRE V7 ........................................................................................... 57
c. hREV1 ........................................................................................... 58
1) Cloning of hREV1 gene .............................................................. 58
2) Presence of an out-of-frame ATG on hREV1 open
reading frame ............................................................................. 58
3) Chromosomal location and functional importance ...................... 59
4) hRev1 as a dCMP transferase ................................................... 59
5) Function of hREV1 is required for human cell mutagenesis ....... 60
d. Interactions of hRev3, hRev7 and hRev1 proteins ........................ 61
e. Polymerase eta, homolog of yeast RAD30 .................................... 63
f. Additional lesion-bypassing DNA polymerases ............................. 64
1) Pol iota ....................................................................................... 64
2) Pol kappa ................................................................................... 64
9. Summary of the kinds of lesions bypassed by error-prone DNA
polymerases .................................................................................. 65

viii

h. Lesion-replicating polymerases could act sequentially in

bypassing lesions .......................................................................... 67

3. Relevance of the damage tolerance pathway to carcinogenesis in
humans ............................................................................................... 68
Ill. References ............................................................................................... 69

CHAPTER 2

Re-expression of Wild-type p53 in a Focus-derived Human Fibroblast Strain that
Can Produce Tumors in Athymic Mice Prevents Focus Formation, Anchorage

Independence, and Tumorigenicity, but Does Not Affect Growth in Culture ....... 94
Abstract ........................................................................................................... 95
Introduction ..................................................................................................... 96
Materials and methods .................................................................................... 98

Cell strain and cell culture ............................................................................ 98
Plasmid construction .................................................................................... 99
Transfection ................................................................................................. 99
Growth curves ............................................................................................ 100
UV-irradiation ............................................................................................. 100
Preparation of cell lysates .......................................................................... 100
Western blot analysis ................................................................................. 101
Focus reconstruction assay ....................................................................... 102
Assay of anchorage-independent growth .................................................. 102
Tumorigenicity test ..................................................................................... 103
Results .......................................................................................................... 104

Determining whether functional wild-type p53 could be successfully re-
introduced and expressed at a normal level into cell strain

MSU-1.1y1-2A1 ...................................................................................... 104
Evidence that the level of expression of exogenous wild-type p53 did not
greatly affect the rate Of growth of the two cell strains ........................... 105
Evidence that the exogenous p53 in the transfected strains was .
functional ................................................................................................ 107
Effect of expression of exogenous p53 on focus formation by the focus-
derived cell strain 2A1 ............................................................................ 111
Expression of p53 also reduced the anchorage-independence ................. 112
Effect of expression of exogenous p53 on the tumorigenicity of cell
strains 88A and 85 ................................................................................. 115
Discussion ..................................................................................................... 121
Acknowledgements ....................................................................................... 126
References .................................................................................................... 126
CHAPTER 3
The Function of the Human Homolog of Saccharomyces cerevisiae RE V1 Is
Required for Mutagenesis Induced by UV Light ................................................ 130
ix

Abstract
lntroduc
Mater‘a‘-

cDNA

 

Refereh

 

Abstract ......................................................................................................... 1 31

Introduction ................................................................................................... 132
Materials and methods .................................................................................. 133
CDNA synthesis, 5' Rapid Amplification of cDNA Ends (RACE), and
Cloning .................................................................................................... 133
DNA purification and sequence analysis ................................................... 134
Preparation of cells that express hRE V1 antisense RNA .......................... 135
Northern blot analysis ................................................................................ 136
Determination of the cytotoxic and mutagenic effects of UV radiation ....... 137
Results and discussion .................................................................................. 138
Human cells possess a homolog of yeast REV1 ....................................... 138
UV-induced mutagenesis is much reduced in human cells expressing
hRE V1 antisense RNA ........................................................................... 143
Acknowledgements ....................................................................................... 151
References .................................................................................................... 151
CHAPTER 4

Expression of Exogenous hRev1 in Human Cells Devoid of Mutations as a
Result of Antisense hRE V1 Restores their Ability to Be Mutated by UV or

Benzo[a]pyrene Diol Epoxide ............................................................................ 154
Abstract ......................................................................................................... 1 55
Introduction ................................................................................................... 156
Materials and methods .................................................................................. 158

Cell strains and cell culture ........................................................................ 158
Plasmids and DNA transfection ................................................................. 158
Analysis for the expression of hRev1 protein ............................................. 159
Determination of the cytotoxic and mutagenic effects of UV radiation ....... 161
Determination of the cytotoxic and mutagenic effects of BPDE ................. 162
Sequencing of mutant HPRT cDNA ........................................................... 163
Results .......................................................................................................... 164
Efforts to determine the cellular level of hRev1 protein .............................. 164
Cell strains expressing exogenous Flag-hRev1 protein ............................. 167
Effects of Flag-hRev1 expression on UV—induced mutagenesis ................ 169
Effects of Flag-hRev1 expression on BPDE-induced mutagenesis ........... 172
hREV1 is required for the majority of UV- and BPDE—induced base
substitutions ........................................................................................... 175
Discussion ..................................................................................................... 178
Acknowledgements ....................................................................................... 182
References .................................................................................................... 183

APPENDIX

Roles of DNA Polymerase zeta and Rev1 Protein in Eukaryotic

Mutagenesis and Translesion Replication ........................................................ 186
PROPERTIES OF YEAST POL zeta AND Rev1p ......................................... 190

 

FUNCTL
mscus
ACKNC.
REFER?

 

 

FUNCTIONS OF POL zeta AND REV1p IN HUMANS .................................. 198

DISCUSSION AND CONCLUSIONS ............................................................ 205
ACKNOWLEDGEMENTS ............................................................................. 210
REFERENCES .............................................................................................. 210

xi

CHAPTEF.
Table I. F

CHAPTER
Table 1. F '
lbepalI
Table 2. SI
hepa
Table 3. TLI
train .

CHAPTER

Table 1. E
cell sf;
expreg

CHAPTER

Table 1, cc
BPDE
antiser
DIOIein.
and fin

AppENbix
Table 1' Ap

IlOm 31
Table 2. By

CI’CIObI
addUct

 

LIST OF TABLES

CHAPTER 1

Table 1. Properties of lesion-replicating DNA polymerases .....................

CHAPTER 2
Table 1. Frequency of focus formation by cell strains 88A, 85, and

the parent strain 2A1 .........................................................................

Table 2. Size distribution of colonies formed by cell strains 88A, 85,

the parent strain 2A1, and two control strains ....................................

Table 3. Tumorigenicity of cell strains 88A, 85, and the parental

strain 2A1 ...........................................................................................

CHAPTER 3

Table 1. Example of UV cytotoxicity and mutagenicity in parental
cell strain MSU-1.2-7AGM and two derivative cell strains

expressing hRE V1 antisense RNA ....................................................

CHAPTER 4

Table 1. Comparison of the kinds of base substitutions induced by
BPDE in the HPRT gene of cells that express hREV1
antisense RNA, but also exogenous Flag-tagged hRev1
protein with the kinds induced in the MSU-1.2 parental strain

and finite life span diploid human fibroblasts. ....................................

APPENDIX
Table 1. Apparent efficiencies for extension by pol zeta or pol alpha

from single terminal base-pair mismatches .......................................

Table 2. Bypass frequencies of an abasic site, T-T cis-syn
cyclobutane dimer, and T-T pyrimidine (6-4) pyrimidinone
adduct in REV, rev1 deletion, rev1-1, and rev3 deletion strains

of yeast ..............................................................................................

xii

page

.......... 66

........ 114

........ 117

........ 119

........ 150

........ 179

........ 192

........ 195

CHAPTER
Figste l. l'.
expres
Figure 2. E
does "
FinreB E
S and 8:".
Figu'ed A
88A a"

Figure 5. P
Strains

Ffigure 6. L5.
Cells de

 

CHAPTER

Figure 1. (A
(BI De;
melanc
locaticr

FIQUIQ 2. 8:
Yeast F

Figure 3. N.
anfiseh

Figure 4_ PA
7AGM
MGM
254 ”It

CHAPIER .

SIIain 7

 

LIST OF FIGURES

CHAPTER 2
Figure 1. Western blot analysis of cell strains 88A and 85 for the

expression of p53 protein ..................................................................

Figure 2. Expression of exogenous p53 in cell strains 88A and 85

does not significantly affect the rate of cell growth ............................

Figure 3. Expression of p53 and p21 proteins in cell strains 88A

and 85 in response to UV irradiation ..................................................

Figure 4. A representative set of foci formed by cell strains 2A1,

88A and 85 .......................................................................................

Figure 5. Photos of representative field of colony formed by cell

strains 88A, 85 and 2A1 in soft agar .................................................

Figure 6. Lack of or markedly reduced expression of p53 protein in

cells derived from tumors formed by 88A cells ..................................

CHAPTER 3

Figure 1. (A) Sequence of the translation product of hREV1 mRNA.
(B) Depiction Of regions of similarity to C. elegans and D.
melanogaster sequences (open boxes) together with the

location of the predicted motifs ..........................................................

Figure 2. Schematic representation of the alignment of hRE V1 and

yeast RE V1 protein sequences ..........................................................

Figure 3. Northern blot analysis of the level of expression of hREV1

antisense RNA in the cell strains .......................................................

Figure 4. Percent survival (A) and frequencies of UV-induced 6-
thioguanine resistant mutants (B) in the parent cell strain
7AGM and the hRE V1 antisense RNA-expressing cell strains
7AGM-17C-R1 and 7AGM-1ZB-R1, plotted against fluence Of

254 nm UV ........................................................................................

CHAPTER 4
Figure 1. Evidence that endogenous hRev1 protein in the parental

strain 7AGM was below the detection limit of Western blotting .........

xiii

page

........ 106

......... 108

........ 110

......... 113

......... 116

........ 120

........ 140

........ 141

........ 145

......... 148

......... 166

FgJeZ
emf:

FgweB
great
IIECL

Fgae4
great
freqo

Fgues I
find

 

APPENC

Fguel.
GsbP
Figure 2.
amm:
Figure 3.
fiom:
Figure 4, F
inuv-
(Alor
flee Cr

 

Figure 2. Western blot analysis of transfected cell strains for the
expression of exogenous Flag-hRev1 protein ............................................ 168

Figure 3. Evidence that expression of Flag-hRev1 protein does not
greatly affect cell survival, but significantly increases the
frequency of UV-induced mutants .............................................................. 170

Figure 4. Evidence that expression of Flag-hRev1 protein does not
greatly affect cell survival but significantly increases the

frequency of BPDE-induced mutants ......................................................... 173
Figure 5. Frequency of mutants induced by BPDE expressed as a

function of percent survival of the target cells ............................................ 176
APPENDIX
Figure 1. Assay of dCMP transferase activity for Gst—Rev1 p and

Gst-Rev1-1 p fusion proteins ...................................................................... 197

Figure 2. Sequence comparisons of conserved regions in the
amino- and carboxy-terminus of Rev3p from various species ................... 199

Figure 3. Sequence comparisons Of conserved regions of Rev1p
from various species. ................................................................................. 201

Figure 4. Percent survival and frequency of 6-thioguanine mutants
in UV-irradiated human cells expressing REV3 antisense RNA
(A) or RE V1 antisense RNA (B), together with their antisense-
free controls. .............................................................................................. 204

xiv

33.:

BPDE:

EST:
FAP:
HNPCC:
HPRT:
LOH:
MMR:
MMS;
MNU:
NER:
ORE
PM;
SIS;
TG:

ch

VEGF;
XPV:

 

a.a.:
AP:

BPDE:

EST:

FAP:

HNPCC:

HPRT:
LOH:
MMR:
MMS:
MNU:
NER:
ORF:
Pol:
STS:
TG:
ch:
UV:
VEGF:
XPV:

LIST OF ABBREVIATIONS

amino acid

apurinic/apyrimidinic

(iI'7B , 8a-dihyd roxy-9a, 1 0a-epoxy—7,8,9, 1 O-tetrahyd ro-
benzo[a]pyrene

expressed sequence tag

familial adenomatous polypopsis
hereditary non-polypopsis colon cancer
hypoxanthine phosphoribosyltransferase
loss of heterozygosity

mismatch repair

methyl methanesulfonate
N-methyl-N-nitrosourea

nucleotide excision repair

open reading frame

DNA polymerase

sequence tagged site

6-thioguanine

ubiquitin-conjugating enzyme

ultraviolet radiation

vascular endothelial growth factor

xeroderma pigmentosum variant

 

 

 

Ca
sefies of
malignant
oncogene
project sit
a potent ‘

broblasts

by DNA da

 

Cha
pursued ir
carcinogen
CaIClnogen
953, The
cerevisiae
IranSlesion
homOIOQs;
QEnes belc

Cha
reseal Ch. I
CanCer Re

"‘9 ability ,

INTRODUCTION

Carcinogenesis is a multistep process, by which a cell accumulates a
series of genetic changes until it finally becomes a cell capable of forming a
malignant tumor. Three classes of genes are involved in this process:
oncogenes, tumor suppressor genes, and DNA repair genes. My dissertation
project studies two aspects of carcinogenesis: the role of loss of function of p53,
a potent tumor suppressor gene, in the malignant transformation of human
fibroblasts and the role of the hREV1 gene in human cell mutagenesis induced
by DNA damaging agents.

Chapter 1 is a review on the literature closely related to the research that I
pursued in my dissertation project. The first section presents a review of
carcinogenesis, including a brief description of the multistep model of
carcinogenesis and a summary of the functioning of the tumor suppressor gene
p53. The second section discusses in greater detail the Saccharomyces
cerevisiae yeast RE V1 gene and its human homolog, hREV1; the mechanisms of
translesion synthesis involving yeast Rev1, Rev3, Rev7 and their human
homologs; and the RAD6/hRAD6 damage tolerance pathway to which these
genes belong.

Chapters 2, 3 and 4, as well as the Appendix, summarize my dissertation
research. Chapter 2 represents a manuscript to be submitted for publication in
Cancer Research. It examines the relationship between loss of p53 function and

the ability of human fibroblasts of the MSU-1 lineage to express characteristics of

 

assessing

Caren-age
ll befits. a
findron or
tumors in h
lite cells to

was to Iran.

 

that had I05
could revere
053 SUDIIre
form tUmors
""0 the rele

IUIIdamema

Chap
Academy (It
be human

hREVI Was

malignant cells, i.e., ability to form foci, colonies in soft-agar, and/or tumors in
athymic mice. Focus formation is a transformation phenotype in which cells in
culture continue replicating, pile up, or form multilayers when the non-
transfonned cells form a confluent monolayer. Anchorage-independence, i.e., the
ability of cells in culture to form colonies in semi-solid medium, reflects the ability
of cells to grow without attachment. Athymic mice are used as animal models for
assessing the tumorigenicity of human cells. It had been shown in the
Carcinogenesis Laboratory that carcinogens can induce focus formation in MSU-
1.1 cells, and that a high percentage of the focus-derived cells have lost the
function of p53. It was also found that only cells that had lost p53 could form
tumors in mice. My research was devoted to testing whether loss of p53 causes
the cells to form foci in culture and malignant tumors in mice. The strategy I used
was to transfect a wild-type p53 gene into a focus-derived malignant cell strain
that had lost both copies of the gene and determine whether expression of p53
could reverse the cell’s transformed phenotypes. The results clearly indicate that
p53 suppresses focus formation, anchorage-independence, and the ability to
form tumors. Terrence P. McManus, the second author, provided critical insight
into the relationship between focus formation and loss of p53 and carried out
fundamental background studies on this important question.

Chapter 3 is a paper published in the Proceedings of the National
Academy of Sciences USA in 2000. It describes the earliest study on the role of
the human REV1 gene in UV-induced mutagenesis. The yeast counterpart of

hRE V1 was known to play an essential role in damage-induced mutagenesis and

 

I0 functi:
|
synthess
Lawrence
test whet'
Usng an
hPEI/l at
mutants. TI

and the Ir:

human Ce

 

elli'v‘ression
Cha
Molecular
determine
Indicate the
0f BPDE?"
mulants in
significenu
Strongly '5
mmagene:
IESIStant C
errogenOU1
damagen

83+

mot! Dr

to function as an auxiliary factor required for DNA polymerase zeta to carry out
synthesis past lesions that block replication. Drs. Peter Gibbs and Christopher
Lawrence Of the University of Rochester Cloned the hREV1 gene. My role was to
test whether hREV1 is required for mutagenesis induced by UV in human cells.
Using an antisense RNA strategy, I found that cells expressing a high level of
hREV1 antisense RNA show a greatly reduced frequency of UV-induced
mutants. The third author, Ziqiang Li, carried out several mutagenesis assays,
and the fourth author, Terrence P. McManus, carried out the transfection of the
human cells and isolation of the transfectant clones to be screened for
expression of the antisense RNA.

Chapter 4 represents a manuscript to be sumitted for publication in
Molecular and Cellular Biology. It summarizes further studies carried out to
determine the role of hREV1 in UV and BPDE-induced mutagenesis. The data
indicate that antisense RNA-expressing cells exhibit a greatly reduced frequency
of BPDE-induced mutants compared to normal cells and that the frequency of
mutants induced by UV or by BPDE in the antisense RNA-expressing cells was
significantly increased by expression of exogenous hRev1 protein. These data
strongly suggest that hRev1 is required for both UV- and BPDE-induced
mutagenesis. Sequencing of the HPRT cDNA amplified from 6-thioguanine-
resistant clones derived from the normal parental cells and cells expressing the
exogenous hRev1 protein show that hREV1 is required for the majority of
damage-induced base substitutions. Dr. Christopher Lawrence, the second

author, provided critical insights into the possible function of the human homolog

of yeast I

Murakurn

 

expressic
I

The

REV1 an:
Dr. Christ:
2000 Col-3

Responses

published 1

 

LXV. My gr

I“ W muta

 

of yeast Rev1 and the polyclonal antibody 468 against hRev1. Dr. Yoshiki
Murakumo, the third author, constructed and provided the plasmid for the
expression of Flag-hRev1 protein.

The Appendix is a review article regarding the roles of yeast and human
REV1 and REV3 in mutagenesis and translesion replication. It was prepared by
Dr. Christopher Lawrence, and he presented a summary of the research in the
2000 Cold Spring Harbor Symposium on Quantitative Biology, entitled Biological
Responses to DNA Damage. The text, which constitues the Appendix, has been
published in the Cold Spring Harbor Symposium on Quantitative Biology Volume
LXV. My contribution to this paper was to provide the data on the role of hREV1

in UV mutagenesis in human cells.

 

 

 

A. Ch
Ca'
conditions
exquisite
without re
to Cancer.
Weinberg
Insensitivrt,
Detential, S.

and Weinbe

3- Evid
Seve
multistep O
Cancers. in:
68CIphagus.

peTIQd aIIQV

CHAPTER 1

Literature Review

I. Carcinogenesis is a multistep process

A. Characteristics of cancer

Cancer is a result of uncontrolled cell proliferation. Under normal
conditions, the multiplication of cells is tightly regulated. Very occasionally, this
exquisite control system breaks down, and a cell begins to grow and divide
without responding to regulation. Ultimately, the indefinite proliferation gives rise
to cancer. At least six essential alterations occur to cancer cells (Hanahan and
Weinberg, 2000). These characteristics include self-sufficiency in growth signals,
insensitivity to anti-growth signals, evasion from apoptosis, unlimited replicative
potential, sustained angiogenesis, and tissue invasion and metastasis (Hanahan

and Weinberg, 2000).

B. Evidence for the multistep nature of carcinogenesis

Several lines of evidence have led to the insight that carcinogenesis is a
multistep or multi-event process. The incidence rate of most common human
cancers, including cancers of the prostate, stomach, skin, rectum, pancreas and
esophagus, increases dramatically with age (Miller, 1980). Apparently a longer
period allows more events to happen. Patients who undergo radiation therapy
often develop new cancers, but the tumors do not form immediately. Neoplasia of
different levels of malignancy can be found in the colon (Kinzler and Vogelstein,

1996). In familial adenomatous polyposis (FAP) patients, one or a few, but not

 

all. of the
sporadic
frequent.
sufficient
of two or
of loci of
(Land et
those th

associarec

 

 

s‘r’stematic

discussed

1- Col

a. Mo

all, of the hundreds of adenomatous polyps eventually progresses to cancer. In
sporadic colorectal tumors, malignant carcinoma and premalignant adenoma are
frequently found adjacent to one another. Transfer of one oncogene was not
sufficient to cause Obvious changes in primary rodent fibroblasts, but co-transfer
of two oncogenes caused abnormal growth of recipient cells, allowing formation
of foci of piled-up cells, and these focus-derived cells formed tumors in mice
(Land et al., 1983). Human papilloma viruses have many subtypes, but only
those whose E6 and E7 proteins respectively bind Rb and p53 proteins are
associated with cervical cancers in human (Munger et al., 1989). Three lines of
systematic research supporting the mutlistep nature of carcinogenesis will be

discussed in a greater detail below.

1. Colorectal tumorigenesis

a. Mutations found in colorectal tumors

Clinical and histopathology studies have identified in the colon of patients
several types of neoplasia, including hyperproliferative epithelium (hyperplasia),
adenoma (benign tumor), carcinoma-in-situ (malignant tumor) and invasive
carcinoma and have suggested that most, if not all, carcinomas arise from
preexisting adenomas (Toribara and Sleisenger, 1995). The availability of tissues
at different steps provides much advantage in studying the genetic and molecular
mechanisms underlying the multistep carcinogenesis.

The first gene identified to involve in colorectal carcinogenesis is the APC
(Adenomatous Polyposis Coli) gene (Bodmer et al., 1987; Leppert et al., 1987). It

was assigned to chromosome 5q21-22 in genetic linkage studies which compare

the frequ-
I

 

(genotype
familial a:
was also _
APC gene
aL 1991
tumors the
mutation :
1992).

The

 

Sequence
tI131 are im
Asef-bindi r
”IICIOlubul.
key IUmOr
tree Bcate
CIUSIer reg
Patients re
ISIeber St

mUIafiOnS

geneS in I

review, Se

compIex VI

the frequency of coinheritance of disease phenotypes and chromosomal markers
(genotypes). Mutation of APC gene is responsible for the hereditary disease
familial adenomatous polyposis (FAP). Loss Of heterozygosity (LOH) in this gene
was also found in 20% of sporadic colorectal tumors (Solomon et al., 1987). The
APC gene was subsequently cloned by the positional cloning strategy (Groden et
al., 1991; Kinzler et al., 1991). Studies indicated that more than 80% of the
tumors that developed in FAP and non-FAP patients had at least one inactivating
mutation in APC gene (Nishisho et al., 1991; Miyoshi et al., 1992; Powell et al.,
1992).

The APC gene encodes a cytoplasmic protein of 2843 amino acids.
Sequence analysis of this large protein has identified several domains and motifs
that are important for APC function. These include homodimerization domain, the
Asef-binding armidillo domain, domains for binding B—catenin, axin and
microtubule, and nuclear import and export sequences (Sieber et al., 2000). The
key tumor suppressor function of APC lies in its ability to bind and destabilize the
free B-catenin protein (Smits et al., 1999). The mutation hot spots or mutation
cluster region identified in APC gene of both FAP and sporadic colorectal cancer
patients reside in the ts-catenin binding and regulation domain of APC protein
(Sieber et al., 2000). In the 15% of colon carcinomas that retain wild-type APC,

mutations were found in B-catenin (Morin et al., 1997). In addtion, mutations in

genes in the same pathway as B-catenin can also initiate tumorigenesis (for a
review, see Bienz and Clevers, 2000). In normal cells, APC protein forms a

complex with axin, which recruits B-Catenin and facilitates the phosphorylation of

ficatenir
targeted
APC and

complex

 

 

modulate:
bansmerr
the activl't

3-Catenin

 

nuclear an,
0nc°9€nes
al., 2000).
Mut
mmorlgen,
K-ras, Ioce
F88 game
Plasma m.
term and ;
”nSIImulat
releaSe Of
interACIS \
GTPase a
IGAp)

. hy,
aqs as a

B-catenin by glycogen synthase kinase 38 (GSK3—B). Phosphorylated B-catenin is
targeted for deconstruction at the proteasome. GSK3-B can also phosphorylate
APC and axin, and thereby enhance the binding of B-catenin to APC-axin
complex and increase B-catenin degradation. The activity of GSK3—B is
modulated by the Wnt-1 pathway. Upon binding of Wnt-1 peptide to its
transmembrane receptor frizzled, the dishevelled protein is activated and inhibits
the activity of GSK3-B. The inhibition Of GSK3-B reduces the phosphorylation of
B-catenin and thereby represses its degradation. The resulting accumulation of

nuclear and cytoplasmic B-catenin up-regulates the transcription of several proto-
oncogenes including cyclin D1 and c-myc by transcription factor ch (Sieber et
al., 2000).

Mutation of a ras gene is another important factor in colorectal
tumorigenesis. There are three members in the res gene family: N-ras, H-ras and
K-ras, located on chromosomes 1, 11 and 12, respectively (Barbacid, 1987). The
ras gene encodes a small GTPase associated with the inner surface of the
plasma membrane. Like all other GTPases, it exists in a GDP-bound, inactive
form and a GTP-bound, active form. The GDP-bound form is the major form in
unstimulated state. Guanine nucleotide exchange factors (GEF) catalyze the
release of GDP from Ras, allowing GTP to bind. The GTP-bound Ras protein
interacts with down stream effectors and activates them. Finally, the intrinsic
GTPase activity of Ras, further enhanced further by GTPase activating proteins
(GAP), hydrolyzes the GTP into GDP and inactivates Ras. In cells, Ras protein

acts as a relay switch that transmits extracellular signal like epidermal growth

. star IEGF
is the mic:
all proliter
effectors i
kinases (P
19%). The
cell cycle
inhibitors. i
Mut
138 gene (
majority 04,
mutations
activated 1
Protein. In
Fortester

frequently

MO

 

 

(BakEI e:
Pctem tu.

(6%”ka

 

factor (EGF) to intracellular signaling cascades. The best characterized cascade
is the mitogen-activated protein kinases (MAPK) pathway involved in controlling
cell proliferation, through the effector protein Raf serine/threonine kinase. Other
effectors include lipid kinases such as phosphoinositide 3-phosphate lipid
kinases (Pl3Ks) and some guanine nucleotide exchange factors (GEF) (Marshall,
1996). The links between activated Ras protein and deregulated cell cycle are
cell cycle regulatory proteins, such as cyclin D1 and cyclin-dependent kinase
inhibitors, including p21 and p27 (Pruitt and Der, 2001).

Mutation of one or other of the res proto-oncogenes or amplification of a
ras gene (Bos, 1988) has been found in many types of human neoplasia. The
majority of alterations are point mutations in codons 12, 13 and 61. The point
mutations abolish the intrinsic GTPase activity and result in a constitutively
activated Ras protein, while gene amplification causes an elevated level of Ras
protein. In colorectal tumors, 40-50% have point mutations (Bos et al., 1987;
Forrester et al., 1987), and among the res gene family, K-ras is the most
frequently mutated ras gene (Vogelstein et al., 1988).

More than 75% of colorectal carcinomas examined showed loss of
Chromosome 17p (Fearon et al., 1987). Chromosome mapping with twenty
Chromosome 17p markers localized the deleted region to 17p12 to 17p13.3
(Baker et al., 1989). This region contains p53 (Isobe et al., 1986), which is a
potent tumor suppressor gene mutated in more than half of human cancers

(Greenblatt et al., 1994). Li-Fraumeni syndrome patients inherit loss of the

   

function of

em th

endencei;
Pancreatic
pancreatic
a role in
mutations
dISOfder C
QBSIrointeg
growth fat:
recelllor,
DIOIeIns! E
and Inlera
Cyme' Suc

abOIISI‘leS .

 

function of one of their p53 genes and retain only one functional copy of that
gene. They are known to be predisposed to various cancers (Malkin et al., 1990).

In addition to Chromosomes 5g and 17p, where the APC gene and p53
gene are located, allelic loss of 18q is also frequently found in colorectal cancers
(Vogelstein et al., 1989). The gene lost in chromosome 18q was originally
believed to be the DCC gene (deleted in colorectal cancer), which encodes for a
protein that shares a significant sequence similarity to cell adhesion molecules
such as N-CAM (Fearon et al., 1990; Cho et al., 1994). However, recent
evidence indicates that loss of an adjacent gene, DPC4/SMAD4 (DPC: deleted in
pancreatic cancer), localized to 18q21 and lost in about 90% of human
pancreatic carcinomas (Hahn et al., 1996), is more likely to be the event playing
a role in colorectal carcinogenesis (Thiagalingam et al., 1996). In addition,
mutations in the SMAD4 gene also cause Juvenile Polyposis Syndrome, a
disorder characterized by a predisposition to hamartomatous polyps and
gastrointestinal cancer (Howe et al., 1998). SMAD4 functions in the transforming
growth factor B (TGF-B) signaling pathway. Upon the binding of TGF-B to its
receptor, the SMAD4 protein forms a heteromeric complex with two other
proteins, SMAD2 and SMAD3, and the complex translocates into the nucleus
and interacts with transcription factors that transactivate inhibitory factors of cell
cycle, such as p15 (Feng et al., 2000). Disruption of SMAD4/DPC4 function

abolishes TGF-B-induced growth control and apoptosis (Derynck et al., 1998)

b. Stages of the occurrence of mutations

1O

e...

 

 

 

in colore-I
componj
adenoma
tumors al
are lesrc
that have
these tr
charater;

At

Similar to

 

Ofadenor
loss of Cf
found in ;
Ilt-‘Ilerozy,S
adenoma
‘038 of17
about 50
timing of
mutation
of them is
18q DTBCE

Benign tumors (adenoma) and malignant tumors (carcinoma) often coexist
in colorectal cancer patients. Based on the size, grade of dysplasia and villous
component of adenomas, Vogelstein et al. (1988) have further divided the
adenomas obtained from patients into three subgroups, Class I, II and Ill. Class I
tumors are small tubular adenomas with low-grade dysplasia. Class II adenomas
are lesions that had not progressed to carcinoma, and Class III are adenomas
that have given rise to small areas of invasive adenocarcinoma. Establishment of
these three benign stages and one malignant stage allows the molecular
Charaterization of genetic changes occurring at each step.

About 50% of Class II and Ill adenomas have K-ras mutations, which is
similar to the frequency of carcinomas. In addition, K-ras mutation occurs in 58%
of adenomas larger than 1 cm, but only in 9% of those under 1 cm in size. Allelic
loss of chromosome 5 is not detected in all Class adenomas examined, but is
found in 29% of either Class II or III adenomas and 36% of carcinomas. Loss of
heterozygosity of chromosome 18q and 17p is not common in Class I and II
adenomas (less than 13%). But it is common in carcinomas (about 75%). While
loss of 17p sequence is found in 24% Class III adenomas, LOH of 18q is found in
about 50% of Class III adenomas. These data revealed a possible order and
timing of genetic changes in colorectal carcinogenesis. That is, Ras gene
mutation and deletion of APC gene happen relatively early but probably neither
of them is the first event. Alterations in 18q and 17p happen later, and deletion in
18q precedes the loss of 17p. These data indicate that carcinogenesis happens

in several steps, and every step involves a genetic change.

11

2. Malignant transformation studies of the MSU-1 cell lineage

To investigate the mechanisms of malignant transformation of human
cells, McCormick and colleagues (Morgan et al., 1991) developed an infinite life
span human fibroblast cell strain, MSU-1.1. This was achieved in part by
transfection of a v-myc oncogene into a diploid normal fibroblast strain
(designated LG1) derived from the foreskin of a normal neonate. The vast
majority of the descendants of this Cloned transfectant eventually entered crisis
and senesced. However, continued feeding and subculturing of the population
yielded an infinite life span cell strain that had escaped senescence. This cell
strain, designated MSU-1.1, has a stable, near-diploid karyotype composed of 45
chromosomes, including two marker Chromosomes. It expresses endogenous
telomerase activity, as shown by the Telomeric Repeat Amplification Protocol
(TRAP) assay (McCormick et al., unpublished data), and grows in 5% serum as
rapidly as early passage finite life span cells grown in 10% serum. Examination
of populations stored frozen at earlier passages revealed the presence of a
diploid precursor strain, designated MSU-1.0, which grows slowly. The MSU-1.0
cells cannot be transformed into malignant cells, whereas the MSU-1.1 cells can
be. The possible role of the two marker Chromosomes present in the MSU-1.1
cell strain in malignant transformation is under vigorous investigation.

In contrast to primary normal human fibroblasts, the nontumorigenic MSU-
1.1 cells can be transformed into cells capable of forming tumors in athymic mice
with a short latency by transfecting them with an activated (mutated) H-ras

(Hurlin et al., 1989), N-ras (Wilson et al., 1990) or K-ras (Fry et al., 1990), or by

12

 

exposingh

al., 199":

 

Examine
overexp'

1990). v.
exhibitec
(Yang at
For exarr
formed

transacti
derived

was IOSt
Plays a
SUmmar
from the
marker
Carcino!
funclior
Imeage

0f Dorrr

expres:

exposing them to a single dose of y-radiation (Reinhold et al., 1996; O'Reilly et
al., 1998) or chemical carcinogen (Yang et al., 1992; Boley et al., 2000).
Examination showed that the cells derived from tumors produced by cells
overexpressing such oncogenes remained diploid (Hurlin et al., 1989; Fry et al.,
1990), whereas the cells transformed to malignancy by carcinogen treatment
exhibited chromosomal Changes additional to those two marker chromosomes
(Yang et al., 1992; Reinhold et al., 1996; O'Reilly et al., 1998; Boley et al., 2000).
For example, a large fraction (240%) of independent cell strains derived from foci
formed by MSU-1.1 cells treated with carcinogens had lost their p53
transactivation function (O'Reilly et al., 1998; Boley et al., 2000). In a focus-
derived cell strain, designated MSU-1.1-y1-2A1, one copy of chromosome 17
was lost. These data suggested that the loss of p53, a tumor suppressor gene,
plays a causal role in transformation of lVISU-1.1 cells by carcinogens. In
summary, in this lineage of cells, as the cells become more tumorigenic, i.e.,
from the normal LG1 cells, the immortal MSU-1.0 cells, the MSU-1.1 cells with
marker chromosomes, to tumorigenic cells transformed by oncogenes or
carcinogens, more genetic alterations occur, and the changes can be gain-of-
function of oncogenes or loss-of-function of tumor suppressor genes. This
lineage of cells provides an excellent model of stepwise malignant transformation

of normal human fibroblasts in culture.

3. Transformation of normal cells with defined steps

Primary rodent cells can be easily transformed into malignant cells by

expressing two oncogenes (Land et al., 1983 and Ruley, 1983), but efforts trying

13

I0 CODVE

 

have yié
success‘
by succe
hesume
aficfi g

(EEnbaa

 

manfiene
Ial’ge-T I

(via R88

to convert primary human cells into tumorigenic cells by transfecting oncogenes
have yielded little success. Very recently, Hahn et al. (1999) reported a
successful tumorigenic conversion of normal human epithelial and fibroblast cells
by successive ectopic expression of the telomerase catalytic subunit (hTERT),
the simian virus 40 large-T antigen and an oncogenic allele of H-ras. This same
set of genes can transform primary human mammary epithelial cells as well
(Elenbaas et al., 2001). These data illustrate elegantly the mutlstep nature of
carcinogenesis and indicate that a minimal of three events, i.e., telomere
maintenance (by hTERT), inactivation of growth control (of p53 and pr by SV40
large-T antigen), and constitutive activation of the mitogen-response pathway

(via Ras) are sufficient to create tumor cells directly from primary human cells.

C. Cancer genes

Three classes of genes are mainly involved in carcinogenesis. They are
oncogenes, tumor suppressor genes and repair genes. These genes will be

briefly discussed.

1. Oncogenes

Oncogenes are genes that are able to transform cells in culture or to
induce tumors in animals. They are often mutated forms of protooncogenes, i.e.,
cellular genes that function to encourage the normal cell growth (Bishop, 1995).
The conversion of a protooncogene into an oncogene involves a gain-of-function
mutation, which could be point mutation, gene amplification or chromosomal

translocation. Oncogenes are often constitutively active, or expressed at an

14

inappro;
uncontrc
mutation ~
enough 2

Tr
importar
cascade

growth if
I

transducI
participa:
example
Many hur
for 8We
oncrigenE
involved i

JUI’)‘ and

2. Tu
Tu
glomh‘ a
tUrTtor Su;
InaCIIVate
mUtathnS

as tlJrTlor

 

inappropriate level. The activation or overexpression of oncogenes promotes
uncontrolled cell growth. Because of this gain-of-function mode of action,
mutation in one copy of the oncogene, rather than mutations in both copies, is
enough to produce its oncogenic effects.

The hallmark of cancer is uncontrolled cell growth. One of the most
important mechanisms of controlling cell growth is the signal transduction
cascade triggered by growth factors. This cascade often consists of extracellular
growth factor, membrane-bound growth factor receptor, intracellular signal
transducers and nuclear transcription factors. Indeed, many oncogene products
participate in these signal transduction cascades (Lodish et al., 2000). For
example, the Sis oncogene, encodes a type of platelet-derived growth factor.
Many human breast cancers overexpress Her2 oncoprotein, which is a receptor
for epidermal growth factor and other related hormones. Ras, the first human
oncogene identified, encodes a small GTPase, one kind of signal transducer
involved in the MAP kinase pathway. Overexpression of transcription factors Fos,

Jun, and Myc can induce transformation (Lodish et al., 2000).

2. Tumor suppressor genes

Tumor suppressor genes are genes that normally function to inhibit cell
growth, and when they are mutated, cells will grow without a restraint. For a
tumor suppressor gene to lose its function, both copies of this gene must be
inactivated. The ways for inactivating tumor suppressor genes can be allelic loss,
mutations or gene siliencing. At least four broad classes of genes are recognized

as tumor suppressor genes (Lodish et al., 2000). They are inhibitors of cell cycle

15

progress
month..
BRCAl
T'
(Friend 6
developo
retina o‘
retinobla
cells in tr
Loss oft
gene is it
Th

 

for G1 to
IS DhOSpr
the Rb is
proQIESSE
Rb “9mm
of ex°ger
inactivate

3. Re

progression, such as RB, p21 and p16; receptors that function to inhibit cell
growth, like TGF-B receptors, genes involved in checkpoint controls, e.g., ATM,
BRCA1 and genes promoting apoptosis, like p53.

The retinoblastoma gene (RB) is the first tumor suppressor gene identified
(Friend et al., 1986; Lee et al., 1987). Loss of function of this gene underlies the
development of retinoblastoma, a malignant tumor arising in the undifferentiated
retina of one or both eyes in early Childhood. Since children with hereditary
retinoblastoma develop tumor from only about one cell among 106 developing
cells in the retina, the inherited mutation of RB is recessive to the wild-type allele.
Loss of the function of the second allele is required for a tumor to form. The Rb
gene is the prototype of tumor suppressor gene.

The Rb protein is a nuclear protein, which normally binds to the E2F family
of transcription factors (E2F) and represses the transcription of genes required
for G1 to S transition of the cell cycle by E2F (Weinberg, 1995). When Rb protein
is phosphorylated by cyclin-dependent kinases complexed with cyclin proteins,
the Rb is inactivated and the transcription by E2F is activated and the cell cycle
progresses (Lundberg and Weinberg, 1998; Harbour et al., 1999). Inactivation of
Rb removes the constraint on cell cycle progression imposed by Rb. Expression
of exogenous Rb protein in retinoblastoma or osteosarcoma cell strains that had
inactivated endogenous RB genes suppresses the growth, anchorage-

independence and tumorigenicity of these tumor cells (Huang et al., 1988).

3. Repair genes

16

P.
function
the mu‘.
suppress
modifica‘

1995). F

suscept :

Tr
illustrate:
accounts
was map.
In IhIS reg
99ne reqr
MUIaIIOns

InCIUding

 

PMS2 IN?
mutL 99”
microSE-Ite
kindreds v
known at
are ”We

50%, 40%

Repair genes are genes involved in various DNA repair mechanisms
functioning in human cells. They do not affect the cell growth directly but reduce
the mutation rate of the entire genome, including oncogenes and tumor
suppressor genes. In mammalian cells, DNA repair pathways repair ~100,000
modifications happening to each DNA molecule every day (Friedberg et al.,
1995). Failure in these genome maintenance mechanisms causes cancer
susceptibility in a variety of tissues (Hoeijmakers, 2001).

The importance of DNA repair in guarding against cancer is best
illustrated in the Hereditary Non-Polyposis Colon Cancer (HNPCC), which
accounts for 5-8% of all colon cancers. The first genetic locus linked to HNPCC
was mapped to chromosome 2p15-16 (Peltomaki et al., 1993). The gene deleted
in this region was found to be a human homologue of bacteria MutS (hMSH2), a
gene required for mismatch repair (MMR) (Leach et al., 1993; Fishel et al., 1993).
Mutations in other genes involved in human MMR were subsequently found,
including hMLH1 (Bronner et al., 1994; Papadopoulos et al., 1994), PMS1 and
PM82 (Nicolaides et al., 1994), all Of which are homologues of the prokaryotic
mutL gene. Indeed, 92% of tumors derived from 74 HNPCC kindreds showed
microsatellite instability, a characteristic of MMR deficiency, and 70% of the 48
kindreds with such instability had mutations in the coding region of 5 MMR genes
known at that time (Liu et al., 1996). Defects in the hMLH1, hMSH2 and hMSH6
are three most common germline mutations among HNPCC, with approximately

50%, 40% and 10% frequency respectively (Peltomaki, 2001).

17

 

exonuc'
loops It
1998). l
Binding
discrimx
removal

heterodii

 

thhZ a
between
hPmsz'
MMR. bI
D
mICIOSat
in DIOIOC
IIISIabilirj
as TGIF)
for hum;

a Dredip,

The MMR mainly deals with mismatches that have escaped the
exonucleolytic proofreading of the replication machinery and insertion/deletion
loops that are caused by slippage of DNA polymerase during replication (Jiricny,
1998). In bacteria, the lesions are recognized by the homodimeric MutS protein.
Binding of MutL homodimer to MutS helps bringing the MutH protein, a strand
discrimination factor and other necessary factors. The outcome of MMR is the
removal of mistakes on the newly synthesized daughter strand. In humans, the
heterodimer of thh2 and thh6 recognizes mismatches and heterodimer of
thh2 and thh3 recognizes insertion/deletion loops. To mediate the interaction
between recognition complex and other factors, hMlh1 forms a heterodimer with
hPms2, hPms1 or hMlh3. The human MMR system is similar to the prokaryotic
MMR, but has a higher complexity (Harfe and Jinks-Robertson, 2000).

Deficiency of MMR causes at least a 100-fold increase in mutation rate of
microsatellite repeat sequences (Parsons et al., 1993), and enhances mutations
in protooncogenes and tumor suppressor genes. In tumor cells with microsatellite
instability, frameshift mutations have been found in tumor suppressor genes such
as TGFflR/I (Markowitz et al., 1995) and PTEN (Guanti et al., 2000). As models
for human disease, mouse knockouts of MSH2, MSH6, PMSZ, or MLH1 showed

a prediposition to diverse types of tumors (Harfe and Jinks-Robertson, 2000).

18

large—T
(SV40)
transfer

Levine,

 

mainten
Subseq-
transtr
(DELeo
had a p
The Iac
IJenkin
tranSfOI
SUSSES
tUnTor

D. The p53 gene

1. From a tumor antigen to an oncogene, and from an oncogene to a

tumor suppressor gene

The protein p53 was first identified as a ~53kD protein interacting with the
large-T antigen protein encoded by the small DNA tumor virus simian virus 40
(SV40) (Lane and Crawford, 1979). Because the level of this p53 protein in the
transformed cells was enhanced by SV40 infection or transformation (Linzer and
Levine, 1979), and SV40 infection was indispensable for the induction and
maintenance of the transformed state, p53 was believed to be a tumor antigen.
Subsequent studies on p53 revealed that many tumor-derived cells or cells
transformed in culture exhibited a higher level of p53 protein than normal cells
(DeLeO et al., 1979) and that p53 protein expressed in SV40-transformed cells
had a much longer half-life than p53 protein in normal cells (Oren et al., 1981).
The fact that cloned p53 genes could immortalize cells of finite lifespan in vitro
(Jenkins et al., 1984) and could cooperate with the activated H-ras oncogene in
transforming normal cells (Eliyahu et al., 1984; Jenkins et al., 1984) strongly
suggested that p53 was an oncogene. It is now well established that p53 is a
tumor suppressor gene. The p53 genes that helped transform normal cells
(Eliyahu et al., 1984; Jenkins et al., 1984) turned out to be mutant forms of p53
(Hinds et al., 1989). It was shown that mutant p53 could interfere with the
function of wild-type p53 by binding to or competing with the wild-type p53, and in
that way cause stabilization of mutant p53 protein (reviewed in Blagosklonny,

2000). What is more, expression of wild-type p53 was found to inhibit oncogene-

19

induce

 

Eliyar
comes
p53 9»:
1988)
wepS;
who ir

(Snvas

1

 

 

 

divided
domam,
reQUIatO
anfino ;
transcrir
Truant E
loos),
beIWSEI‘
COnSen S
SeCIUenC
(eI‘Deiry
dQwDStre

300-393

induced focus formation by primary rat embryo fibroblasts (Finlay et al., 1989;
Eliyahu et al., 1989). Additional proof that p53 acts as a tumor suppressor gene
comes from genetic studies showing that the chromosomal region harboring the
p53 gene is lost at very high frequency in colorectal tumors (Vogelstein et al.,
1988). It is now known that more than half of human cancers have mutations in
the p53 gene (Greenblatt et al., 1994), and that Li-Fraumeni syndrome patients,
who inherit one mutant allele of p53, are predisposed to various tumors

(Srivastava et al., 1990).

2. Functional domains of p53

The human p53 protein consists of 393 amino acids. It can be roughly
divided into three major functional domains, the N-terminal transactivation
domain, the central sequence-specific DNA-binding domain, and the C-terminal
regulatory domain (Ko and Prives, 1996). The transactivation domain lies within
amino acid residues 1-42 (Unger et al., 1992). This domain recruits basic
transcription factors like TATA box-binding protein (T BP) (Seto et al., 1992;
Truant et al., 1993) and TBP-associated factors (Thut et al., 1995; Farmer et al.,
1996). The sequence-specific binding domain was mapped to the region
between ~80-300 amino acids (Wang et al., 1993; Pavletich et al., 1993). The
consensus sequence that this domain binds to consists of two inverted
sequences of 5'-PuPuPuC(Afl')(T/A)GPyPyPy-3', separated by 0-13 base pairs
(eI-Deiry et al., 1992). Binding of p53 to this sequence in the promoter region of
downstream target genes initiates the transcription of these genes. Residues

300-393 is the C-tenninal regulatory domain. In this domain, there are two

20

subd:

 

(Fade

Khan;
I

adst:

mnb]

”(Hut

ofarr

dneds

 

2500)
tumors
cluster
1994)

transcr

realativ

 

subdomains. One, the tetramerization domain, lying within residues ~320-360

(Pavletich et al., 1993), can promote DNA binding and enhance transcription
(Stenger et al., 1994). The other, consisting of the last 30 amino acid residues,
acts to keep the DNA binding activity of the whole protein latent. This inhibition
can be alleviated by deletion of the C-terminus, mild proteolysis of p53, addition
of a monoclonal antibody against this region, or phosphorylation by casein kinase
II (Hupp et al., 1992). In addition, p53 has a nuclear localization domain, which
directs the transport of p53 protein into the nucleus. Compilation of more than
2,500 p53 gene mutations that were found to have occurred in various human
tumors and tumor cell lines revealed that the vast majority of mutations are
clustered in the central sequence-specific DNA binding domain (Hollstein et al.,
1994), suggesting that p53 exerts its tumor suppression function mainly as a

transcription factor.

3. Activation of p53

In normal or unstressed cells, the level and the activity of p53 are both
relatively low (Giaccia and Kastan, 1998). This is achieved mainly by two
mechanisms, the action of Mdm2 protein and the allosteric inhibition by the C-
terrninal domain of p53. As a transcription factor, p53 transactivates the
expression of its own negative regulator Mdm2 (Barak et al., 1993). The Mdm2
protein can form a complex with p53 protein (Momand et al., 1992) at the
transactivation domain of p53 (Oliner et al., 1993) and thereby conceal the
transactivation domain from basic transcription factors and reduce the level of

transcription induced by p53 (Momand et al., 1992; Oliner et al., 1993). In

21

 

addit
1997
1998
et at
D53 i:
norma
dosed

1353 pt

shess

UV ra

addition, the Mdm2 protein also has a ubiquitin-Iigase activity (Honda et al.,
1997). Binding of Mdm2 protein to p53 causes ubiquitination Of p53 (Fuchs et al.,
1998) and therefore, targets p53 for degradation via the 26S proteasome (Haupt
et al., 1997; Kubbutat et al., 1997). This latter activity of Mdm2 keeps the level of
p53 protein low. The latent activity of p53 is achieved by the C-tenninus, which
normally folds back and keeps the sequence-specific DNA binding domain in a
closed conformation. Together, the actions of Mdm2 and the C-terminus of the
p53 protein keep the p53 function dormant.

The cryptic function of p53 can be activated when the cells are under
stress. A number Of factors can activate p53. These include ionizing radiation,
UV radiation, hypoxia, cell adhesion and decreased ribonucleotides. Similarly, a
number of mechanisms can activate p53, for example, phosphorylation,
acetylation and glycosylation are employed. The complexity in the modulation of
p53 has been reviewed in many articles (see for example, KO and Prives, 1996;
Giaccia and Kastan, 1998). The activation of p53 mainly involves stabilization of
p53 protein by preventing Mdm2-mediated p53 ubiquitination and induction of the
exposure of the sequence specific DNA-binding domain by changing the
conformation of the C-terminus. As an example, in response to ionizing
radiation, Ser15 of p53 protein is phosphorylated (Shieh et al., 1997; Siliciano et
al., 1997), and Lys382 of p53 becomes acetylated (Sakaguchi et al. 1998).
Phosphorylation of Ser15 leads to an inhibition in the binding of p53 to Mdm2
protein in vitro and in vivo (Shieh et al., 1997) and acetylation of Lys382

enhances the sequence-specific DNA binding of p53 (Sakaguchi et al. 1998). At

22

prese

  

other

relate

953 9,,
most V
Inhibitc
Ior Cdl
doing .

SIIding

least two kinases are able to catalyze the phosphorylation at Ser15, i.e., DNA-
activated protein kinase (DNA-PK) (Shieh et al., 1997; Woo et al., 1998) and
ATM, the product of the gene mutated in patients with ataxia telangiectasia
(Siliciano et al., 1997; Banin et al., 1998; Canman et al., 1998). Conceivably, the
presence Of multiple serine, threonine residues on p53 protein, the availability of
other protein kinases, such as checkpoint kinase (Chk1 and Chk2), ATM-rad3-
related kinase (ATR), and the alternative ways of modification no doubt make the
modulation of p53 activity much more complicated (for review, see Giaccia and
Kastan, 1998).

In addition to being activated by stress factors, oncogenes, such as myc,
can also activate p53, as a result of reduction of Mdm2 activity by turning on
expression of p14ARF, a negative regulator of Mdm2 (Prives, 1998). The cellular
localization of p53 is another way of regulating p53 activity in the cell (Liang and

Clarke, 2001).

4. Tumor suppression functions of p53

a. Growth arrest

As a transcription factor, p53 can activate the transcription of many genes.
p53 executes its functions mainly through these downstream genes. p21 is the
most well studied down stream gene (el-Deiry et al., 1993). p21 protein is an
inhibitor of many cyclin-dependent kinases (Cdk) (Xiong et al., 1993), especially
for Cdk2, the kinase required for G1 to S phase transition (Harper et al., 1993). In
doing this, p21 forms a quarternary complex with cyclin/Cdks and PCNA, the

sliding clamp for DNA replication (Zhang et al., 1993). In response to DNA

23

damag

|
1994::

61 ar'l
cells rl
mutatl
increal
Introd.
tumor
expres
SUpprE

tumOrg

mamrr

 

damage, p21 is upregulated by p53 and G1 arrest is induced (eI-Deiry et al.,
1994). Embryonic fibroblasts derived from p21 knock-out mice are defective in
G1 arrest in response to DNA damage (Deng et al., 1995). The G1 arrest allows
cells more time to carry out DNA repair and reduces the chance of introducing
mutations into the genome during DNA synthesis at S phase. In addition, the
increased function of p21 alone appears sufficient for tumor suppression.
Introduction of p21 cDNA suppressed the growth of human brain, lung, and colon
tumor cells in culture (eI-Deiry et al., 1993). Adenoviral vector-transduced
expression of p21 in a p53-deficient mouse prostate cancer cell line significantly
suppressed the growth of the cells in culture and the ability of the cells to form
tumors in mice (Eastham et al., 1995), and this was also the case for a mouse

mammary tumor cell line (Shibata et al., 2001).

b. Apoptosis

A second mechanism by which p53 acts as a tumor suppressor is by
inducing apoptosis, a form of programmed cell death. Apoptosis is characterized
by distinct morphological phenotypes, including blebbing of the plasma
membrane, condensation of the cytoplasm and nucleus, and cellular
fragmentation into membrane apoptotic bodies, and by the biochemical hallmark
of chromosomal degradation into monomers or multimers of 200 base pairs
(Steller, 1995). In response to cellular stress, stabilization and activation of p53
activates the expression of a number of genes involved in apoptosis. Bax, an
apoptosis-inducing factor, was the first one identified as participating in p53-

dependent pathway (Miyashita and Reed, 1995). By using subtractive

24

hybridization or differential display to compare tumor cell lines lacking p53 and
corresponding cells expressing endogenous or ectopic wild-type p53, it has been
possible to identify a number of other genes that play a role in p53-dependent
apoptotic pathway (Moll and Zaika, 2001). Examples include PERP, a member of
the PMP-22Igas3 tetraspan membrane protein family, cloned after subtracting
G1 arrest-associated p53-transactivated messages (Attardi et al., 2000), as well
as p53AlP1 and p53DINP1, two factors induced when p21 protein is not induced
(Oda et al., 2000; Okamura et al., 2001). PERP, p53AlP1 and p53DINP1 are
presumed to be more specific to and more decisive in p53-dependent apoptosis
than other factors. The effect of the apoptotic factors can vary depending on the
types of tissues or cells, and/or the types of cellular stress. For example, mice
lacking a functional Bax gene displayed hyperplasia in thymocytes and B cells,
massive cell death in multinucleated giant cells and dysplastic cells, and
aberrations in other cell lineages (Knudson et al., 1995). What is more, different
factors can act in synergy. Bax deficiency only partially reduced the p53-
dependent apoptosis in mouse primary fibroblasts expressing the adenovirus
E 1A oncogene, a cellular setting in which it has been shown that in response to
DNA damage cells undergo apoptosis, but not G1 arrest (McCurrach et al.,
1997). Apoptotic factors induced by p53 activate the caspase cascade and lead
to the characteristic apoptotic phenotypes (Schuler and Green, 2001). In addition
to transcription of apoptotic genes, mechanisms such as transrepression and
direct protein-protein interaction involving p53 have been reported as well (Moll

and Zaika, 2001).

25

c. Inhibition of angiogenesis

When a tumor reaches a certain size, it needs a greater supply of
nutrients and oxygen to support its further growth. This is achieved by stimulating
angiogenesis. Angiogenesis is controlled locally by stimulatory factors such as
vascular endothelial growth fatcor (VEGF) and inhibitory factors such as
thrombospondin (lrueIa-Arispe and Dvorak, 1997). p53 appears to regulate
angiogenesis as well. Li-Fraumeni patients inherit one normal allele and one
mutant allele of p53. Fibroblasts derived from such patients would retain this
characteristic at early passage, but would spontaneously lose the wild-type copy
of p53 in later stage (Yin et al., 1992). Concomitant with the loss of wild-type p53
in continued culture, the level of thrombospondin-1 (T sp-1) protein secreted into
the media was found reduced, and the anti-angiogenic activity of the media
tested in vitro and in vivo was decreased as well (Dameron et al., 1994). The
modulation of Tsp-1 expression occurred at the mRNA level and was caused by
loss of p53 (Dameron et al., 1994). In addition, human bladder cancers with low
expression level of Tsp-1 exhibited high microvessel density counts, and were
significantly associated with loss of p53 as well (Grossfeld et al., 1997). These
data suggest that p53 can inhibit tumor growth by transactivating inhibitory
factor(s) of angiogenesis. To the same end, the expression of VEGF, the major
angiogenic inducer, is negatively regulated by wild-type p53, and loss of p53
function exhibited an increase in the expression level of VEGF (Kieser et al.,
1994; Mukhopadhyay et al., 1995; Bouvet et al., 1998; Pal et al., 2001). The p53

regulates the expression of VEGF at the transcription level (Kieser et al.,1994;

26

Zhang
humar
repres.
repress
suppre:

factor(s

p:
cells are
morpholc
e.g., exp]
Human fil
Tantigen
Senesceni
DIESmId’ t
bindlng dc
fibroblast
the contrc

 

Cells aDpr
IIanSactiva

SUgrUe et

 

bladder Ca.

 

Zhang et al., 2000c). Deletion of one of the four Sp1-binding elements in the
human VEGF promoter region completely abolished the transcriptional
repression activity of wild-type p53, but how p53 utilizes this Sp1 element in
repressing transcription is not Clear (Zhang et al., 2000c). Thus, tumor
suppressor p53 appears to inhibit angiogenesis by increasing anti-angiogenic

factor(s) and decreasing pro-angiogenic factor(s) simultaneously.

d. Senescence

p53 appeared to play a role in cellular senescence as well. Senescent
cells are characterized by irreversible growth arrest, resistance to apoptosis,
morphological Changes, e.g., cellular enlargement, and biochemical alterations,
e.g., expression of B-galatosidase with optimum pH Of 6 (Itahana et al., 2001).
Human fibroblasts in culture that had been immortalized with an inducible SV40
T-antigen underwent senescence following the de—induction of T-antigen, and the
senescence could be rescued by re-expressing intact T-antigen from a second
plasmid, but not by re-expressing a T-antigen deletion mutant lacking the p53-
binding domain (Shay et al., 1991). By using an early passage, human diploid
fibroblast stable transfectant cell strain, which expressed B-galatosidase under
the control of p53-responsive element, Bond et al. (1996) found a steady
increase of B-galatosidase activity and steady decrease in DNA synthesis as
cells approached senescence, clearly indicating the involvement of p53
transactivation function in cellular senescence (Bond et al., 1996). Indeed,
Sugrue et al. (1997) showed that induced overexpression of wild-type p53 in EJ

bladder carcinoma cells lacking functional p53 triggered an onset of irreversible

27

growth an
senescen
strong cal
al., 1999;
genes in
recombine
et al., 19E
factor, wh
transcripti:
reoressed
963 and

Senescenc

e. Rol
Sev

and home
GADD45 g
Zhan et a
interacting I

to DNA 1’.

 

acceSSibilir

 

growth arrest within 2-3 days, with cells exhibiting morphological and biochemical
senescent phenotypes. In addition to its essential role in G1 arrest, p21 is also a
strong candidate for p53-dependent senescence (Fang et al., 1999; Sayama et
al., 1999; Chang et al., 1999). Normal human diploid fibroblasts with the p21
genes inactivated by two successive rounds of targeted homologous
recombination escaped senescence during continuous growth in culture (Brown
et al., 1997). Recently, Jung et al. (2001) showed that the NF-Y transcription
factor, which is inactivated in senescent human fibroblasts and is essential for
transcription of the cdk1 and cyclin B genes, which are also irreversibly
repressed in senescent cells, was inactivated by p53 and its two homologues,
p63 and p73, suggesting an alternative mechanism of p53—dependent

senescence.

e. Role in DNA repair and homologous recombination

Several properties of p53 allow p53 to have a potential role in DNA repair
and homologous recombination. As a transcription factor, p53 transactivates
GADD45 gene expression in response to DNA damage (Kastan et al., 1992;
Zhan et al., 1994). In addition to inhibiting entry of cells into S-phase by
interacting with PCNA (Smith et al., 1994), GADD45 protein has activities related
to DNA repair, including recognizing damaged chromatin, modifying DNA
accessibility to other proteins (Carrier et al., 1999) and stimulating excision repair
(Smith et al., 1994). Studies with fibroblasts heterozygous and homozygous for
p53 mutations (Ford and Hanawalt, 1995), normal fibroblasts, and fibroblasts

expressing human papillomavirus 16 E6 protein (Ford et al., 1998) showed that

28

p53 is n
banscnb
depende
p53-depe
Smith et I

Be
with prote
with the t
Zhou et a

endonucle

POIymeras
DNA.Ba&
D53 was i
Possesses
aCtivity is ,
binding do
Specific D

eXOnUCIeaS

 

Tfar
DOIymerast
I

(NER) (SI/r

TFllH COmI

 

p53 is required for nucleotide excision repair of UV damage in non-actively-
transcribing genomic regions. The p48 gene, whose expression is p53-
dependent, and also GADD45 gene, has been implicated as playing a role in this
p53-dependent global genomic nucleotide excision repair (Hwang et al., 1999;
Smith et al., 2000).

Besides transactivating genes involved in DNA repair, p53 also interacts
with proteins physically executing DNA repair. Direct involvement of p53 protein
with the base excision repair machinery has been reported (Offer et al., 1999;
Zhou et al., 2001). These investigators showed that p53 could interact with AP
endonuclease and DNA polymerase B, and that association of p53 with DNA
polymerase [I stabilized the interaction between DNA polymerase B and abasic
DNA. Base excision repair capacity of the cell extracts was much reduced when
p53 was immunodepleted (Zhou et al., 2001). Interestingly, p53 protein also
possesses a 3’-to-5’ exonuclease activity (Mummenbrauer et al., 1996). This
activity is carried out by a domain overlapping with the sequence-specific DNA
binding domain, but it is differentially affected by factors that activate sequence-
specific DNA binding activity (Janus et al., 1999b). The exact role of the
exonuclease activity of p53 in vivo remains to be elucidated.

Transcription factor IIH (TFIIH) is a basal transcription factor for RNA
polymerase II, and its function is also essential for nucleotide excision repair
(NER) (Svejstrup et al., 1996). p53 protein has been found to interact with two
TFIIH complex-associated NER factors, XPB and XPD, and a third NER factor,

CSB (Wang et al., 1995). Although modulation of NER activity by p53 has been

29

suggesh
hanscnt
activity v
Ford et :
activatior
apoptotic
belong to
XPD patie
apOptosis
exhibited
damaging
highly puri-
can PhOSp
Specific D
terminus c
double-stre
IUDCIIOI‘ts a

(Janus et a

D53

 

recombinail
counterpar
I’eCCImbinaT

activities 0

 

 

suggested (Wang et al., 1995), the role of p53 in NER in repair of actively-
transcribing genes seems to be limited. Only defective global genomic NER
activity was found in fibroblasts lacking p53 function (Ford and Hanawalt, 1995;
Ford et al., 1998). These data suggest that association with TFIIH is a way for
activation of p53 protein itself. In contrast to the wild-type p53-dependent
apoptotic response in normal cells, primary fibroblasts derived from patients that
belong to xeroderma pigmentosum complementation group B or D, i.e., XPB or
XPD patients, but not XPA or XPC patients, have an attenuated p53-dependent
apoptosis (Wang et al., 1996). Lymphoblastoid cell lines with XPD defect
exhibited reduced and delayed apoptosis induced by doxorubicin, a DNA-
damaging agent and topoisomerase II inhibitor (Robles et al., 1999). Indeed, the
highly purified kinase components of TFIIH, the CDK7-cycH-p36 trimeric complex
can phosphorylate the inhibitory C-terminus of p53 and activate the sequence-
specific DNA binding activity of p53 in vitro (LLI et al., 1997). The ability of the C-
terrninus of p53 protein to bind various DNA structures, such as non-specific
double-stranded or single stranded DNA, double-strand breaks, Holliday
junctions and insertion/deletion mismatches may provide ways for p53 activation
(Janus et al., 1999a).

p53 has been shown to exert an inhibitory role on homologous
recombination. Wild-type p53 can interact with hRad51 protein and its prokaryotic
counterpart, RecA protein, both of which are key factors of homologous
recombination (Sturzbecher et al., 1996). Human p53 protein inhibits the

activities of RecA required for in vitro recombination (Sturzbecher et al., 1996),

30

and this
well. Inc
p53 an
homolog
(Mekeel
intrachrc
expressi
strains 63
of p53 in
(Saintign

P53 prote

ll. Th
A. Ce

1. So
UN.

is IEIaliveli
SOUrCeS II”
CIaSSified
Ellteratjons
deamlnatio

deIIUrinatio

 

SpeCles. D

compOUnd

and this suggests that p53 inhibits homologous recombination in eukaryotes as
well. Indeed, inactivation of wild-type p53 function by a dominant negative form of
p53 and various viral proteins causes a dramatic increase in the rate of
homologous recombination as indicated by an engineered reporter plasmid
(Mekeel et al., 1997). An elevated rate of homologous recombination between
intrachromosomal direct repeat sequences was also observed in mouse cells
expressing mutant p53 (Bertrand et al., 1997). Subsequent studies with cell
strains expressing p53 defective in transactivation revealed that the involvement
of p53 in homologous recombination does not require its transactivation function
(Saintigny et al., 1999; Willers et al., 2000), suggesting that direct participation of

p53 protein is involved.

II. The RAD6 pathway for dealing with DNA damage
A. Cellular responses to DNA damage

1. Sources of DNA damage

DNA is the genetic material for essentially all living organisms. Although it
is relatively stable, it is still quite structurally dynamic and chemically active. The
sources that cause alterations in DNA, or DNA damage can be generally
classified into two categories, spontaneous or environmental. Spontaneous
alterations include incorporation of incorrect nucleotides during replication,
deamination Of cytosine, adenine or guanine bases, loss of bases via
depurination or depyrimidination, and damage caused by reactive oxygen
species. DNA reacts easily with a variety of physical radiation or chemical

compounds from the environment as well. Ionizing radiation like X-ray or y-ray

31

I
causes be

radiation I
like N-met

used for ca

 

reactive n
chemicals I
chemistry
maintain I;

deal with ti

 

2- DN.

3. Thr

A d
Sensitivity
agents of
gene may
mutant Ce
SECond S
synergism
SEDSltive
theSE tW<
Imitation
theSe IVs/c

Strategy

causes base damage and single— or double-strand breaks. Ultraviolet (UV)
radiation induces dimerization of adjacent pyrimidines. DNA alkylating agents,
like N-methyl-N-nitrosourea (MNU) and methyl methanesulfonate (MMS), drugs
used for cancer chemotherapy, such as platinum derivatives or mitomycin C, and
reactive metabolites of a myriad of other naturally occurring or man-made
chemicals, such as aflatoxin and benzo[a]pyrene all react with and modify the
chemistry of DNA in one way or the other (Friedberg et al., 1995). Fortunately, to
maintain the integrity of the genome, organisms have evolved mechanisms to

deal with these DNA damage.

2. DNA repair and damage tolerance

a. Three major pathways in dealing with DNA damage

A defect in pathways dealing with DNA damage often causes increased
sensitivity to agents causing such damage. Thus, sensitivity to DNA damaging
agents of a cell that has an inactivating mutation in a gene indicates that this
gene may function in DNA repair. Comparison of such sensitivity between this
mutant cell and a cell containing an inactivating mutation in this gene and a
second gene can reveal the relationship of these two genes, i.e., either
synergism or epistasis. If having mutations in two genes makes the cell more
sensitive than cells with only one gene mutated, i.e., causes a synergistic effect,
these two genes can be said to function in different pathways. If the second
mutation does not increase sensitivity, that gene is epistatic to the other, i.e.,
these two genes are considered to function in the same pathway. By using this

strategy. scientists have identified three major pathways dealing with DNA

32

damag
(Fnedt
in their
epistati
exhibit

demons
RADSZ

least un
that allor
al., 1995
humans,

thorough

b. Ex
Exc
Ornucleot,‘
Includes th
excision Is
“their utiliz

AP endor

 

deoxyrlbos

”UCIGOtide A
menarche

damage in yeast S. cerevisiae. They are: RADB, RAD52, and RAD6 pathways
(Friedberg et al., 1995). RAD3, RAD52, and RADG are the most important genes
in their own respective pathways because mutations in these three genes are
epistatic in radiation-sensitivity to all the other mutants in the same pathway and
exhibit the most extreme sensitivity in the same pathway as well. It is now well
demonstrated that the RAD3 pathway is the excision repair pathway and the
RAD52 pathway carries out homologous recombination. The RAD6 pathway is
least understood, but in principle, it represents a damage tolerance mechanism
that allows continuation of DNA replication without lesion removal (Friedberg et
al., 1995). The yeast RAD3 and RAD52 pathways are largely conserved in
humans, and will be described very briefly in the following sections, followed by a

thorough description of the RAD6 pathway.

b. Excision repair

Excision repair refers to the excision of damaged or inappropriate bases
or nucleotides and subsequent repair synthesis of correct bases or nucleotides. It
Includes three closely related but mechanistically distinct subpathways, i.e., base
excision repair, mismatch repair and nucleotide excision repair. Base excision
repair utilizes DNA glycosylase to excise the damaged or inapporiate bases, 5'
AP endonuclease and DNA deoxyribophosphodiesterase to remove the
deoxyribose-phosphate moiety, and DNA polymerase and ligase to fill the single
nucleotide gap and to Iigate the nicked DNA. Mismatch repair deals with
mismatched base pairs and small insertion/deletion loops by specifically

removing the single mispaired nucleotide or few affected nucleotides. The third

33

one.nU<
of DNA l
of ~25-I
polymer;
is evolut

1995).

c. Hc

HO
strand br
Jasin, 201
First, blnc
breaks 6X;
assembly‘
strand exc
resolved b.
fUrlction of
tElanglecta
reconIbinat

 

 

 

 

 

 

 

 

one, nucleotide excision repair recognizes lesions that distort the helical struture
Of DNA and recruits a complex enzymatic machinery to excise an oligonucleotide
of ~25-30nt containing the damage. The resulting gap is filled in by DNA
polymerases and the repair is finished by DNA ligase. The excision repair system
is evolutionarily conserved from prokaryotes to eukaryotes (Friedberg, et al.,

1995).

c. Homologous recombination

Homologous recombination is the major pathway for repairing DNA double
strand breaks caused by exogenous or endougenous agents (Johnson and
Jasin, 2001). Homologous recombination takes place in a stepwise manner.
First, binding of the Rad50/Mre11/Nbs1 complex to the 3' ends of the strand
breaks exposes the 3'-OH groups, and promotes strand invasion. Then, filament
assembly through Rpa, Rad51, Rad51-related proteins and Rad52 allows single
strand exchange and DNA synthesis to occur. Finally, the Holliday junction is
resolved by resolvase. The activation of homologous recombination requires the
function of upstream protein kinases including ATM, a kinase mutated in ataxia
telangiectasia and ATR (ATM- and Rad3—related). Defects in homologous
recombination as a result of mutations in these upstream kinases predispose

patients to lymphomas and other types of cancer (Hoeijmakers, 2001).

B. Damage tolerance: RAD6 pathway in Saccharomyces cerevisiae
1. Principal genes involved

a. RAD6 and RAD18

34

(Lawre
Mutants
a broa-
nitroquir
bifunctic
unable t
DNA ter
greatly ir
dSMaya
etal., 19;
(Jones et
and induc
(Kern and
RAD5 gen
the RADr
bmmmla
PlOteins 10
protein CO
'” additio,
reliuired fc
for the C

completely

 

At least 15 genes have been assigned to the RAD6 pathway in yeast
(Lawrence, 1994). RAD6 and RAD18 are the founding members of the pathway.
Mutants for RAD6 and RAD18 genes are very sensitive to the cytotoxic effect of
a broad range of DNA damaging agents, including UV, X-ray, y-ray, 4-
nitroquinoline-N-oxide, trimethoprim, bleomycin, and monofunctional and
bifunctional alkylating agents (Friedberg, et al., 1995). A rad6 mutant strain is
unable to convert the low molecular weight DNA synthesized on UV-damaged
DNA templates into normal high molecular weight form, and rad18 strain is
greatly inhibited in this process (Prakash, 1981). Unlike rad6 mutants, which
display a loss of UV- and chemical carcinogen-induced mutagenesis (Lawrence
et al., 1974, 1976 and Prakash, 1974), rad18 mutants show normal mutagenesis
(Jones et al., 1988). The rates of spontaneous mutation and both spontaneous
and induced mitotic recombination are increased in both rad6 and rad18 strains
(Kern and Zimmermann, 1978). Biochemical analyses indicate that the yeast
RADB gene encodes a ubiquitin-conjugating enzyme (Jentsch et al., 1987) and
the RAD18 gene encodes a protein possessing DNA binding and nucleotide
binding activities (Jones et al., 1988). The ability of yeast Rad6 and Rad18
proteins to form a heterodimer (Bailly et al., 1994 and 1997) suggest that Rad6
protein could be targeted to damage-containing DNA regions by Rad18 protein.
In addition, the ubiquitin-conjugating activity of Rad6 protein is absolutely
required for its function. The substitution of residue Cy588, an essential residue
for the conjugating activity of Rad6 protein, with either alanine or valine

completely abolished the functions of wild-type Rad6 in DNA repair and

35

mutage

conferre

8
effect of
by a RA
Uohnsor
to the R
mutations
gene revs
Contains :
COUId fom

motif (Joh

c. RA

Am
8' 09revisi
Shares hOr

IKUIaeva ELI

gene was

 

and Rousr
(MCDODan

CI’IOtoXiC E

 

mutagenesis. The defects caused by this point mutation are equivalent to those

conferred by rad6 null mutants (Sung et al., 1990).

b. RAD5

Strains mutant for the RAD5 gene are more sensitive to the cytotoxic
effect of UV and other agents than wild-type strains, but the sensitivity conferred
by a RAD5 mutation is less than those caused by RAD6 or RAD18 mutations
(Johnson et al., 1992). Epistasis analysis for UV-sensitivity assigns RADS gene
to the RADS pathway (Johnson et al., 1992). RAD5 plays virtually no role in
mutations caused by UV (Lawrence and Christensen 1978). Cloning of the RAD5
gene reveals that this gene encodes a protein of 1,169 amino acids. The protein
contains seven domains characteristic of helicases, one cysteine-rich motif that
could form DNA binding zinc-finger domain, and one leucine zipper DNA binding

motif (Johnson et al., 1992).

c. RAD30 and PCNA

Amino acid sequence alignment analysis of the complete genome of yeast
S. cerevisiae revealed one gene, (SCD9461.8; YDR419W), whose gene product
shares homology with bacteria UmUC, DinB proteins and eukaryotic Rev1 protein
(Kulaeva et al., 1996). The gene was designated as RAD30 when deletion of this
gene was found to confer mild sensitivity to UV radiation (McDonald et al., 1997
and Roush et al., 1998). Epistasis analysis places RAD30 in the RAD6 pathway
(McDonald et al., 1997). RAD30 disruption strains are more sensitive to the

cytotoxic effect of UV than wild-type strains, but rad30 rad6 and rad30 rad18

36

double Ir
mutants. I
RAD30 m

mutations

 

REV3, Rf
rev7, andl
are single
mutants a
rev3, rev?
rad30 mu

functions

RAD30 agi

 

error-free .

demonstr

double mutant strains are no more sensitive to UV than rad6 or rad18 single
mutants. RAD30 does not belong to the RAD3 or RAD52 epistatis group, since
RAD30 mutation is additive to either RAD1, a gene in the RAD3 group, or RAD52
mutations in UV sensitivity. RAD30 does not work in the same subpathways as
REV3, REV7, REV1 or as RAD5 (McDonald et al., 1997). rad30 rev3, rad30
rev7, and rad30 rev1 double mutants are all more sensitive to killing by UV than
are single mutants for either rad30, rev3, rev7, or rev1. rad30 rad5 double
mutants are more sensitive to either single mutant as well. Interestingly, unlike
rev3, rev7 and rev1 mutants, which are all deficient for UV-induced mutagenesis,
rad30 mutants show an increase in UV-mutagenesis, indicating that RAD30
functions in an error-free manner in dealing with UV damage. In summary,
RAD30 appears to function in a RADB/RAD18-dependent, REV-independent,
error-free mechanism that does not involve RAD5. Biochemical analysis
demonstrated that Rad30 protein is a translesion DNA polymerase that can
replicate efficiently and faithfully past a thymine-thymine cis-syn cyclobutane
dimer, incorporating two correct adenines opposite the misinstructional thymine-
thymine dimer template (Johnson et al., 19993). Rad30 has, therefore, been
named DNA polymerase eta. Yeast Pol eta also has been shown to bypass
cyclobutane dimers and (6-4) photoproducts formed at CC and TC sites in error-
free manner (YU et al., 2001 ). Kinetic analysis (Washington et al., 2001) and
structural analysis (Trincao et al., 2001) also support the error-prone mode of
action of yeast Pol eta. The DNA polymerase activity of Rad30 is essential for its

biological function, since Rad30 protein carrying an inactivating Asp-Glu to Ala-

37

Ala mut.
rad30 de

P
shape c
major re
polymer;
et al., 15
mutation
of DNA I
Simultan
to two at
and pot:
IAYYagar
mutants 3
exhibits a
degree 0
mutant pr
loader RF
epsilon. E

 

Arr
the (3thle

type Pol

 

Ala mutation in the highly conserved SIDE sequence cannot complement the
rad30 deletion mutation (Johnson et al., 1999c).

Proliferating cell nuclear antigen (PCNA), as a homotrimer, forms a ring-
shape conformation and serves as sliding clamp or processivity factor for the
major replicative DNA polymerase, Pol delta, during DNA replication and for DNA
polymerase epsilon filling the gap in DNA during nucleotide excision repair (Shivji
et al., 1992). The gene encoding S. cerevisiae PCNA is POL30. Because a null
mutation of POL30 is lethal to the yeast, the role of PCNA in the RAD6 pathway
of DNA repair could not be determined until the mutant pol30-46 was identified.
Simultaneous mutations of two adjacent charged residues (Asp, Glu, Lys or Arg)
to two alanines in the PCNA protein gave rise to two mutant proteins (pol30-9
and pol30-22) that allow normal growth and confer modest UV sensitivity
(Ayyagari et al., 1995). Combination of the mutations in these two double
mutants yielded the pol30-46 mutant (Torres-Ramos et al., 1996). This mutant
exhibits a higher degree of UV sensitivity than either double mutant, but the
degree of cytotoxicity, compared to wild-type yeast, is still mild. The pol30-46
mutant protein is as efficient as wild-type PCNA in being loaded by the clamp
loader RFC protein and in mediating processlve DNA synthesis by Pol delta or
epsilon. Epistasis analysis clearly places PCNA into a REV3-independent RAD6
pathway, and not in the RADB or RAD52 pathways.

Amino acid sequence analysis reveals a potential PCNA-binding motif on
the C-terminus of yeast Pol eta protein (Haracska et al., 2001a). Indeed, wild-

type Pol eta protein forms a complex with PCNA since they are coeluted from

38

sizeexclc
extension
On the c
mutant P:
acids or .
PCNA pr
polymeras
expressici
mutants c
Pol eta r
essential

eI3) funct

 

size-exclusion columns. Also, addition of PCNA protein to the in vitro primer
extension reactions greatly enhances the DNA synthesis catalyzed by Pol eta.
On the other hand, evidence from a yeast two-hybrid system indicated that
mutant Pol eta protein carrying either a deletion of the C-terminal eight amino
acids or a substitution of two conserved amino acids loses the ability to bind
PCNA protein. Although the mutant Pol eta proteins still retain robust DNA
polymerase and T-T dimer in vitro bypass activities identical to wild-type protein,
expression of either of these two mutant proteins in rad5 rad30 double deletion
mutants cannot increase the UV survival of the strain to the level that wild-type
Pol eta protein achieves. These data suggest that interaction with PCNA is
essential for Pol eta function in vivo. Therefore, it is very likely that Rad30 (Pol

eta) functions with PCNA in one branch of error-free RADG-mediated pathway.

d. Ub, MMSZ and UBC13

Ubiquitin is a small peptide of 76 amino acids. It is best known as a
covalent degradation signal for cellular proteins. The Ubiquitin molecules in the
multiubiquitin Chain for efficient degradation of substrate protein via the 26S
proteasome are usually linked by Lys48-Gly76 isopeptide bonds (Hochstrasser,
1996). In a study looking at the possibility of multiubiquitin chain formation via
alternate lysine residues, Spence et al. (1995) found that substitution of Lys-63
with arginine (UbK63R) causes disappearance of a family of abundant
multiubiquitin protein conjugates. Interestingly, the overall protein turnover in the
UbK63R mutant strain is not affected, as evidenced by the normal growth rate,

normal levels of Ubiquitin-protein conjugates, and fully efficient turnover of short-

39

lived p
showec
the pre
further
RAD52
linkage
1
sensitivi
MMSZ,
the orig
protein .
almost a
Cysteine
°°niudarr
UPC activ
analysiss
01 RAD4

leCOmbin‘

 

These (1

pathwayS ‘
MMS? dc
REV3 in

REL/find

 

lived proteins in such mutant yeast (Spence et al., 1995). Cytotoxicity assays
showed that the UbK63R mutation confers sensitivity to MMS and UV, indicating
the presence of a defect in DNA repair in UbK63R mutant. Epistatic analyses
further assign the UbK63R mutation to the RAD6 group instead of the RAD3 and
RADSZ groups (Spence et al., 1995). These data implicate a role for Lys-63 as a
linkage site in the formation of multiubiquitin chains in the RAD6 repair pathway.
The MMSZ gene was first characterized in a yeast mutant (mm32-1) strain
sensitive to methyl methanesulfonate (MMS) (Prakash and Prakash, 1977).
MMSZ, recently cloned from a yeast genomic library by its ability to complement
the original mmsZ-1 mutant phenotype (Broomfield et al., 1998), encodes a
protein of 137 amino acids, which shares significant sequence homology to
almost all known Ubiquitin-conjugating (ch) proteins. However, since it lacks a
cysteine residue critical for ch activity, MmsZ appears to be a ubiquitin-
conjugating enzyme variant, i.e., a protein looks like ch but does not have the
ch activity. mmsZ null mutants are sensitive to both MMS and UV. Epistasis
analysis shows that mutant MMSZ is additive in UV sensitivity to deletion mutants
of RAD4, a gene for nucleotide excision repair, or of RAD50, a gene for
recombination repair, but it is epistatic to both rad6 and rad18 deletion mutants.
These data indicate that MMSZ does not belong to the RAD3 and RAD52
pathways, but rather belongs to the RAD6 pathway. In addition, a mutation in
MMSZ does not impair the UV-mutagenesis and is synergistic to a mutation in
REV3 in both UV and MMS sensitivity, suggesting that MMS2 functions in a

REV3-independent pathway. Furthermore, because the spontaneous mutation

4O

rate of m
the mutar
defect in
function |.'
In
enzyme.
that ch1

protein M

 

 

 

form 3 Ci
showed 1
double, a
COmparat
(Hofman,

TF
(2000) U,
‘3 epista
that UB(
mutant t‘
PEI/ad
I09Ethe

rate of mmsZ mutant strains is elevated, and mutations in MMSZ are epistatic to
the mutant Rad6 lacking the first nine amino acids, which presumably causes a
defect in the error-free but not the mutagenesis pathway, MMS2 is considered to
function in the error-free branch of RAD6 pathway (Broomfield et al., 1998).

In 1999, Hofmann and Pickart (1999) showed that a Ubiquitin-conjugating
enzyme, ch13 mediates the assembly of K63-linked polyubiquitin chains and
that ch13 does this with the help of the Ubiquitin-conjugating enzyme variant
protein Mms2 (Hofmann and Pickart, 1999). Mms2 protein and ch13 protein
form a complex with very high affinity. Genetic data in the same study also
showed that a ubc13 mutant yeast strain is sensitive to killing by UV, and single,
double, and triple mutants of the UBC13, MMSZ, and UbK63R genes display a
comparable phenotype, suggesting that these three genes function together
(Hofmann and Pickart, 1999).

The role of UBC13 in RAD6 pathway was further defined by Brusky et al.
(2000) using genetic epistasis analyses. Their data show that the ubc13 mutation
is epistatic to mmsZ and rad6, and that ubc13 is synergistic to rev3, indicating
that UBC13 is in a pathway alternative to RE V3 but related to MMSZ. The ubc13
mutant is fully functional in UV-induced mutagenesis and displays up to a 30-fold
REV3-dependent increase in the spontaneous mutation rate. All these results
together demonstrate that Ub, MMSZ and UBC13 are members of the error-free

branch of the RAD6 pathway.

e. Genes for mutagenesis: REV3, REV7 and REV1

41

In
in yeast
al., 2001 '
are slight
virtually
spontane

Mutants l

 

 

 

levels of
discussec
(Nelsone
et al,‘ 1
translesii
0f introd

fUDCIIOns

 

In addition to RADG, a number of other genes are involved in mutagenesis
in yeast (Reviewed in Prakash et al., 1993; Lawrence, 1994 and Broomfield et
al., 2001). Mutant REV3, REV7, REV1 strains (collectively referred to as REVS)
are slightly more sensitive to DNA damage than the wild-type yeast, but exhibit
virtually no mutagen-induced mutagenesis. In rev3 mutant strains, the
spontaneous mutation rates are 50-75% lower than in REV3-wiId-type strains.
Mutants for other genes such as REV6, NGM2, REV4 and REV5 show varied
levels of increased sensitivity and reduced mutagenesis. As will be further
discussed below, yeast Rev3 and Rev7 interact and form DNA polymerase zeta
(Nelson et al., 1996a), and Rev1 protein is an auxiliary factor for Pol zeta (Nelson
et al., 1996b). Rev3, Rev7, Rev1, and probably other proteins employ a
translesion synthesis mechanism to deal with a stalled replication fork, at a price
of introducing mutations into the genome. Much less is known about the

functions of RE V6, NGM2, RE V4 and RE V5.

f. Damage avoidance and mutagenesis subpathways

RAD6 mediates at least two subpathways (Lawrence, 1994; Xiao et al.,
2000). The first one is the mutagenic pathway dependent on the functions of
REV3, REV7, REV1, and possibly other genes. The functions of REV3, REV1 or
RADG are required for more than 90% of base substitutions and frameshift
mutations induced by UV (Lawrence and Hinkle, 1996). The mechanism of this
pathway is translesion synthesis. The second pathway is a damage avoidance
mechanism that requires the functions of RAD5, MMSZ and other genes (Xiao et

al., 2000), and contributes the major portion of the ability of yeast to tolerate DNA

42

“—14.4."_‘ -_'

damag
screen
convert
into no
rad18 r
normal

reflect tl

well Undi
weight C
Although
pathway

meChanis
Pathway.
proposed

 

b. Co

MI

 

damage (Watkins et al., 1993). This mechanism is relatively error-free. In a
screen for yeast strains defective in postreplication repair, i.e., the ability to
convert low molecular weight DNA synthesized on UV-damaged DNA templates
into normal high molecular weight DNA, Prakash (1981) found that rad6 and
rad18 mutants are unable to carry out this repair, whereas rev3 mutants are
normal in this repair. The process of converting small DNA into large DNA might

reflect the execution of this damage avoidance mechanism.

2. Damage avoidance mechanisms

a. Recombination-dependent mechanism in the error-free damage

tolerance pathway

The mechanism(s) for the error-free damage avoidance pathway are not
well understood. The conversion of low molecular weight DNA to high molecular
weight DNA might be an intermediate step of this process (Prakash, 1981).
Although homologous recombination is carried out by the RAD52 pathway, a
pathway distinct from the RAD6 pathway, a recombination-dependent
mechanism is believed to play a role in the error-free branch of the RAD6
pathway. Two models, i.e., strand exchange or template switching, have been
proposed (reviewed in Broomfield et al., 2001). More evidence is needed in order

to elucidate the mechanism(s) underlying in the damage avoidance pathway.

b. Coordination of two Ubiquitin-conjugating enzymes Rad6 and

Mmlech13

43

It

 

comugat
can atta:
ligase (Je
with Rac
ch13 c
protein it'
catalyzesl
chain in tl
are Red!
whose fu

Th
Isahoge
eliistaticr
Ramos e'
or MMS;
MWMar n
mOIeCUIa
BY Using

(2000) h

It is now known that the RAD6 pathway has at least two ubiquitin-
conjugating units. The Rad6 protein is a Ubiquitin-conjugating enzyme (ch) that
can attach Ubiquitin to a substrate protein with or without the help of a Ubiquitin
ligase (Jentsch et al., 1987; Sung et al., 1991). Rad6 protein forms a complex
with Rad18 protein. The second Ubiquitin-conjugating unit is the stable Mms2-
Ub013 complex (Hofmann and Pickart, 1999). As discussed above, Mmsz
protein itself is a ch variant and it is the ch13 protein in this complex that
catalyzes ubquitination. The MmsZ-ch13 complex can assemble multiubiquitin
chain in the absence of a substrate protein (Hofmann and Pickart, 1999). How
are Rad6, whose function is required for the whole pathway, and Mms2/ch13,
whose function is required only for the error-free subpathway coordinated?

The function of MMSZ-UBC13 appeared to be dependent on RADS, which
is also genetically placed in the error-free subpathway, since mutation of RAD5 is
epistatic to mutation in MMS2 in UV sensitivity (Ulrich and Jentsch, 2000; Torres-
Ramos et al., 2002). In addition, yeast single mutant strains defective for RAD5
or MMSZ, and a double mutant strain defective in RAD5 and MMSZ exhibited
similar reduced ability in converting low-molecular weight DNA into high-
molecular weight DNA fragments after UV treatment (T orres-Ramos et al., 2002).
By using yeast two-hybrid system and coimmunoprecipitation, Ulrich and Jentch
(2000) found that Rad5 interacts with ch13 and Rad18 using two distinct
domains. By using yeast three-hybrid system and colocalization assays, they
further showed that interaction between Rad5 and Rad18 can bring Mms2-

ch13, via association with Rad5, and Rad6 protein, via association with Rad18,

44

into cont
were plO
(Ulrich aI
assembi

protein.

   

MmsZ-Ut
RADO p

coordinat

3. MI

3. Mt

Th
mUIBQEnE
protein is
dEletion c
(Monmon
CYCLE dep
that Revgl

REV7 Qer

I

Strain (TO
ablIlty to
Gene (“Sr

REV7 Oer

 

into contact and promote the formation of a multimeric complex. Two models
were proposed for the coordination of the activity of Rad6 and Mms2-ch13
(Ulrich and Jentsch, 2000). One, Rad6 might attach multiubiquitin chains pre-
assembled by MmsZ-ch13 to the substrate protein. The other, the substrate
protein, conjugated with one ubiquitin moiety by Rad6, is further ubiquitinated by
Mms2-Ub013. The identification of one or more substrates of ubiquitination in
RADG pathway will no doubt greatly help elucidate the mechanism of

coordination.

3. Mutagenic mechanisms involving REVs

a. Molecular cloning of yeast REV3, REV7 and REV1 genes

The yeast REV3 gene was cloned by the ability to restore UV-
mutagenesis in a yeast rev3 mutant strain. Sequence analysis predicts that Rev3
protein is a DNA polymerase. Since haploid yeast cells carrying a complete
deletion of REV3 are viable, the putative Rev3 polymerase is not essential for life
(Morrison et al., 1989). In addition, the expression of REV3 does not show cell
cycle dependence, a pattern that many replication enzymes have, suggesting
that Rev3, as a polymerase, has specialized function (Singhal et al., 1992).
REV7 gene was cloned by functional complementation as well in a rev7 mutant
strain (Torpey et al., 1994). The REV1 gene of S. cerevisiae was isolated by its
ability to complement the yeast rev1-1 mutant, and its identity was verified by
gene disruption using the cloned DNA sequence (Larimer et al., 1989). The
REV1 gene encodes a protein of 985 amino acid residues, with a 152-residue

internal segment sharing 25% identity with bacteria UmuC protein, a protein that

45

W35 {80'

translesi:

rename:

 

b. Rt.

Tr
by Nelsc

1989). V.

 

 

yeast cD
frame in
overeXpr.
0f Glutat
were be
REV3 8!
activity

alone, E
Rev3-t:
a “On;
an in \
‘33s].
Dhgm

b‘ilba

\ESS

was recently determined to be a specialized polymerase that functions in
translesion synthesis in E. coli during the SOS response. This protein has been

re-named E. coli DNA Pol V (Tang et al., 1999).

b. Rev3 and Rev? proteins form Pol zeta

The interaction between Rev3 and Rev7 proteins was first demonstrated
by Nelson et al. (1996a) using the yeast two-hybrid system (Fields and Song,
1989). When LexA-Rev3 was used as bait to screen a GAL4 fusion library of
yeast cDNA, only two positive clones were obtained, and both of these were in-
frame fusions with REV7 (Nelson et al., 1996a). When Rev7 protein was
overexpressed in a yeast strain overexpressing Rev3 protein fused with the tag
of Glutathione S-Transferase (Gst), i.e., Gst—Rev3, the Rev7 and Rev3 proteins
were both present in the glutathione-sepharose fraction, indicating that yeast
Rev3 and Rev? form a complex (Nelson et al., 1996a). The DNA polymerase
activity of the Gst-Rev3zRev7 complex was 20-30 times the activity of Gst—Rev3
alone, as measured by in vitro primer extension assays. Based on this result, the
Rev3zRev7 complex was named DNA Pol zeta (Nelson et al., 1996a). Pol zeta is
a nonprocessive polymerase that also lacks 3’ to 5’ exonuclease activity. But, in
an in vitro assay, this polymerase was found to replicate past a thymine-thymine
cis-syn cyclobutane dimer with an efficiency of ~10%. This major UV—induced
photoproduct normally severely inhibits replication. In contrast, the efficiency of
bypass replication past this photoproduct with yeast DNA polymerase alpha is

less than 1% (Nelson et al., 1996a).

0. Studies on Rev1

46

fl

dCTPt
transfer
transfer
uracil 0
al.. 199
abasic 5
protein t
detectat

(Nebon.

3 R.
Tr

Was initie
yeast 8.
IOCate-d (
TEplicated
adCMP
freqUencii

cytOSlne

 

alld LaWr

traHSfErag

1) The Rev1 protein has a deoxycytidyl transferase activity

Purified recombinant Rev1 protein can transfer a dCMP from its precursor
dCTP to the 3' end of a DNA primer in a template-dependent manner. Efficient
transfer was observed opposite a template abasic site, but the efficiencies of
transfer opposite a template guanine was only ~20%, opposite an adenine or
uracil only ~10%. and opposite a thymine or cytosine, less than 1% (Nelson et
al., 1996b). In the absence of Rev1, Pol zeta inserts a nucleotide opposite an
abasic site and further extends it with an efficiency of 7%, but addition of Rev1
protein to the reaction increases the bypass efficiency to 30-40%. In contrast, no
detectable bypass was seen with Pol alpha, in the absence or presence of Rev1

(Nelson et al., 1996b).

2) Rev1 as a dCMP transferase in abasic site bypassing

The role of Rev1 as a dCMP transferase in abasic site bypassing in vivo
was initially recognized by Gibbs and Lawrence (1995). By transforming the
yeast S. cerevisiae with a vector containing a single abasic site specifically
located within a 28-nucleotide single-stranded region, and sequencing the
replicated vectors from transfonnants, they found that the yeast prefers to insert
a dCMP opposite the abasic site. In three different sequence contexts, the
frequencies of dCMP insertion were 83%, 62% and 85%. A similar bias for
cytosine insertion was also found with entirely single-stranded vectors (Gibbs
and Lawrence, 1995). The yeast rev1-1 strain, which expresses a Rev1 protein
with a glycine to arginine mutation at position 193, and retains substantial dCMP

transferase activity, showed a reduced ability to bypass abasic sites on

47

enginee
Neverth
them he
The abc
byyeas
different
gene C/
defective
base exc
at the N
glycosyle
mutation:
mutations
SUggesur
further as
mutant F

Substitutir

 

ConSlstlng
alanines

gene into

 

 

Can1' mu
mUtant S.

dgpensat

 

engineered constructs, i.e., 7%, compared to 25% by wild-type yeast.
Nevertheless, sequencing of those replicated constructs showed that 75% of
them had a dCMP insertion opposite the original abasic site (Nelson et al., 2000).
The above data indicate that the dCMP transferase activity of Rev1 is employed
by yeast in bypassing abasic sites. However, Haracska et al. (2001b) reported a
different observation. They studied the mutation spectrum of the chromosomal
gene CANs induced by DNA alkylating agent MMS in yeast double mutant strains
defective for AP endonucleases APN1 and APN2, and therefore incapable of
base excision repair. MMS can methylate adenine at the N3 position and guanine
at the N7 position. The removal of alkylated bases by an N-methyl purine DNA
glycosylase results in abasic sites. These investigators found that ~70% of the
mutations obtained were base substitutions, and ~30% were +1 or —1 frameshift
mutations. Among these base substitutions, the majority (64%) were adenine,
suggesting that dAMP is preferentially inserted across from the abasic sites. To
further assess the role of the dCMP transferase activity of Rev1 in vivo, a Rev1
mutant protein lacking the dCMP transferase activity was generated by
substituting the Asp 467 and Glu 468 residues in the highly conserved motif III
consisting of serine, isoleucine, aspartate, and glutamate residues (SIDE) with
alanines (Haracska et al., 2001b). Introduction of the rev1 Nam-Ala"68 mutant
gene into apn1 apn2 rev1 triple deletion mutant strain restored MMS-induced
can1’ mutations to the level seen in the parental apn1 apn2 double deletion
mutant strain, indicating that the deoxycytidyl transferase activity of Rev1 is

dispensable for mutagenesis induced by abasic sites (Haracska et al., 2001b).

48

 

Althoug"
abasic l

endence

3) E
In

mutagen

mutagen

 

insertion
transfera
T-T CIs-s
also aba
mutation
Confers

mUtatior
Centrally
mutant 3
from tra
the WP:
reduCed
in few,
the ”Mt
data 8U

aCtjvlty

Although these data might reflect the difference of the sequence context of the
abasic sites, i.e., an exogenous vector versus a chromosomal gene, other

evidence is needed in order to clarify this discrepancy.

3) Evidence for a second function of Rev1

In addition to the bypassing of abasic sites, Rev1 function is required for
mutagenesis induced by UV, ionizing radiation, and a variety of chemical
mutagens (Lawrence, 1994). Since the latter events usually do not involve the
insertion of dCMP, Rev1 may possess a function other than acting as a
transferase. To obtain more evidence, Nelson et al. (2000) studied the bypass of
T-T cis-syn cyclobutane dimers, T-T pyrimidine (6-4) pyrimidone adducts, and
also abasic sites in yeast strains carrying REV1 or REV3 deletions or the rev1-1
mutation. rev1-1 mutant protein retains substantial dCMP transfearse activity, but
confers UV—sensitivity and impaired mutagenesis to yeast strains carrying this
mutation. Double-stranded vectors containing one of these lesions located
centrally within a 28-nucleotide single-stranded region were transformed into the
mutant and wild-type strains, and the proportion of replicated plasmids resulting
from translesion replication was calculated (Nelson et al., 2000). As expected,
the bypass frequency of abasic sites in rev1 or rev3 deletion mutants was greatly
reduced (1-3%), as compared to wild-type strains (25%). The bypass frequency
in rev1-1 mutant was significantly reduced as well (7%). Nevertheless, 75% of
the mutations generated in rev1-1 mutant resulted from a dCMP insertion. These
data suggest at least in part, that function of Rev1 other than the transferase

activity is needed for bypassing abasic sites. Interestingly, these investigators

49

also
4) a
T—T
mute
ind ic
bypa

depe

and F
essen
melJ\
etal(
me NH.
mutatio,
debfion
Sensitivil
mdhafing
mutage”,
zeta Was
finanw
primer, E
delta Was

”OI Sllmu

 

also found that REV1 and REV3 functions are required for the bypass of T-T (6-
4) adducts but not T-T cyclobutane dimers. The wild-type strain bypassed 19% of
T-T (6-4) adduct-containing vector molecules used, but rev1 or rev3 deletion
mutants or rev1-1 mutant bypassed only 1-2%. The data from the rev1-1 mutant
indicates that a function other than dCMP transferase activity is involved in
bypassing T-T (6-4) adducts. The bypass of T-T cyclobutane dimers might

depend on Pol eta.

Yeast Pol delta consists of three subunits encoded by the POL3, POL31,
and POL32 genes respectively. It has been shown that POL3 and POL31 are
essential for viability, but the POL32 gene is not and that pol32 deletion mutants
are UV—sensitive and defective in UV mutagenesis (Gerik et al. 1998). Haracska
et al. (2001b) studied the role of replicative polymerase Pol delta in bypassing
the MMS-induced abasic sites by determining the effect of the pol32 deletion
mutation on MMS-induced CAN1s to can1’ mutations in the apn1 apn2 double
deletion mutant strain. Their data showed that pol32 deletion mutants exhibit mild
sensitivity to MMS, and complete abolishment of MMS-induced mutations,
indicating a requirement of the Pol32 subunit and probably Pol delta for the
mutagenic bypass of abasic sites. In in vitro primer extension assays, purified Pol
zeta was unable to insert a nucleotide opposite this lesion, but Pol delta or Rev1
protein was able to insert a nucleotide but could not extend from the resulting
primer. Efficient bypass of the AP site, however, could be achieved when Pol
delta was combined with Pol zeta, or Rev1 was combined with Pol zeta. Rev1 did

not stimulate AP bypass when it was combined with Pol delta. The bypassing

5O

efficie
of a

oppos
major
state I
nucleo
nucleor
Haracs
requires
step d(
Predomi
polymer.
bl'IJass i

lesion,

C- D:

1- D

a. h
H

all known
member
(Schneida
are logate

1W0 152‘8

efficiency with a Pol delta/Pol zeta combination (55%) was a little higher than that
of a Rev1/Pol zeta combination (32%). The nucleotide preferentially inserted
opposite AP sites by Pol delta in vitro was found to be dAMP, the same as the
major nucleotide (64%) being inserted in vivo (Haracska et al., 2001b). Steady-
state kinetic analyses show that Pol zeta was highly inefficient in inserting
nucleotides opposite the AP sites, but was efficient in extending from
nucleotides, particularly an A, inserted opposite this lesion. In light of these data,
Haracska et al. (2001b) proposed that in eukaryotes, bypass of an AP site
requires the sequential action of two DNA polymerases, wherein the extension
step depends upon Pol zeta, and the insertion step involves either the
predominant action of Pol delta, or the minor role of various translesional
polymerases, like Rev1 or Pol eta, and that the predominant role of Rev1 in AP
bypass is likely to be structural, i.e., to mediate the access of Pol zeta to the

lesion.

C. Damage tolerance RADG pathway in humans
1. Damage avoidance mechanisms

a. hHR6A, hHRGB and hRAD18

Humans have a conserved RAD6 pathway. Human homologs for almost
all known yeast genes of the RAD6 pathway have been found. For the founding
member RAD6, two human homologs, hHR6A and hHR6B, have been identified
(Schneider et al., 1990; Woffendin et al., 1991 and Koken et al., 1991), and they
are located on Xq24-q25 and 5q23-q31, respectively (Koken et al., 1992). These

two 152-amino acid Rad6 proteins share 95% sequence identity with each other

51

and ~7C
are ubic
level int
mice 08.
hHR6B
cerevisia
(Koken 6
human F

2000). s

 

purified
Rad6 pr
hRad18
the Cons
effect of

reWICatio

b. hill

A t
Well (Xiac
encodes a
alSo Share
idEntjfie d |
a tandEm

s50% ider

and ~70% identity with the yeast Rad6 protein (Koken et al., 1991). Both of them
are ubiquitously expressed in various tissues and cell types, with an elevated
level in testis (Koken et al., 1996). Inactivation of the hHR6B-homologous gene in
mice causes male infertility (Roest et al., 1996). In addition, both hHR6A and
hHR6B can complement the damage tolerance functions of RAD6 in S.
cerevisiae rad6 deletion mutants including damage avoidance and mutagenesis
(Koken et al., 1991), suggesting that the function of RAD6 is conserved. The
human RAD18 has been cloned as well (Tateishi et al., 2000 and Xin et al.,
2000). Stable hRad18-hHR6A or hRad18-hHR6B protein complexes can be
purified when hRad18 protein is co-expressed in yeast cells with either human
Rad6 protein (Xin et al., 2000). Human fibroblasts which stably express a mutant
hRad18 protein carrying a cysteine to phenylalanine substitution at residue 28 in
the conserved Rad6-binding ring-finger motif become sensitive to the cytotoxic
effect of UV, methyl methanesulfonate, and mitomycin C, and are defective in the

replication of UV-damaged DNA (Tateishi et al., 2000).

b. hMMSZ and UBC13

A human homolog of the yeast MMSZ gene (hMMSZ) has been cloned as
well (Xiao et al., 1998b). Like the yeast MMSZ gene, the hMMSZ gene also
encodes a ubiquitin-conjugating variant protein. Interestingly, the hMms2 protein
also shares >90% amino acid sequence identity with Croc—1, a protein previously
identified by its ability to transactivate c-fos expression in cells in culture through
a tandem repeat enhancer sequence. Because hMmsZ and Croc-1 also share

~50% identity and ~75% similarity with the yeast MmsZ protein, and both the

52

hMMSZ
mms2 .
spontan
two hon
with hur
(VanDer
ubiquitin

A:
human h
genes h
pathway
more the

the Yeast

2- Tr
a. hF
1) Ctr

Thi
et al. (15
disCoVere
enCOded

length CD

 

 

Amplificah.

hMMSZ and the CROC-1 genes are able to functionally complement the yeast
mmsZ defects with regard to sensitivity to DNA damaging agents and
spontaneous mutagenesis, the hMMSZ and CROC-1 genes are considered to be
two homologs of yeast MMSZ. hMmsZ protein forms a complex preferentially
with human ch13 (Moraes et al., 2001), just like its yeast counterpart does
(VanDemark et al., 2001). Association of MmsZ protein with ch13 facilitates the
ubiquitin chain assembly by ch13 (McKenna et al., 2001).

As discussed later, the REV3, REV7, REV1 and RAD30 genes all have
human homologs. In summary, except for RAD5, all major yeast RAD6 pathway
genes have identified human counterparts. Therefore, the RAD6 DNA repair
pathway is very likely conserved in humans, and since some human genes have
more than one homolog, the human RAD6 pathway might be more complex than

the yeast system.

2. Translesion synthesis mechanisms
a. hREV3

1) Cloning of hREV3 gene

The human homolog of the yeast REV3 gene was cloned in 1998 by Xiao
et al. (1998a) and independently by Gibbs et al. (1998). The latter group
discovered an expressed sequence tag (EST) in the dBEST database that
encoded a peptide with sequence homology to yeast Rev3 protein. The full
length cDNA of the hREV3 gene was then acquired by combining the EST
sequence and 5' upstream sequence obtained by the method of 5' Rapid

Amplification of cDNA Ends (RACE). The deduced hRev3 protein consists of

53

3.130 a
39% ide
residue

hRev3

polymer
byUnet
CNOmos
hybridize
HOWEVEr
gene or‘

confirmir

2) Tr

 

In

hREV3 g.

from the i

3,130 amino acid residues and shares with the yeast Rev3 protein 29%, 29%,
39% identity in one N-terminal region of ~340 residues, one middle region of 55-
residue and a C-terminal region of ~850 residues, respectively. In addition, the
hRev3 protein contains all six motifs characteristic of eukaryotic DNA
polymerases in the right order. The hRE V3 gene was also cloned independently
by Lin et al. (1999a). The hREV3 gene was originally considered to be located at
chromosome 1p32—33 as a result of studies using fluorescence in situ
hybridization with chromosome metaphase spreads (Xiao et al., 1998a).
However, a second report of the same year (Morelli et al., 1998) localized hREV3
gene on chromosome 6q21. A more recent study also provided evidence

confirming that hRE V3 is located on chromosome 6q21 (Kawamura et al., 2001).

2) The level of hRev3 protein in human cells might be low

In addition to the start codon ATG for the main open reading frame, the
hRE V3 gene has an out-of-frame ATG at position -58 nt. The translation initiated
from the additional ATG terminates at a stop codon within the main hREV3 open
reading frame, and would generate a peptide unrelated to hRev3 protein (Gibbs
et al., 1998). Moreover, the translation efficiency from these two ATG start
codons are expected to be comparable since the sequence context of these two
ATG’s are almost equally good in supporting translation. An out-of-frame ATG is
also found in the yeast REV3 gene. Examination of the 5' untranslated region of
the hREV3 mRNA also revealed a segment with potential to form a stem-loop
(hairpin) secondary structure (Lin et al., 1999a). The presence of an out-of-frame

ATG and a hairpin structure in the 5’ untranslated region suggest that the

54

Uansk

proteir

3)

functio
examir
mRNA.
was ub
that the

1
Qefierati
et al., 2c
(2000) r
Motifs w,‘
the COntn
mid‘geste
markedly

Were SIT

 

de"‘S'Opm

 

Was first

HSSUGS of

death Coin

translation of full-length hRev3 protein is very inefficient, and the level of hRev3

protein in human cells may be very low.

3) Importance of the function of hREV3 in mammals

The presence of the mRNA transcript is often used as an indicator of the
functioning of a gene in certain tissues. By using RT-PCR, Lin et al. (1999a)
examined 16 different human tissues and found that all expressed hREV3
mRNA. In an independent study, Kawamura et al. (2001) reported that hREV3
was ubiquitously expressed in all 27 normal human tissues tested and showed
that the expression is not dysregulated in malignant human tissues.

To study the function of RE V3 in mammals, mREV3 knock-out mice were
generated independently by three groups at almost the same time (Wittschieben
et al., 2000; Bemark et al., 2000; and Esposito et al., 2000). Wittschieben et al.
(2000) replaced two mREV3 exons containing conserved DNA polymerase
motifs with a cassette encoding G418 resistance and beta-galactosidase, under
the control of the mRE V3 promoter. They found that disruption of mRE V3 caused
mid-gestation embryonic lethality, with the frequency of embryos declining
markedly between 9.5 and 12.5 days post coitum. mREV3 knock-out embryos
were smaller than their heterozygous littemtates and showed retarded
development. Tissues in many areas were disorganized, with significantly
reduced cell density. mRev3 expression, traced by beta-galactosidase staining,
was first detected during early somitogenesis and gradually expanded to other
tissues of mesodermal origin, including extraembryonic membranes. Embryonic

death coincided with the period of more widely distributed mRev3 expression. No

55

 

 

signifies

reducec
out a fu
also pla

(Zan et

 

 

hUman [
CD4+ T'
bcl-s 9e
oligonUCl
mutagen
immUniza
pornon o
and term
antibome

VH genes

haematopoietic cells, fibroblast cells and embryonic cells could be derived in
mREV3(-/-) embryos (Esposito et al., 2000). These data indicate that REV3 is
essential for embryonic development in mammals. The exact mechanism

causing embryonic lethality is not known.

4) The role of hREV3 in mutagenesis

The requirement of hRE V3 gene for mutagenesis was demonstrated with
human cells (Gibbs et al., 1998). Human fibroblasts expressing high levels of an
hREV3 antisense RNA to a 5’ portion of hREV3 mRNA grow normally, are not
significantly more sensititve than normal cells to killing by UV, but show greatly
reduced UV-induced mutagenesis. The human gene, therefore, appears to carry
out a function similar to that of its yeast counterpart (Gibbs et al., 1998). hREV3
also plays a role in somatic mutations in the immunoglobulin and bcI-6 genes
(Zan et al., 2001; Diaz et al., 2001). hREV3 was constitutively expressed in
human B cells. Upon B cell receptor engagement and coculture with activated
CD4+ T cells, these lymphocytes upregulated Pol zeta, and mutated the Ig and
bcI-6 genes. Inhibition of the hREV3 by specific phosphorothioate-modified
oligonucleotides decreased lg and bcI-6 hypermutation and UV damage-induced
mutagenesis, without affecting cell cycle or viability (Zan et al., 2001). Following
immunization with antigens, transgenic mice that expressed antisense RNA to a
portion of mREV3 gene could induce vigorous antibody response, switch to lgG
and form germinal center, but showed a delay in the generation of high affinity
antibodies and a decrease in the accumulation of somatic hypermutation in the

VH genes of memory B cells (Diaz et al., 2001). These data suggest that

56

mamr
dame;

lgGrn

l

 

idenfifle
mhuak.
53% 5;
mRNA

testis to
importar

t0 ChrOn

 

freQuen
1992; S
mUltiple
tranSCrip
Cennnes
DIOtein S
DRNenyl
beMeEn
The Sigr
aumdafia

ConSeWe

mammalian REV3 is required not only for the mutagenesis induced by DNA
damaging agents, but also for natural process that requires mutagenesis such as

lgG maturation.

b. hREV7

In 2000, a candidate human homolog of S. cerevisiae REV7 (hREV7), was
identified in a yeast two-hybrid screen by using fragments of hREV3 as bait
(Murakumo et al., 2000). The hREV7 gene product displays 23% identity and
53% similarity with yeast Rev7. Northern blot analysis showed that hREV7
mRNA was ubiquitously expressed in various tissues, with the highest level in
testis followed by thymus, spleen, and peripheral blood leukocyte, indicating the
importance of the function of hREV7 in humans. The hREV7 gene was localized
to chromosome 1p36, a region that had been previously shown to have high
frequency of loss of heterozygosity in some types of human tumors (Moley et al.,
1992; Simon et al., 1995). However, Northern blot analysis using a human
multiple tissue blot did not reveal any alterations in the length of hREV7
transcript. Furthermore, sequence analysis of hREV7 cDNA in 50 tumor-derived
cell lines and 33 clinical tumor samples did not reveal any mutations. The hRev7
protein shares 23% identity and 54% similarity with the human mitotic checkpoint
protein hMad2 (MAD: mitotic arrest-deficient). What is more, an interaction
between hRev7 and hMad2 proteins was identified in in vitro pull-down assay.
The significance of the interaction between hRev7 and hMad2 remains to be
elucidated, and also, it has not yet been demonstrated that the hRev7 homolog

conserves the function of yeast Rev7 in mutagenesis.

57

1)

the sar
human
sequen
aligning
(2000)s
terminus
then ob
cDNA e

laborato

 

 

 

amino 3
Pretein, |
0fYeast,
31% and
One regic
respectiv.

2) p,

Th.

ATG inrtr.

c. hRE V1

1) Cloning of hREV1 gene

The hREV1 gene was cloned independently by two laboratories at virtually
the same time (Lin et al., 1999b; Gibbs et al., 2000). Lin et al. (1999b) screened
human bone marrow and leukocyte cDNA libraries with a human expressed
sequence tag (EST) as probe and derived the hREV1 cDNA sequence by
aligning the clones generated, based on the overlapping sequences. Gibbs et al.
(2000) started by identifying an EST encoding a peptide with similarity to the C-
terminus of yeast Rev1 protein, followed this by sequencing of the clone, and
then obtained the remaining cDNA by the method of 5' rapid amplification of
cDNA ends (Gibbs et al., 2000). The hREV1 genes identified in these two
laboratories are the same. The gene encodes an expected protein of 1,251
amino acid residues, compared with 985 residues in the corresponding yeast
protein. Evidence supporting the hypothesis that this human gene is a homolog
of yeast RE V1 comes from comparing these two proteins. They share 41%, 20%,
31% and 83% identity in two N-terminal regions of approximately 100 residues,
one region of approximately 320 residues, and one central motif of 13 residues,
respectively (Gibbs et al., 2000). The calculated molecular weight of the hRev1

protein is ~140kD.

2) Presence of an out-of-frame ATG on hREV1 open reading frame

The hREV1 cDNA has a 5’ out-of-frame ATG codon at position —35nt
upstream of the initiator ATG codon of the main open reading frame (ORF). This

ATG initiates a small reading frame that terminates at a TGA stop codon

58

overlap
ot-trarr.
this alte

is likely

3) t

E
hREV1
these tv
2q11.l a
20kb up:
chain re
2am.
examiner

al., 1999l

 

testis he

examined

4) hR
Th

Wthh ins
allUlinlc/a
We“ Pro

e)derlSIOn

 

overlapping the main ORF ATG (Gibbs et al., 2000). Translation from the 5' out-
of-frame ATG would lead to synthesis of an unrelated peptide. The presence of
this alternate open reading frame predicts that the level of hRev1 protein in cells

is likely to be low.

3) Chromosomal location and functional importance

By using the hREV1 cDNA as a probe, Lin et al. (1999b) isolated two
hREV1 genomic clones from a human placenta genomic DNA library. One of
these two clones contained a sequence tagged site (STS) localized between
2q11.1 and 2q11.2 on the cytogenetic ideogram. The STS was approximately
20kb upstream from the 5’ end of the hREV1 gene, as estimated by polymerase
chain reaction. Therefore, the hREV1 gene is located between 2q11.1 and
2q11.2. The mRNA of hREV1 gene was present in all 16 human tissues
examined, suggesting that the function of hREV1 is ubiquitous in humans (Lin et
al., 1999b). In an independent study, Murakumo et al. (2001) reported that the
testis has a much higher level of hREV1 mRNA than does other normal tissues

examined and that hREV1 expression is not abnormal in human cancer cell lines.

4) hRev1 as a dCMP transferase

The yeast Rev1 protein has an intrinsic deoxycytidyl transferase activity,
which inserts a dCMP at the 3’ end of a DNA primer opposite a template G or an
apurinic/apyrimidinic (AP) site (Nelson et al., 1996). To determine whether the
hRev1 protein possesses this activity, Lin et al. (1999b) performed in vitro primer

extension experiments with purified recombinant hRev1 protein. Their results

59

showe
(1 bu
sugge
abkeh
stern
at3.l
fanMy
and d.
1999a
al,19
DfilBl
conser
faWMy,
depen(

for the

 

pohu
that h
powme

SUQQES

”08er

5)i

damag

showed that human Rev1 protein specifically inserts a dCMP opposite a template
G, but does not insert any deoxynucleotide opposite a template A, C or T,
suggesting that hRev1 is a dCMP transferase. In addition, the hRev1 protein is
able to efficiently and specifically insert a dCMP opposite a DNA template abasic
site or a uracil residue, but cannot extend the synthesis if the next template is not
a.G. The hRev1 protein, and its yeast counterpart, are members of a growing
family of translesion DNA polymerases that includes umuC (Tang et al., 1999)
and dinB/dinP (Wagner et al., 1999) of Escherichia coli, Rad30 (Johnson et al.,
1999a) of the yeast Saccharomyces cerevisiae, and XPV/Rad30A (Johnson et
al., 1999b and Masutani et al., 1999a,b), Rad3OB (McDonald et al., 1999), and
DINB1 (Gerlach et al., 2001) of humans. Although hRev1 protein contains the
conserved domain required for the polymerase activity of other members in the
family, it does not possess such polymerase activity. Instead, it has a template-
dependent transferase activity. To determine the domains that are responsible
for the transferase activity, Masuda et al. (2001) generated various deletion and
point mutation mutants of the hRev1 protein. Primer extension analyses showed
that hRev1 proteins that carried mutations in the conserved domain of
polymerases lost the deoxycytidyl transferase activity and DNA binding ability,
suggesting that the structure of the active site of the deoxycytidyl transferase

closely resembles the active site of other members of the family.

5) Function of hREV1 is required for human cell mutagenesis

In yeast, the function of REV1 is absolutely required for virtually all

damage-induced mutagenesis (Lawrence, 1994). My research for the Ph.D.

60

degree
was t'
antiser
The de
indicat
that h?
BPDE

prepar

0f the
SystEm
binding
fused \
protein
actiVatE
Cells t)

galacto

 

evaant
Under

respect
hRev7.

SeqUen

degree involved the role of hREV1 in human cell mutagenesis. My hypothesis
was that hREV1 is required for mutagenesis. To test this, I employed an
antisense RNA strategy to decrease the function of hRev1 in human fibroblasts.
The data presented in Chapter 3, Chapter 4, and Appendix of this dissertation
indicate that hREV1 is required for mutagenesis induced by UV and BPDE, and
that hRev1 is involved in the generation of all base substitutions induced by
BPDE (Gibbs et al., 2000; Lawrence et al., 2000; Wang et al., manuscript in

preparation).

d. Interactions of hRev3, hRev7 and hRev1 proteins

A yeast two-hybrid system was extensively used to study the interactions
of the hRev1, hRev3 and hRev7 proteins (Murakumo et al., 2000, 2001). In this
system, a bait protein or fragment of a protein is fused with the GAL4 DNA
binding domain and another protein, or fragment of a protein, under study is
fused with the GAL4 transcription activation domain. If the bait protein and the
protein under study bind each other sufficiently, the hybrid transactivator will
activate the expression of two reporter genes, i.e., the gene that allows the yeast
cells to grow in medium without histidine and the gene coding for B-
galactosidase. These two reporter genes allow qualitative and quantitative
evaluation of the degree of binding between the bait protein and the protein
under study, by using colony growth assay and B-galactosidase assay,
respectively. Using this strategy, Murakumo et al. (2001) found that hRev3 binds
hRev7. The minimal domain for hRev3 to bind hRev7 is within amino acid

sequence 1847-1892 and the minimum region for hRev7 to interact with hRev3 is

61

whhm

pmfieu
pmneu'
respec
with th
rayon
Zland
hRev3
confint
2001)

detect

 

Neven
human

not be

that thi
$30)”
Aha Q
term)”i
Shows
difiEFEI
etal(;

hOmOC

within 21-155aa. These investigators also showed that the hRev1 and hRev7
proteins interact with each other. The minimum sequences of hRev1 and hRev7
proteins for such binding are amino acid sequences 1130-1251 and 21-155,
respectively. Interestingly, the hRev7 protein binds hRev3 and hRev1 protein
with the same region. hRev7 protein was also shown to form homodimer, and the
region of hRev7 for homodimerization is located between amino acid residues
21 and 155, the same region required for its interaction with hRev1 or with
hRev3. The authors used pull-down assays and coimmunoprecipitation assays to
confirm the interactions identified by yeast two-hybrid system (Murakumo et al.,
2001). However, in their in vitro pull-down assays, these investigators failed to
detect hRev1, hRev3, and hRev7 proteins forming a stable complex.
Nevertheless, the possibility that hRev3, hRev7, and hRev1 function together in
human cells cannot be ruled out just because the complex of three proteins could
not be identified in one kind of in vitro pull-down assay.

Sequence comparisons between human and yeast Rev proteins indicated
that the regions homologous to the interaction domains of hRev3 and hRev7 are
also present in yeast Rev3 and Rev7 proteins, suggesting that DNA polymerase
zeta complex is functionally conserved from yeast to human. However, the C-
terminal region of hRev1, which is important for interaction with hRev7 protein,
shows no homology with yeast Rev1, suggesting that hRev1 may somewhat
different from Rev1 in its enzymatic properties. Based on their results, Murakumo
et al. (2001) proposed a model in which, prior to DNA damage, hRev7 exists as a

homodimer, and hRev1 or hRev3 exists in the form of monomers in human cells,

62

andthr
hRev7
hRev3

order ti

mutate
(XPV)i
to the
(Maher
(ENS a
indudn
a SV4l
Masutt
contple
'ength
DFObes

et al.,

an 0Vi

 

(MaSut

 

 

and that when DNA damage occurs and translesion synthesis activity is needed,
hRev7 dissociates from the homodimers and forms the complex with hRev1 or
hRev3, allowing them to execute their functions. More studies are needed in

order to gain more insight into this pathway.

e. Polymerase eta, homolog of yeast RAD30

The human homolog of yeast RAD30 is found to be the gene that is
mutated in the cells of patients with the variant form of xeroderma pigmentosum
(XPV) (Masutani et al., 1999a, b). Cells derived from XPV patients are sensitive
to the cytotoxic effect of UV and show an elevated UV—induced mutagenesis
(Maher et al., 1976). In vitro studies with XPV cell extracts revealed that XPV
cells are less efficient than normal cells in bypassing most bulky lesions,
including UV photoproducts (reviewed in Cordonnier and Fuchs, 1999). By using
a SV40 origin-based plasmid containing a site-specific T-T cyclobutane dimer,
Masutani et al. (1999a) isolated a protein from HeLa cells capable of
complementing the T-T dimer bypassing defect of the XPV cell extracts. A full
length cDNA was subsequently identified from a HeLa cDNA library using DNA
probes deduced from four partial amino acid sequences of this protein (Masutani
et al., 1999b). Sequence homology searching revealed that this protein shares
an overall 19.6% identity and 31.9% similarity with yeast Rad30 protein
(Masutani et al., 1999b), which had just been identified as DNA polymerase eta
(Johnson et al., 1999a). Thus, the XPV gene is a human homolog of yeast
RAD30. The XPV gene was designated as hRAD30A as a second homolog,

hRAD3OB, to yeast RAD30 was found later (see below). Because XPV protein

63

(Pole‘
Masun

memn

1)

coHeag
the yea
1999):
second
thathE
etal,

hRad3
thereto
POlio
incomc
The I
Dh0top

aCehda

2I

 

CO/i Di

 

(Pol eta) inserts dAMP across T-T dimers very efficiently (Wang et al., 1991;
Masutani et al., 2000 and Johnson et al., 2000b), i.e., in an error-free manner,

the function of yeast RAD30 is conserved in humans.

f. Additional lesion-bypassing DNA polymerases
1) Pol iota

The yeast RAD30 gene was first identified by Woodgate and his
colleagues (McDonald et al., 1997). In an attempt to isolate human homolog(s) of
the yeast RAD30, the same group identified a novel homolog (McDonald et al.,
1999) different from the first homolog, hRAD30A, coding for human Pol eta. The
second homolog was designated as hRAD3OB. Northern blot analyses showed
that hRAD3OB is expressed in many human tissues, but at a low level (McDonald
et al., 1999). In vitro primer extension experiments with purified recombinant
hRad3OB protein revealed that it possesses a DNA polymerase activity, and
therefore, hRad3OB protein was named Pol iota (T issier et al., 2000a). Although
Pol iota replicates DNA in a template—dependent manner, it frequently
incorporates wrong nucleotides, even on undamaged DNA (Tissier et al., 2000a).
This polymerase can insert nucleotides opposite lesions, including UV
photoproducts (T issier et al., 2000b), abasic sites, 8-oxoguanine and N-2-

acetylaminofluorene-adduct in vitro (Zhang et al., 2001).

2) Pol kappa

Sequence homology searching for a human peptide with homology to E.

coli DinB protein (E. coli Pol IV), a UmuC homolog involved in untargeted

64

mutagi
tflDNVE
uduch

nuclec
abasm
error-p
dhner

humar
N2~dG
aninu
lesknu

Wang

bYPass
Obtaini

thChE

 

DOIym

CtrCUnr

 

mutagenesis, identified hDinB (Gerlach et al., 1999; Ogi et al., 1999). The
hDIN81 gene was found to encode a DNA polymerase (designated Pol kappa),
which synthesizes DNA with extraordinarily low fidelity (~1 error per 200
nucleotides) (Zhang et al., 2000b; Ohashi eta l., 2000). Pol kappa can bypass an
abasic site, an N-2-acetylaminofluorene (AAF)-adduct, or an 8-oxoguanine in an
error-prone manner, but it does not bypass a cis-syn or (6-4) thymine-thymine
dimer or a cisplatin-adduct (Zhang et al., 2000a; Ohashi eta I., 2000). In addition,
human Pol kappa effectively bypasses a template (-)-trans-anti-benzo[a]pyrene-
Nz-dG lesion in an error-free manner (Zhang et al., 2000a). These data implicate
an important role for Pol kappa in the mutagenic bypass of certain types of DNA
lesions. Recently, Pol kappa was found to be overexpressed in lung cancer (0-

Wang et al., 2001).

9. Summary of the kinds of lesions bypassed by error-prone DNA

polymerases

Table 1 summarizes the ability of error-prone DNA polymerases in
bypassing various kinds of DNA lesions. This is mainly based on the data
obtained from in vitro primer extension assays. Genetic evidence is used when
biochemical data are not available. It should be noted that the ability of a purified
polymerase found in vitro might not be used in vivo, and that in some

circumstances, more than one DNA polymerase could act together.

65

   

.. 3L FIF ~
E _ 3.3 h... E C?
-uCo
-u<<~
mcEZ —

l\ .t.
‘4

or

 

. Bog.
QOEQ h m.
o mmzto
m:.~mo.:QogiCO..mo~ k
boa <20 .
mom—meme:
Q

.w_nm__m>m .2. 2m 901: :0 San mm 55m 63> co Emu a
.Coom coca—2 ucm 8:233 ”room .mcm>> .6 955. 9: co woman 25...... e

 

 

 

 

 

 

 

 

 

 

as. 22.2%
«9.0.x.» ..o..$ Fr. :o_mco..xo
.0A<AA0:. 6:36 3:88 .o. .P a memo: 02380... $38. an:
2.8:. .0 9.35 30.85 69.85 .< 9.09.. So .< 9.35 229305. 5:5;
229%?
._.0 EB.
www.mEboa c2223
2.0.9.25 c2338 coficfixo 55:35 0: Sn
new m>_SnEm_u o: o: umxoofi 3.603 9. .0A<A._.A0 F 27.330 5.2 an:
boEoExo c< .0 3.35 .< 3.35 >393 2:.onch atoms 0 £30.... momnmm
Ame..—
3.8.3
29:6 Acetate. mucofio coo—.936 3:336
._.._. mcfimmgn mucofio .0\0A._.A< 0: .<< mucous. c2238 9. Em an:
.8 60536on .0 mtpmc. 3.35 .0 atoms $.35 :40 3.09: .02. $.an 02880.... <omnmm
m _on_ .o Ema
£533... .3 62.35
.o 32.9 map 32820::
m .3 pecans -commSE 55650 EB.
£850.82: 93 c_ .8 $3 53> 9.2.8 cmo €>om+v
.o. “.958. um._:cmm_ mommmgm Sn 69.85 6252.65 99mm
9m C>om+v $359232. c2336 ca 03565..
m>om .Eom 93 c_ .8 6256mm .0 m 9.85 9206 gem
mmmEmn c
33 osmEEEEaa. :23.
9968 33m 8385293 >3 2:225 225559... $2 .6 83
.926
m£cm=0 055:0 .056 2.23 3.539 <20
8.02 -u_<< -momm To. ._.-._. -226 ._..._. -oxo-w mam 239.. 8883::

 

 

 

 

 

 

 

 

 

 

«momma—Econ <20 m:_.mo__am.-co_mo_ .0 $3.30... ... 038.

66

 

acri
(Ate
CHVA
DbtA
Syntt
Oppo:
exten.
eukan
Whoa)
Pol z
2000a
abasl
photo
Pol z
mhnr

emce

 

h. Lesion-replicating polymerases could act sequentially in bypassing

lesions

To bypass a replication-blocking lesion and allow continuation of DNA
synthesis, the polymerase(s) must accomplish two steps, the insertion of a
deoxynucleotide opposite the lesion and the extension of synthesis from the
incorporated deoxynucleotide (Woodgate, 2001). This two-step bypassing can be
carried out by one DNA polymerase. For example, human Pol eta inserts AA
across from T-T dimer and extends from the inserted AA very efficiently
(Masutani et al., 1999b). In some circumstances, at least in vitro, error-prone
DNA polymerases have been shown to cooperate in bypassing lesions that block
DNA synthesis (Johnson et al., 2000a). Human Pol iota is error-prone in DNA
synthesis even on undamaged DNA. It can efficiently insert a deoxynucleotide
opposite an abasic site or the 3’ T of a T-T (6-4) photoproduct, but can not
extend the synthesis from inserted deoxynucleotides (Johnson et al., 2000a). In
eukaryotes, including yeast and humans, DNA polymerase zeta is essential for
virtually all damage-induced mutagenesis (Lawrence and Maher, 2001). Although
Pol zeta bypasses a T-T dimer with a frequency of 3%-10% (Johnson et al.,
2000a; Nelson et al., 1996), it does not bypass a T-T (6-4) photoproduct and
abasic site (Johnson et al., 2000a). However, efficient bypass of a T-T (6-4)
photoproduct and an abasic site was achieved by the combination of Pol iota and
Pol zeta (Johnson et al., 2000a). Indeed, yeast Pol zeta extends from
mismatched primer-template ends on undamaged or damaged DNA very

efficiently (Johnson et al., 2000a). Other examples of polymerase coordination

67

iscau
sugge

Mmon

Dwnma
Skin c,
that )

transf

mode

 

ehen
Conta
addu
mout

elids

 

include bypassing of abasic sites by Rev1 and Pol zeta (Nelson et al., 1996b),
and bypassing of bulky adducts by hRev1 and Pol kappa (Zhang et al., 2002), if
Rev1 or hRev1 is considered a DNA polymerase as suggested by Haracska et
al. (2002). Although the cooperation between lesion-replicating polymerases has

only been demonstrated in vitro so far, it is possible to be employed in vivo.

3. Relevance of the damage tolerance pathway to carcinogenesis in

humans

Cancer is a disease charaterized by uncontrolled cell proliferation, which
is caused by mutations in oncogenes and/or tumor suppressor genes. Evidence
suggests that the human RAD6 pathway plays an important role in
tumorigenesis.

Mutations in the hRAD30A gene cause the variant form of xeroderma
pigmentosum (XPV) (Masutani et al., 1999a, b). XPV patients are predisposed to
skin cancer (Mamada et al., 1992). In vitro transformation studies also showed
that XP variant cells are more sensitive than normal cells to UV-induced
transformation to anchorage independence (McCormick et al., 1986).

As discussed above, Pol kappa is a low-fidelity polymerase with a
moderate processivity when replicating undamaged DNA. In vitro primer
extension assays also showed that recombinant Pol kappa can replicate DNA
containing lesions such as an abasic site, a N—2-acetylaminofluorene guanine
adduct or 8-oxoguanine. Transient expression of this polymerase in cultured
mouse cells caused a 10-fold increase in the mutation frequency of the

endogenous HPRT gene. In an attempt to explore the potential involvement of

68

Poll
level

semii
and I
cance
adeni
ovens
kian

smnm
analys

develc

Attardi

At’yage

Bailly .

Bailly

Baker

 

Pol kappa in carcinogenesis, O-Wang et al. (2001) compared the expression
level of Pol kappa in tumors and adjacent nontumorous tissues by Northern blot,
semiquantitative RT-PCR, and Western blot analyses. Among 29 pairs of tumor
and normal specimens from patients with stages I to lllb non-small cell lung
cancer, including 13 adenocarcinomas, 15 squamous cell cancers, and 1
adenosquamous carcinoma, 21 tumor samples were found to have
overexpression of Pol kappa. The elevated Pol kappa expression was likely due
to an activated transcription, because gene amplification was not found in tumor
samples exhibiting higher expression of Pol kappa, as shown by Southern blot
analysis. These data suggests that Pol kappa may contribute to lung tumor

development by accelerating the accumulation of mutations.

Ill. References

Attardi LD, Reczek EE, Cosmas C, Demicco EG, McCurrach ME, Lowe SW,
Jacks T. (2000) PERP, an apoptosis-associated target of p53, is a novel
member of the PMP-22/ga53 family. Genes Dev., 14:704-18.

Ayyagari R, lmpellizzeri KJ, Yoder BL, Gary SL, Burgers PM. (1995) A mutational
analysis of the yeast proliferating cell nuclear antigen indicates distinct
roles in DNA replication and DNA repair. Mol Cell Biol., 15:4420-9.

Bailly V, Lamb J, Sung P, Prakash S, Prakash L. (1994) Specific complex
formation between yeast RAD6 and RAD18 proteins: a potential
mechanism for targeting RAD6 ubiquitin-conjugating activity to DNA
damage sites. Genes Dev., 8:811-20.

Bailly V, Lauder S, Prakash S, Prakash L. (1997) Yeast DNA repair proteins
Rad6 and Rad18 form a heterodimer that has ubiquitin conjugating, DNA
binding, and ATP hydrolytic activities. J Biol Chem., 272:23360-5.

Baker SJ, Fearon ER, Nigro JM, Hamilton SR, Preisinger AC, Jessup JM,
vanTuinen P, Ledbetter DH, Barker DF, Nakamura Y, White R, Vogelstein
B. (1989) Chromosome 17 deletions and p53 gene mutations in colorectal
carcinomas. Science, 244:217-21.

69

Banin

Barak

Barbe

Bemar

Bertra

Bienz

Bisho;

Bbgog

BOdnK

B°'ev.

80nd \

808 JL

 

Banin S, Moyal L, Shieh S, Taya Y, Anderson CW, Chessa L, Smorodinsky NI,
Prives C, Reiss Y, Shiloh Y, Ziv Y. (1998) Enhanced phosphorylation of
p53 by ATM in response to DNA damage. Science, 281 :1674-7.

Barak Y, Juven T, Haffner R, Oren M. (1993) mdm2 expression is induced by
wild type p53 activity. EMBO J., 12:461-8.

Barbacid M. (1987) ras genes. Annu Rev Biochem, 56:779-827.

Bemark M, Khamlichi AA, Davies SL, Neuberger MS. (2000) Disruption of mouse
polymerase zeta (Rev3) leads to embryonic lethality and impairs
blastocyst development in vitro. Curr Biol., 10:1213-6.

Bertrand P, Rouillard D, Boulet A, Levalois C, Soussi T, Lopez BS. (1997)
Increase of spontaneous intrachromosomal homologous recombination in
mammalian cells expressing a mutant p53 protein. Oncogene. 14:1117—
22.

Bienz M, Clevers H. (2000) Linking colorectal cancer to Wnt signaling. Cell,
103:311-20.

Bishop JM. (1995) Cancer: the rise of the genetic paradigm. Genes Dev., 9:1309-
15.

Blagosklonny MV. (2000) p53 from complexity to simplicity: mutant p53
stabilization, gain-of-function, and dominant-negative effect. FASEB J.,
14:1901-7.

Bodmer WF, Bailey CJ, Bodmer J, Bussey HJ, Ellis A, Gorman P, Lucibello FC,
Murday VA, Rider SH, Scambler P, Sheer D, Solomon E, Spurr NK.
(1987) Localization of the gene for familial adenomatous polyposis on
chromosome 5. Nature, 328:614-6.

Boley, S.E., McManus, T.P., Maher, V.M., McCormick, J.J. (2000) Malignant
transformation of human fibroblast cell strain MSU-1.1 by N-methyl-N-
nitrosourea: evidence of elimination of p53 by homologous recombination.
Cancer Res., 60: 4105-4111.

Bond J, Haughton M, Blaydes J, Gire V, Wynford-Thomas D, Wyllie F. (1996)
Evidence that transcriptional activation by p53 plays a direct role in the
induction of cellular senescence. Oncogene, 13:2097-104.

Bos JL, Fearon ER, Hamilton SR, Verlaan-de Vries M, van Boom JH, van der Eb
AJ, Vogelstein B. (1987) Prevalence of ras gene mutations in human
colorectal cancers. Nature, 327:293-7.

Bos JL. (1988) The ras gene family and human carcinogenesis. Mutat. Res,
195:255-71.

7O

Bouve

Bronni

Broom

Broom‘

Brown

Brusky

Canm;

Carrie;

 

 

Chang

 

Cho l<

Comet

 

 

Bouvet M, Ellis LM, Nishizaki M, Fujiwara T, Liu W, Bucana CD, Fang B, Lee JJ,
Roth JA. (1998) Adenovirus—mediated wild-type p53 gene transfer down-
regulates vascular endothelial growth factor expression and inhibits
angiogenesis in human colon cancer. Cancer Res., 58:2288-92.

Bronner CE, Baker SM, Morrison PT, Warren G, Smith LG, Lescoe MK, Kane M,
Earabino C, Lipford J, Lindblom A, Tannergard P, Bollag RJ, Godwin AR,
Ward DC, Nordenskjold M, Fishel R, Kolodner R, Liskay RM (1994)
Mutation in the DNA mismatch repair gene homologue hMLH1 is
associated with hereditary non-polyposis colon cancer. Nature, 368:258-
61.

Broomfield S, Chow BL, Xiao W. (1998) MMSZ, encoding a ubiquitin-conjugating-
enzyme-like protein, is a member of the yeast error-free postreplication
repair pathway. Proc Natl Acad Sci U S A., 95:5678-83.

Broomfield S, Hryciw T, Xiao W. (2001) DNA postreplication repair and
mutagenesis in Saccharomyces cerevisiae. Mutat. Res., 486:167-84.

Brown JP, Wei W, Sedivy JM. (1997) Bypass of senescence after disruption of
p21CIP1/WAF1 gene in normal diploid human fibroblasts. Science,
277:831-4.

Brusky J, Zhu Y, Xiao W. (2000) UBC13, a DNA-damage-inducible gene, is a
member of the error-free postreplication repair pathway in Saccharomyces
cerevisiae. Curr. Genet, 37:168-74.

Canman CE, Lim DS, Cimprich KA, Taya Y, Tamai K, Sakaguchi K, Appella E,
Kastan MB, Siliciano JD. (1998) Activation of the ATM kinase by ionizing
radiation and phosphorylation of p53. Science, 281:1677-9.

Carrier F, Georgel PT, Pourquier P, Blake M, Kontny HU, Antinore MJ, Gariboldi
M, Myers TG, Weinstein JN, Pommier Y, Fornace AJ Jr. (1999) Gadd45, a
p53-responsive stress protein, modifies DNA accessibility on damaged
chromatin. Mol Cell Biol., 19:1673-85.

Chang BD, Xuan Y, Broude EV, Zhu H, Schott B, Fang J, Roninson lB. (1999)
Role of p53 and p21waf1/cip1 in senescence-like terminal proliferation
arrest induced in human tumor cells by chemotherapeutic drugs.
Oncogene, 18:4808-18.

Cho KR, Oliner JD, Simons JW, Hedrick L, Fearon ER, Preisinger AC, Hedge P,
Silverman GA, Vogelstein B. (1994) The DOC gene: structural analysis
and mutations in colorectal carcinomas. Genomics, 19:525-31.

Cordonnier AM, Fuchs RP. (1999) Replication of damaged DNA: molecular
defect in xeroderma pigmentosum variant cells. Mutat Res., 435:111-9.

71

Dame
DeLe
Deng

Dewr

[haz.

Easth

el‘DEi

el-Dei

Et-Dej

Elenb;

 

Dameron KM, Volpert OV, Tainsky MA, Bouck N. (1994) Control of angiogenesis
in fibroblasts by p53 regulation of thrombospondin-1. Science, 26521582-
4.

DeLeo AB, Jay G,'Appella E, Dubois GC, Law LW, Old LJ. (1979) Detection of a
transfon'nation-related antigen in chemically induced sarcomas and other
transformed cells of the mouse. Proc Natl Acad Sci U S A, 76:2420-4.

Deng C, Zhang P, Harper JW, Elledge SJ, Leder P. (1995) Mice lacking
p21CIP1NVAF1 undergo normal development, but are defective in G1
checkpoint control. Cell, 822675-84.

Derynck R, Zhang Y, Feng XH. (1998) Smads: transcriptional activators of TGF-
beta responses. Cell, 95:737-40.

Diaz M, Verkoczy LK, Flajnik MF, Klinman NR. (2001) Decreased frequency of
somatic hypermutation and impaired affinity maturation but intact germinal
center formation in mice expressing antisense RNA to DNA polymerase
zeta. J lmmunol., 167:327-35.

Eastham JA, Hall SJ, Sehgal I, Wang J, Timme TL, Yang G, ConneII-Crowley L,
Elledge SJ, Zhang W, Harper JW, Thompson TC (1995) In vivo gene
therapy with p53 or p21 adenovirus for prostate cancer. Cancer Res.,
55:5151-5.

eI-Deiry WS, Harper JW, O'Connor PM, Velculescu VE, Canman CE, Jackman J,
Pietenpol JA, Burrell M, Hill DE, Wang Y, Wiman KG, Mercer WE, Kastan
MB, Kohn KW, Elledge SJ, Kinzler KW, Vogelstein B. (1994) WAF1/CIP1
is induced in p53-mediated G1 arrest and apoptosis. Cancer Res.,
54:1169-74.

eI-Deiry WS, Kern SE, Pietenpol JA, Kinzler KW, Vogelstein B. (1992) Definition
of a consensus binding site for p53. Nat Genet, 1:45-9.

el-Deiry WS, Tokino T, Velculescu VE, Levy DB, Parsons R, Trent JM, Lin D,
Mercer WE, Kinzler KW, Vogelstein B. (1993) WAF1, a potential mediator
of p53 tumor suppression. Cell, 752817-25.

Elenbaas B, Spirio L, Koerner F, Fleming MD, Zimonjic DB, Donaher JL,
Popescu NC, Hahn WC, Weinberg RA. (2001) Human breast cancer cells
generated by oncogenic transformation of primary mammary epithelial
cells. Genes Dev., 15:50-65.

Eliyahu D, Michalovitz D, Eliyahu S, Pinhasi-Kimhi O, Oren M. (1989) Wild-type
p53 can inhibit oncogene-mediated focus formation. Proc Natl Acad Sci U
S A, 86:8763-7.

72

 

Eh

Es;

Far

Fan

Fea

Fear

Feng

Field

Finla

 

FShe

Ford

F0rd

 

Eliyahu D, Raz A, Gruss P, Givol D, Oren M. (1984) Participation of p53 cellular
tumour antigen in transformation of normal embryonic cells. Nature,
312:646-9.

Esposito G, Godindagger l, Klein U, Yaspo ML, Cumano A, Rajewsky K. (2000)
Disruption of the Rev3l-encoded catalytic subunit of polymerase zeta in
mice results in early embryonic lethality. Curr Biol., 10:1221-4.

Fang L, lgarashi M, Leung J, Sugrue MM, Lee SW, Aaronson SA. (1999)
p21Waf1/Cip1/Sdi1 induces permanent growth arrest with markers of
replicative senescence in human tumor cells lacking functional p53.
Oncogene, 18:2789-97.

Farmer G, Colgan J, Nakatani Y, Manley JL, Prives C. (1996) Functional
interaction between p53, the TATA-binding protein (T BP), and TBP—
associated factors in vivo. Mol Cell Biol., 16:4295—304.

Fearon ER, Cho KR, Nigro JM, Kern SE, Simons JW, Ruppert JM, Hamilton SR,
Preisinger AC, Thomas G, Kinzler KW, Vogelstein B. (1990) Identification
of a chromosome 18q gene that is altered in colorectal cancers. Science,
247:49-56.

Fearon ER, Hamilton SR, Vogelstein B. (1987) Clonal analysis of human
colorectal tumors. Science, 238:193-7.

Feng XH, Lin X, Derynck R. (2000) Smad2, Smad3 and Smad4 cooperate with
Sp1 to induce p15(lnk4B) transcription in response to TGF-beta. EMBO
J., 19:5178-93.

Fields S and Song 0. (1989) A novel genetic system to detect protein-protein
interactions. Nature, 340:245-6.

Finlay CA, Hinds PW, Levine AJ. (1989) The p53 proto-oncogene can act as a
suppressor of transformation. Cell, 57:1083-93.

Fishel R, Lescoe MK, Rao MR, Copeland NG, Jenkins NA, Garber J, Kane M,
Kolodner R. (1993) The human mutator gene homolog MSH2 and its
association with hereditary nonpolyposis colon cancer. Cell, 75:1027-38.

Ford JM, Baron EL, Hanawalt PC. (1998) Human fibroblasts expressing the
human papillomavirus E6 gene are deficient in global genomic nucleotide

excision repair and sensitive to ultraviolet irradiation. Cancer Res.,
58:599-603.

Ford JM, Hanawalt PC. (1995) Li-Fraumeni syndrome fibroblasts homozygous
for p53 mutations are deficient in global DNA repair but exhibit normal
transcription-coupled repair and enhanced UV resistance. Proc Natl Acad
Sci U S A, 92:8876-80.

73

Forh

Fnei

Fnel

an

Fucr

Gent

Gene

Gene

Ghoc

(fibbs

Gibbs

Gibbs

 

 

Forrester K, Almoguera C, Han K, Grizzle WE, Perucho M. (1987) Detection of
high incidence of K-ras oncogenes during human colon tumorigenesis.
Nature, 327:298-303.

Friedberg EC, Walker GC and Siede W. (1995) DNA Repair and Mutagenesis,
American Society for Microbiology Press, Washington DC.

Friend SH, Bernards R, Rogelj S, Weinberg RA, Rapaport JM, Albert DM, Dryja
TP. (1986) A human DNA segment with properties of the gene that
predisposes to retinoblastoma and osteosarcoma. Nature, 323:643—6.

Fry, DG, Milam, LD, Dillberger, JE, Maher, VM, McCormick, JJ (1990) Malignant
transformation of an infinite life span human fibroblast cell strain by
transfection with v-Ki-ras. Oncogene, 521415-1418.

Fuchs SY, Adler V, Buschmann T, Wu X, Ronai Z. (1998) Mdm2 association with
p53 targets its ubiquitination. Oncogene, 17:2543-7.

Gerik, K.J., Li, X., Pautz, A., and Burgers, P.M.J. (1998) Characterization of the
two small subunits of Saccharomyces cerevisiae DNA polymerase 6. J.
Biol. Chem, 273: 19747-19755.

Gerlach VL, Aravind L, Gotway G, Schultz RA, Koonin EV, Friedberg EC. (1999)
Human and mouse homologs of Escherichia coli DinB (DNA polymerase
IV), members of the UmuC/DinB superfamily. Proc Natl Acad Sci U S A,
96:1 1922-7.

Gerlach VL, Feaver WJ, Fischhaber PL, Friedberg EC. (2001) Purification and
characterization of pol kappa, a DNA polymerase encoded by the human
DINB1 gene. J Biol Chem., 276:92-8.

Giaccia AJ, Kastan MB. (1998) The complexity of p53 modulation: emerging
patterns from divergent signals. Genes Dev., 12:2973-83.

Gibbs PE and Lawrence CW. (1995) Novel mutagenic properties of abasic sites
in Saccharomyces cerevisiae. J Mol Biol, 251:229-36.

Gibbs PE, McGregor WG, Maher VM, Nisson P, Lawrence CW. (1998) A human
homolog of the Saccharomyces cerevisiae REV3 gene, which encodes
the catalytic subunit of DNA polymerase zeta. Proc Natl Acad Sci U S A,
95:6876-80.

Gibbs PE, Wang XD, Li Z, McManus TP, McGregor WG, Lawrence CW, Maher
VM. (2000) The function of the human homolog of Saccharomyces
cerevisiae REV1 is required for mutagenesis induced by UV light. Proc
Natl Acad Sci U S A, 97:4186-91.

74

Cheer

(Node

Choss

Guan

Hahn

Hahr

Hana

Halal

Halal

Hag.

Herb

 

Greenblatt MS, Bennett WP, Hollstein M, Harris CC. (1994) Mutations in the p53
tumor suppressor gene: clues to cancer etiology and molecular
pathogenesis. Cancer Res., 54:4855-78.

Groden J, Thliveris A, Samowitz W, Carlson M, Gelbert L, Albertsen H, Joslyn G,
Stevens J, Spirio L, Robertson M, Sargeant L, Krapcho K, Wolff E, Burt R,
Hughes JP, Warrington J, McPherson J, Wasmuth J, Lepaslier D,
Abderrahim H, Cohen D, Leppert M, White R. (1991) Identification and
characterization of the familial adenomatous polyposis coli gene. Cell,
66:589—600.

Grossfeld GD, Ginsberg DA, Stein JP, Bochner BH, Esrig D, Groshen S, Dunn
M, Nichols PW, Taylor CR, Skinner DG, Cote RJ. (1997)
Thrombospondin-1 expression in bladder cancer: association with p53
alterations, tumor angiogenesis, and tumor progression. J Natl Cancer
Inst., 892219-27.

Guanti G, Resta N, Simone C, Cariola F, Demma I, Fiorente P, Gentile M. (2000)
Involvement of PTEN mutations in the genetic pathways of colorectal
cancerogenesis. Hum Mol Genet, 92283-7.

Hahn SA, Schutte M, Hoque AT, Moskaluk CA, da Costa LT, Rozenblum E,
Weinstein CL, Fischer A, Yeo CJ, Hruban RH, Kern SE. (1996) DPC4, a
candidate tumor suppressor gene at human chromosome 18q21.1.
Science, 271 :350-3.

Hahn WC, Counter CM, Lundberg AS, Beijersbergen RL, Brooks MW, Weinberg
RA. (1999) Creation of human tumour cells with defined genetic elements.
Nature, 400:464-8.

Hanahan D, Weinberg RA. (2000) The hallmarks of cancer. Cell, 100257-70.

Haracska L, Kondratick CM, Unk I, Prakash S, Prakash L. (2001a) Interaction
with PCNA is essential for yeast DNA polymerase eta function. Mol Cell,
8:407-15.

Haracska L, Unk I, Johnson RE, Johansson E, Burgers PM, Prakash S, Prakash
L. (2001b) Roles of yeast DNA polymerases delta and zeta and of Rev1 in
the bypass of abasic sites. Genes Dev., 152945-54.

Haracska L, Prakash S, Prakash L. (2002) Yeast Rev1 Protein Is a G Template-
specific DNA Polymerase. J Biol Chem., 277:15546-51.

Harbour JW, Luo RX, Dei Santi A, Postigo AA, Dean DC. (1999) Cdk
phosphorylation triggers sequential intramolecular interactions that
progressively block Rb functions as cells move through G1. Cell, 98:859-
69.

75

Hart

Harr

Hau

Hind

Hocl
Hoei‘

Hofrr

Holls:

Hond

How!

HUan

 

HUpp

Hurlit

 

 

Harfe BD, Jinks-Robertson S. (2000) DNA mismatch repair and genetic
instability. Annu Rev Genet, 34:359-399.

Harper JW, Adami GR, Wei N, Keyomarsi K, Elledge SJ. (1993) The p21 Cdk-
interacting protein Cip1 is a potent inhibitor of G1 cyclin-dependent
kinases. Cell, 752805-16.

Haupt Y, Maya R, Kazaz A, Oren M. (1997) Mdm2 promotes the rapid
degradation of p53. Nature, 387:296-9.

Hinds P, Finlay C, Levine AJ. (1989) Mutation is required to activate the p53
gene for cooperation with the ras oncogene and transformation. J Virol,
632739-46.

Hochstrasser M. (1996) Ubiquitin-dependent protein degradation. Annu Rev
Genet, 302405-39.

Hoeijmakers JH. (2001) Genome maintenance mechanisms for preventing
cancer. Nature, 411:366-74.

Hofmann RM and Pickart CM. (1999) Noncanonical MMSZ-encoded ubiquitin-
conjugating enzyme functions in assembly of novel polyubiquitin chains
for DNA repair. Cell, 962645-53.

Hollstein M, Rice K, Greenblatt MS, Soussi T, Fuchs R, Sorlie T, Hovig E, Smith-
Sorensen B, Montesano R, Harris CC. (1994) Database of p53 gene

somatic mutations in human tumors and cell lines. Nucleic Acids Res.,
22:3551-5.

Honda R, Tanaka H, Yasuda H. (1997) Oncoprotein MDM2 is a ubiquitin ligase
E3 for tumor suppressor p53. FEBS Lett, 420:25-7.

Howe JR, Roth S, Ringold JC, Summers RW, Jarvinen HJ, Sistonen P,
Tomlinson IP, Houlston RS, Bevan S, Mitros FA, Stone EM, Aaltonen LA.
(1998) Mutations in the SMAD4/DPC4 gene in juvenile polyposis.
Science, 280:1086-8.

Huang HJ, Yee JK, Shew JY, Chen PL, Bookstein R, Friedmann T, Lee EY, Lee
WH. (1988) Suppression of the neoplastic phenotype by replacement of
the RB gene in human cancer cells. Science, 242:1563-6.

Hupp TR, Meek DW, Midgley CA, Lane DP. (1992) Regulation of the specific
DNA binding function of p53. Cell, 712875-86.

Hurlin, PJ, Maher, VM and McCormick, JJ (1989) Malignant transformation of
human fibroblasts caused by expression of a transfected T24 HRAS
oncogene. Proc. Natl Acad. Sci. USA, 86:187-191.

76

Hw;

hue

Isot

hah.

Jani

JanL

Jenk

Jent

hncn

John

JOhn

John

JOhn

 

Hwang BJ, Ford JM, Hanawalt PC, Chu G. (1999) Expression of the p48
xeroderma pigmentosum gene is p53-dependent and is involved in global
genomic repair. Proc Natl Acad Sci U S A, 96:424-8.

Iruela-Arispe ML, Dvorak HF. (1997) Angiogenesis: a dynamic balance of
stimulators and inhibitors. Thromb Haemost., 78:672-7.

Isobe M, Emanuel BS, Givol D, Oren M, Croce CM. (1986) Localization of gene
for human p53 tumour antigen to band 17p13. Nature, 320:84-5.

ltahana K, Dimri G, Campisi J. (2001) Regulation of cellular senescence by p53.
Eur J Biochem., 268:2784-91.

Janus F, Albrechtsen N, Dornreiter I, Wiesmuller L, Grosse F, Deppert W.
(1999a) The dual role model for p53 in maintaining genomic integrity. Cell
Mol Life Sci, 55:12-27.

Janus F, Albrechtsen N, Knippschild U, Wiesmuller L, Grosse F, Deppert W.
(1999b) Different regulation of the p53 core domain activities 3'-to-5'
exonuclease and sequence-specific DNA binding. Mol Cell Biol., 19:2155-
68.

Jenkins JR, Rudge K, Currie GA. (1984) Cellular immortalization by a cDNA
clone encoding the transformation-associated phosphoprotein p53.
Nature, 312:651-4.

Jentsch S, McGrath JP, Varshavsky A. (1987) The yeast DNA repair gene RAD6
encodes a ubiquitin-conjugating enzyme. Nature, 329:131-4.

Jiricny J. (1998) Replication errors: cha(lle)nging the genome. EMBO J.,
17:6427-36.

Johnson RD, Jasin M (2001) Double-strand-break-induced homologous
recombination in mammalian cells. Biochem Soc Trans., 29:196-201.

Johnson RE, Henderson ST, Petes TD, Prakash S, Bankmann M, Prakash L.
(1992) Saccharomyces cerevisiae RAD5-encoded DNA repair protein
contains DNA helicase and zinc-binding sequence motifs and affects the
stability of simple repetitive sequences in the genome. Mol Cell Biol.,
1223807-18.

Johnson RE, Prakash S, Prakash L. (1999a) Efficient bypass of a thymine-
thymine dimer by yeast DNA polymerase, Pol eta. Science, 283:1001-4.

Johnson RE, Kondratick CM, Prakash S, Prakash L. (1999b) hRAD30 mutations
in the variant form of xeroderma pigmentosum. Science, 285:263-5.

77

Job

Job

Johr

Jone

Jung

Kast

Kaw;

Kern

KtESe

Kinzle’

 

Johnson RE, Prakash S, Prakash L. (19990) Requirement of DNA polymerase
activity of yeast Rad30 protein for its biological function. J Biol Chem.,
274:15975-7.

Johnson RE, Washington MT, Haracska L, Prakash S, Prakash L. (2000a)
Eukaryotic polymerases iota and zeta act sequentially to bypass DNA
lesions. Nature, 406:1015-9.

Johnson RE, Washington MT, Prakash S, Prakash L. (2000b) Fidelity of human
DNA polymerase eta. J Biol Chem., 275:7447-50.

Jones JS, Weber S, Prakash L. (1988) The Saccharomyces cerevisiae RAD18
gene encodes a protein that contains potential zinc finger domains for

nucleic acid binding and a putative nucleotide binding sequence. Nucleic
Acids Res., 16:7119-31.

Jung MS, Yun J, Chae HD, Kim JM, Kim SC, Choi TS, Shin DY. (2001) p53 and
its homologues, p63 and p73, induce a replicative senescence through
inactivation of NF-Y transcription factor. Oncogene, 20:5818-25.

Kastan MB, Zhan Q, eI-Deiry WS, Carrier F, Jacks T, Walsh WV, Plunkett BS,
Vogelstein B, Fornace AJ Jr. (1992) A mammalian cell cycle checkpoint
pathway utilizing p53 and GADD45 is defective in ataxia-telangiectasia.
Cell, 712587-97.

Kawamura K, O-Wang J, Bahar R, Koshikawa N, Shishikura T, Nakagawara A,
Sakiyama S, Kajiwara K, Kimura M, Tagawa M. (2001) The error-prone
DNA polymerase zeta catalytic subunit (Rev3) gene is ubiquitously
expressed in normal and malignant human tissues. Int J Oncol., 18297-
103.

Kern R, Zimmermann FK. (1978) The influence of defects in excision and error
prone repair on spontaneous and induced mitotic recombination and
mutation in Saccharomyces cerevisiae. Mol Gen Genet, 161:81-8.

Kieser A, Weich HA, Brandner G, Marrne D, Kolch W. (1994) Mutant p53
potentiates protein kinase C induction of vascular endothelial growth
factor expression. Oncogene, 92963-9.

Kinzler KW, Nilbert MC, Su LK, Vogelstein B, Bryan TM, Levy DB, Smith KJ,
Preisinger AC, Hedge P, McKechnie D, Finniear R, Markham A, Groffen J,
Boguski MS, Altschul SF, Horii A, Ando H, Miyoshi Y, Miki Y, Nishisho I,
Nakamura Y. (1991) Identification of FAP locus genes from chromosome
5q21. Science, 253:661-5.

Kinzler KW, Vogelstein B. (1996) Lessons from hereditary colorectal cancer. Cell,
872159-70.

78

KnL

Kol

Kok
Koke
Koke
Kubb
Ififlae

Land

Lane

Larime

Lat/life)
Lame)

LaVl(fer

 

Knudson, C.M., K.S.K. Tung, W.G. Tourtellotte, G.A.J. Brown, and SJ.
Korsmeyer. (1995). Bax-deficient mice with lymphoid hyperplasia and
male germ cell death. Science, 270: 96-99

Ko LJ, Prives C. (1996) p53: puzzle and paradigm. Genes Dev., 10:1054-72.

Koken MH, Hoogerbrugge JW, Jasper-Dekker I, de Wit J, Willemsen R, Roest
HP, Grootegoed JA, Hoeijmakers JH. (1996) Expression of the ubiquitin-
conjugating DNA repair enzymes HHR6A and B suggests a role in
spermatogenesis and chromatin modification. Dev Biol., 173:119-32.

Koken MH, Reynolds P, Jaspers-Dekker I, Prakash L, Prakash S, Bootsma D,
Hoeijmakers JH. (1991) Structural and functional conservation of two
human homologs of the yeast DNA repair gene RAD6. Proc Natl Acad Sci
U S A, 88:8865-9.

Koken MH, Smit EM, Jaspers-Dekker l, Oostra BA, Hagemeijer A, Bootsma D,
Hoeijmakers JH. (1992) Localization of two human homologs, HHR6A and
HHR6B, of the yeast DNA repair gene RAD6 to chromosomes Xq24-q25
and 5q23-q31. Genomics, 122447-53.

Kubbutat MH, Jones SN, Vousden KH. (1997) Regulation of p53 stability by
Mdm2. Nature, 387:299-303.

Kulaeva OI, Koonin EV, McDonald JP, Randall SK, Rabinovich N, Connaughton
JF, Levine AS, Woodgate R. (1996) Identification of a DinB/UmuC
homolog in the archeon Sulfolobus solfataricus. Mutat Res., 357:245-53.

Land H, Parada LF, Weinberg RA. (1983) Tumorigenic conversion of primary
embryo fibroblasts requires at least two cooperating oncogenes. Nature,
304:596-602.

Lane DP, Crawford LV. (1979) T antigen is bound to a host protein in SV40-
transformed cells. Nature, 278:261-3.

Larimer FW, Perry JR, Hardigree AA. (1989) The REV1 gene of Saccharomyces
cerevisiae: isolation, sequence, and functional analysis. J Bacteriol.,
171:230-7.

Lawrence C. (1994) The RAD6 DNA repair pathway in Saccharomyces
cerevisiae: what does it do, and how does it do it? Bioessays, 16:253-8.

Lawrence CW, Christensen R. (1976) UV mutagenesis in radiation-sensitive
strains of yeast. Genetics, 822207-32.

Lawrence CW, Christensen RB. (1978) Ultraviolet-induced reversion of cyc1
alleles in radiation sensitive strains of yeast. ll. rev2 mutant strains.
Genetics, 902213-26.

79

La

La

Lav

Lav.

Lea

Lee

Lepr

Lin \

 

Lin V

 

Lawrence CW and Hinkle DC. (1996) DNA polymerase zeta and the control of
DNA damage induced mutagenesis in eukaryotes. Cancer Surv., 28:21-
31.

Lawrence CW, Maher VM. (2001) Eukaryotic mutagenesis and translesion
replication dependent on DNA polymerase zeta and Rev1 protein.
Biochem Soc Trans, 29:187-91.

Lawrence CW, Stewart JW, Sherman F, Christensen R. (1974) Specificity and
frequency of ultraviolet-induced reversion of an iso-1-cytochrome c ochre
mutant in radiation-sensitive strains of yeast. J Mol Biol., 852137-62.

Lawrence CW, Gibbs PEM, Murante RS, Wang XD, Li Z, McManus TP,
McGregor WG, Nelson JR, Hinkle DC, Maher VM (2000) Roles of DNA
polymerase zeta and REV1 protein in eukaryotic mutagenesis and
translesion replication. Cold Spring Harbor Symposia on Quantitative
Biology, 65:61-69.

Leach FS, Nicolaides NC, Papadopoulos N, Liu B, Jen J, Parsons R, Peltomaki
P, Sistonen P, Aaltonen LA, Nystrom-Lahti M, Guan XY, Zhang J, Meltzer
PS, Yu JW, Kao FT, Chen DJ, Cerosaletti KM, Fournier REK, Todd S,
Lewis T, Leach RJ, Naylor SL, Weissenbach J, Mecklin JP, Jarvinen H,
Petersen GM, Hamilton SR, Green J, Jass J, Watson P, Lynch HT, Trent
JM, Delachapelle A, Kinzler KW, Vogelstein B. (1993) Mutations of a mutS
homolog in hereditary nonpolyposis colorectal cancer. Cell, 7521215-25.

Lee WH, Bookstein R, Hong F, Young LJ, Shew JY, Lee EY. (1987) Human
retinoblastoma susceptibility gene: cloning, identification, and sequence.
Science, 23521394-9.

Leppert M, Dobbs M, Scambler P, O'Connell P, Nakamura Y, Stauffer D,
Woodward S, Burt R, Hughes J, Gardner E, Lathrop M, Wasmuth J,
Lalouel JM, White R. (1987) The gene for familial polyposis coli maps to
the long arm of chromosome 5. Science, 238:1411-3.

Liang SH, Clarke MF. (2001) Regulation of p53 localization. Eur J Biochem,
268:2779-83.

Lin W, Wu X, Wang Z. (1999a) A full-length cDNA of hREV3 is predicted to
encode DNA polymerase zeta for damage-induced mutagenesis in
humans. Mutat Res., 433:89-98.

Lin W, Xin H, Zhang Y, Wu X, Yuan F, Wang Z. (1999b) The human REV1 gene
codes for a DNA template-dependent dCMP transferase. Nucleic Acids
Res., 27:4468-75.

8O

Li

Li

Lc

LL

Lu

Ma

Ma

Mar

Mar

Mar
Mas

 

Mas

 

Linzer DI, Levine AJ. (1979) Characterization of a 54K dalton cellular SV40 tumor
antigen present in SV40-transformed cells and uninfected embryonal
carcinoma cells. Cell, 17:43-52.

Liu B, Parsons R, Papadopoulos N, Nicolaides NC, Lynch HT, Watson P, Jass
JR, Dunlop M, Wyllie A, Peltomaki P, de la Chapelle A, Hamilton SR,
Vogelstein B, Kinzler KW. (1996) Analysis of mismatch repair genes in
hereditary non-polyposis colorectal cancer patients. Nat Med., 22169-74.

Lodish HF, Berk A, Zipursky SL, Matsudaira P, Baltimore D, and Darnell, J
(2000) Molecular Cell Biology (4"1 edition), WH Freeman and Company.

Lu H, Fisher RP, Bailey P, Levine AJ. (1997) The CDK7-cycH-p36 complex of
transcription factor IIH phosphorylates p53, enhancing its sequence-
specific DNA binding activity in vitro. Mol Cell Biol., 17:5923-34.

Lundberg AS, Weinberg RA. (1998) Functional inactivation of the retinoblastoma
protein requires sequential modification by at least two distinct cyclin-Cdk
complexes. Mol Cell Biol., 182753-61.

Maher VM, Ouellette LM, Curren RD, McCormick JJ. (1976) Frequency of
ultraviolet light-induced mutations is higher in xeroderma pigmentosum
variant cells than in normal human cells. Nature, 261:593-5.

Malkin D, Li FP, Strong LC, Fraumeni JF Jr, Nelson CE, Kim DH, Kassel J,
Gryka MA, Bischoff FZ, Tainsky MA, Friend SH. (1990) Germ line p53
mutations in a familial syndrome of breast cancer, sarcomas, and other
neoplasms. Science, 250:1233-8.

Mamada A, Miura'K, Tsunoda K, Hirose I, Furuya M, Kondo S. (1992)
Xeroderma pigmentosum variant associated with multiple skin cancers
and a lung cancer. Dermatology, 184:177-81.

Markowitz S, Wang J, Myeroff L, Parsons R, Sun L, Lutterbaugh J, Fan RS,
Zborowska E, Kinzler KW, Vogelstein B, Brattain M, Willson JKV. (1995)
Inactivation of the type II TGF-beta receptor in colon cancer cells with
microsatellite instability. Science, 268:1336-8.

Marshall CJ. (1996) Ras effectors. Curr. Opin. Cell Biol., 81197-204.

Masuda Y, Takahashi M, Tsunekuni N, Minami T, Sumii M, Miyagawa K, Kamiya
K. (2001) Deoxycytidyl transferase activity of the human REV1 protein is
closely associated with the conserved polymerase domain. J Biol Chem.,
276:15051-8.

Masutani C, Araki M, Yamada A, Kusumoto R, Nogimori T, Maekawa T, lwai S,
Hanaoka F. (1999a) Xeroderma pigmentosum variant (XP-V) correcting

81

protein from HeLa cells has a thymine dimer bypass DNA polymerase
activity. EMBO J., 18:3491-501.

Masutani C, Kusumoto R, Yamada A, Dohmae N, Yokoi M, Yuasa M, Araki M,
lwai S, Takio K, Hanaoka F. (1999b) The XPV (xeroderma pigmentosum
variant) gene encodes human DNA polymerase eta. Nature, 3992700-4.

Masutani C, Kusumoto R, lwai S, Hanaoka F. (2000) Mechanisms of accurate
translesion synthesis by human DNA polymerase eta. EMBO J., 1923100-
9.

McCormick, J.J. (1996) Malignant transformation of human fibroblasts by ionizing
radiation. Int J Radiat Biol, 692 707-715.

McCormick JJ, Kateley-Kohler S, Watanabe M, Maher VM. (1986) Abnormal
sensitivity of human fibroblasts from xeroderma pigmentosum variants to

transformation to anchorage independence by ultraviolet radiation. Cancer
Res., 462489-92.

McCurrach, M.E., T.M.F. Connor, C.M. Knudson, S.J. Korsmeyer, and SW.
Lowe. (1997) Bax-deficiency promotes drug resistance and oncogenic

transformation by attenuating p53-dependent apoptosis. Proc. Natl. Acad.
Sci USA, 94: 2345-2349.

McDonald JP, Levine AS, Woodgate R. (1997) The Saccharomyces cerevisiae
RAD30 gene, a homologue of Escherichia coli dinB and umuC, is DNA
damage inducible and functions in a novel error-free postreplication repair
mechanism. Genetics, 14721557-68.

McDonald JP, Rapic-Otrin V, Epstein JA, Broughton BC, Wang X, Lehmann AR,
Wolgemuth DJ, Woodgate R. (1999) Novel human and mouse homologs
of Saccharomyces cerevisiae DNA polymerase eta. Genomics, 60:20-30.

McDonald JP, Tissier A, Frank EG, lwai S, Hanaoka F, Woodgate R. (2001) DNA
polymerase iota and related rad30-like enzymes. Philos Trans R Soc Lond
B Biol Sci., 356253-60.

McKenna S, Spyracopoulos L, Moraes T, Pastushok L, Ptak C, Xiao W, Ellison
MJ. (2001) Noncovalent interaction between ubiquitin and the human
DNA repair protein MmsZ is required for ch13-mediated
polyubiquitination. J Biol Chem, 276:40120-6.

Mekeel KL, Tang W, Kachnic LA, Luo CM, DeFrank JS, Powell SN. (1997)
Inactivation of p53 results in high rates of homologous recombination.
Oncogene, 14:1847-57.

Miller DG (1980) On the nature of susceptibility to cancer. The presidential
address. Cancer, 46:1307-1318.

82

Miyashita T, Reed JC. (1995) Tumor suppressor p53 is a direct transcriptional
activator of the human bax gene. Cell, 80:293-9.

Miyoshi Y, Nagase H, Ando H, Horii A, Ichii S, Nakatsuru S, Aoki T, Miki Y, Mori
T, Nakamura Y. (1992) Somatic mutations of the APC gene in colorectal

tumors: mutation cluster region in the APC gene. Hum Mol Genet, 12229-
33.

Moley, J. F., Brother, M. B., Fong, C.-T., White, P. S., Baylin, S. B., Nelkin, 8.,
Wells, S. A., and Brodeur, G. M. (1992) Consistent association of 1p loss
of heterozygosity with pheochromocytomas from patients with multiple
endocrine neoplasia type 2 syndromes. Cancer Res., 52:770-774.

Moll UM, Zaika A. (2001) Nuclear and mitochondrial apoptotic pathways of p53.
FEBS Lett, 493:65-9.

Momand J, Zambetti GP, Olson DC, George D, Levine AJ. (1992) The mdm-2
oncogene product forms a complex with the p53 protein and inhibits p53-
mediated transactivation. Cell, 69:1237-45.

Moraes TF, Edwards RA, McKenna S, Pastushok L, Xiao W, Glover JN, Ellison
MJ. (2001) Crystal structure of the human ubiquitin conjugating enzyme
complex, hMms2-hch13. Nat Struct Biol., 82669-73.

Morelli C, Mungall AJ, Negrini M, Barbanti-Brodano G, Croce CM. (1998)
Alternative splicing, genomic structure, and fine chromosome localization
of REV3L. Cytogenet Cell Genet, 83:18-20.

Morgan, T.L., Yang, D.J., Fry, D.G., Hurlin, P.J., Kohler, S.K., Maher, V.M.,
McCormick, J.J. (1991) Characteristics of an infinite life span diploid
human fibroblast cell strain and a near-diploid strain arising from a clone

of cells expressing a transfected v-myc oncogene. Exp. Cell Res., 197:
125-136.

Morin PJ, Sparks AB, Korinek V, Barker N, Clevers H, Vogelstein B, Kinzler KW.
(1997) Activation of beta-catenin-ch signaling in colon cancer by
mutations in beta-catenin or APC. Science, 275:1787-90.

Morrison A, Christensen RB, Alley J, Beck AK, Bernstine EG, Lemontt JF,
Lawrence CW. (1989) REV3, a Saccharomyces cerevisiae gene whose
function is required for induced mutagenesis, is predicted to encode a
nonessential DNA polymerase. J Bacteriol., 171:5659-67.

Mukhopadhyay D, Tsiokas L, Sukhatme VP. (1995) Wild-type p53 and v-Src
exert opposing influences on human vascular endothelial growth factor
gene expression. Cancer Res., 55:6161-5.

83

Mummenbrauer T, Janus F, Muller B, Wiesmuller L, Deppert W, Grosse F.
(1996) p53 Protein exhibits 3'-to-5' exonuclease activity. Cell, 85:1089-99.

Munger K, Phelps WC, Bubb V, Howley PM, Schlegel R. (1989) The E6 and E7
genes of the human papillomavirus type 16 together are necessary and

sufficient for transformation of primary human keratinocytes. J Virol,
63:4417-21.

Murakumo Y, Roth T, Ishii H, Rasio D, Numata S, Croce CM, Fishel R. (2000) A
human REV7 homolog that interacts with the polymerase zeta catalytic
subunit hREV3 and the spindle assembly checkpoint protein hMAD2. J
Biol Chem., 275:4391-7.

Murakumo Y, Ogura Y, Ishii H, Numata S, lchihara M, Croce CM, Fishel R,
Takahashi M. (2001) Interactions in the error-prone postreplication repair
proteins hREV1, hREV3, and hREV7. J Biol Chem., 276:35644-51.

Nelson JR, Gibbs PE, Nowicka AM, Hinkle DC, Lawrence CW. (2000) Evidence
for a second function for Saccharomyces cerevisiae Rev1 p. Mol.
Microbiol., 372549-54.

Nelson JR, Lawrence CW, Hinkle DC. (1996a) Thymine-thymine dimer bypass
by yeast DNA polymerase zeta. Science, 272:1646-9.

Nelson JR, Lawrence CW, Hinkle DC. (1996b) Deoxycytidyl transferase activity
of yeast REV1 protein. Nature, 382:729-31.

Nicolaides NC, Papadopoulos N, Liu B, Wei YF, Carter KC, Ruben SM, Rosen
CA, Haseltine WA, Fleischmann RD, Fraser CM, Adams MD, Venter JC,
Dunlop MG, Hamilton SR, Petersen GM, Delachapelle A, Vogelstein B,
Kinzler KW (1994) Mutations of two PMS homologues in hereditary
nonpolyposis colon cancer. Nature, 371 :75-80.

Nishisho l, Nakamura Y, Miyoshi Y, Miki Y, Ando H, Horii A, Koyama K,
Utsunomiya J, Baba S, Hedge P. (1991) Mutations of chromosome 5q21
genes in FAP and colorectal cancer patients. Science, 253:665-9.

Oda K, Arakawa H, Tanaka T, Matsuda K, Tanikawa C, Mori T, Nishimori H,
Tamai K, Tokino T, Nakamura Y, Taya Y. (2000) p53AlP1, a potential
mediator of p53-dependent apoptosis, and its regulation by Ser-46-
phosphorylated p53. Cell, 102:849-62.

Offer H, Wolkowicz R, Matas D, Blumenstein S, Livneh Z, Rotter V. (1999) Direct
involvement of p53 in the base excision repair pathway of the DNA repair
machinery. F EBS Lett, 4502197-204.

84

Ogi T, Kato T Jr, Kato T, Ohmori H. (1999) Mutation enhancement by DINB1, a
mammalian homologue of the Escherichia coli mutagenesis protein dinB.
Genes Cells, 42607-18.

Ohashi E, Ogi T, Kusumoto R, lwai S, Masutani C, Hanaoka F, Ohmori H. (2000)
Error-prone bypass of certain DNA lesions by the human DNA polymerase
kappa. Genes Dev., 14:1589-94.

Okamura S, Arakawa H, Tanaka T, Nakanishi H, Ng CC, Taya Y, Monden M,
Nakamura Y. (2001) p53DINP1, a p53-inducible gene, regulates p53-
dependent apoptosis. Mol Cell, 8:85-94.

Oliner JD, Pietenpol JA, Thiagalingam S, Gyuris J, Kinzler KW, Vogelstein B.
(1993) Oncoprotein MDM2 conceals the activation domain of tumour
suppressor p53. Nature, 362:857-60.

O'Reilly, 3., Walicka, M., Kohler, S.K., Dunstan, R., Maher, V.M., McCormick,
J.J. (1998) Dose-dependent transformation of cells of human fibroblast
cell strain MSU-1.1 by cobalt-60 gamma radiation and characterization of
the transformed cells. Radiat Res., 150: 577-584.

Oren M, Maltzman W, Levine AJ. (1981) Post-translational regulation of the 54K
cellular tumor antigen in normal and transformed cells. Mol Cell Biol,
12101-10.

O-Wang J, Kawamura K, Tada Y, Ohmori H, Kimura H, Sakiyama S, Tagawa M.
(2001) DNA polymerase kappa, implicated in spontaneous and DNA
damage-induced mutagenesis, is overexpressed in lung cancer. Cancer
Res., 61:5366-9.

Pal S, Datta K, Mukhopadhyay D. (2001) Central role of p53 on regulation of
vascular permeability factor/vascular endothelial growth factor
(VPFNEGF) expression in mammary carcinoma. Cancer Res., 61 :6952-7.

Papadopoulos N, Nicolaides NC, Wei YF, Ruben SM, Carter KC, Rosen CA,
Haseltine WA, Fleischmann RD, Fraser CM, Adams MD, Venter JC,
Hamilton SR, Petersen GM, Watson P, Lynch HT, Peltomaki P, Mecklin
JP, Delachapelle A, Kinzler KW, Vogelstein B. (1994) Mutation of a mutL
homolog in hereditary colon cancer. Science, 263:1625-9.

Parsons R, Li GM, Longley MJ, Fang WH, Papadopoulos N, Jen J, de la
Chapelle A, Kinzler KW, Vogelstein B, Modrich P. (1993) Hypen'nutability
and mismatch repair deficiency in RER+ tumor cells. Cell, 75:1227-36.

Pavletich NP, Chambers KA, Pabo CO. (1993) The DNA-binding domain of p53
contains the four conserved regions and the major mutation hot spots.
Genes Dev., 722556-64.

85

Peltomaki P, Aaltonen LA, Sistonen P, Pylkkanen L, Mecklin JP, Jarvinen H,
Green JS, Jass JR, Weber JL, Leach FS, Petersen GM, Hamilton SR,
Delachapelle A, Vogelstein B. (1993) Genetic mapping of a locus
predisposing to human colorectal cancer. Science, 260:810-2.

Peltomaki P. (2001) Deficient DNA mismatch repair: a common etiologic factor
for colon cancer. Hum Mol Genet, 102735-40.

Powell SM, Zilz N, Beazer-Barclay Y, Bryan TM, Hamilton SR, Thibodeau SN,
Vogelstein B, Kinzler KW. (1992) APC mutations occur early during
colorectal tumorigenesis. Nature, 359:235-7.

Prakash L. (1974) Lack of chemically induced mutation in repair-deficient
mutants of yeast. Genetics, 78:1101-18.

Prakash L. (1981) Characterization of postreplication repair in Saccharomyces
cerevisiae and effects of rad6, rad18, rev3 and rad52 mutations. Mol Gen
Genet, 184:471-8.

Prakash L and Prakash S. (1977) Isolation and characterization of MMS-
sensitive mutants of Saccharomyces cerevisiae. Genetics, 86:33-55.

Prakash S, Sung P, Prakash L. (1993) DNA repair genes and proteins of
Saccharomyces cerevisiae. Annu Rev Genet, 27:33-70.

Prives C. (1998) Signaling to p53: breaking the MDM2-p53 circuit. Cell, 9525-8.

Pruitt K, Der CJ. (2001) Ras and Rho regulation of the cell cycle and
oncogenesis. Cancer Lett, 171 :1-10

Reinhold, D.S., Walicka, M., Elkassaby, M., Milam, L.D., Kohler, S.K., Dunstan,
R.W., McCormick, J.J. Malignant transformation of human fibroblasts by
ionizing radiation. Int J Radiat Biol, 69: 707-715, 1996.

Robles Al, Wang XW, Harris CC. (1999) Drug-induced apoptosis is delayed and
reduced in XPD lymphoblastoid cell lines: possible role of TFIIH in p53-
mediated apoptotic cell death. Oncogene, 18:4681-8.

Roest HP, van Klaveren J, de Wit J, van Gurp CG, Koken MH, Vermey M, van
Roijen JH, Hoogerbrugge JW, Vreeburg JT, Baarends WM, Bootsma D,
Grootegoed JA, Hoeijmakers JH. (1996) Inactivation of the HR6B
ubiquitin-conjugating DNA repair enzyme in mice causes male sterility
associated with chromatin modification. Cell, 86:799-810.

Roush AA, Suarez M, Friedberg EC, Radman M, Siede W. (1998) Deletion of the
Saccharomyces cerevisiae gene RADBO encoding an Escherichia coli
DinB homolog confers UV radiation sensitivity and altered mutability. Mol
Gen Genet. 2572686-92.

86

Ruley, H. (1983)Adenovirus early region 1A enables viral and cellular
transforming genes to transform primary cells in culture. Nature, 3042602-
606.

Saintigny Y, Rouillard D, Chaput B, Soussi T, Lopez BS. (1999) Mutant p53
proteins stimulate spontaneous and radiation-induced intrachromosomal
homologous recombination independently of the alteration of the
transactivation activity and of the G1 checkpoint. Oncogene, 1823553-63.

Sakaguchi K, Herrera JE, Saito S, Miki T, Bustin M, Vassilev A, Anderson CW,
Appella E. (1998) DNA damage activates p53 through a phosphorylation-
acetylation cascade. Genes Dev., 12:2831-41.

Sayama K, Shirakata Y, Midorikawa K, Hanakawa Y, Hashimoto K. (1999)
Possible involvement of p21 but not of p16 or p53 in keratinocyte
senescence. J Cell Physiol., 179:40-4.

Schneider R, Eckerskorn C, Lottspeich F, Schweiger M. (1990) The human
ubiquitin carrier protein E2 (Mr = 17,000) is homologous to the yeast DNA
repair gene RAD6. EMBO J., 9:1431-5.

Schuler M, Green DR. (2001) Mechanisms of p53-dependent apoptosis.
Biochem Soc Trans., 292684-8.

Seto E, Usheva A, Zambetti GP, Momand J, Horikoshi N, Weinmann R, Levine
AJ, Shenk T. (1992) Wild-type p53 binds to the TATA-binding protein and
represses transcription. Proc Natl Acad Sci U S A, 89:12028-32.

Shay JW, Pereira-Smith OM, Wright WE. (1991) A role for both RB and p53 in
the regulation of human cellular senescence. Exp Cell Res., 196:33-9.

Shibata MA, Yoshidome K, Shibata E, Jorcyk CL, Green JE. (2001) Suppression
of mammary carcinoma growth in vitro and in vivo by inducible expression
of the Cdk inhibitor p21. Cancer Gene Ther., 8:23-35.

Shieh SY, lkeda M, Taya Y, Prives C. (1997) DNA damage-induced
phosphorylation of p53 alleviates inhibition by MDM2. Cell, 912325-34.

Shivji KK, Kenny MK, Wood RD. (1992) Proliferating cell nuclear antigen is
required for DNA excision repair. Cell, 69:367-74.

Sieber OM, Tomlinson IP, Lamlum H. (2000) The adenomatous polyposis coli
(APC) tumour suppressor—genetics, function and disease. Mol Med
Today, 62462-9.

Siliciano JD, Canman CE, Taya Y, Sakaguchi K, Appella E, Kastan MB. (1997)
DNA damage induces phosphorylation of the amino terminus of p53.
Genes Dev., 11:3471-81.

87

 

Simon, M., von Deimling, A., Larson, J. J., Wellenreuther, R., Kaskel, P., Waha,
A., Wamick, R. E., Tew, J. M., Jr., and Menon, A. G. (1995) Allelic losses
on chromosomes 14, 10, and 1 in atypical and malignant meningiomas: a
genetic model of meningioma progression.Cancer Res., 55:4696-4701.

Singhal RK, Hinkle DC, Lawrence CW. (1992) The REV3 gene of
Saccharomyces cerevisiae is transcriptionally regulated more like a repair
gene than one encoding a DNA polymerase. Mol Gen Genet, 236217-24.

Smith ML, Chen IT, Zhan Q, Bae l, Chen CY, Gilmer TM, Kastan MB, O'Connor
PM, Fornace AJ Jr. (1994) Interaction of the p53-regulated protein
Gadd45 with proliferating cell nuclear antigen. Science, 266:1376-80.

Smith ML, Ford JM, Hollander MC, Bortnick RA, Amundson SA, Seo YR, Deng
CX, Hanawalt PC, Fornace AJ Jr. (2000) p53-mediated DNA repair
responses to UV radiation: studies of mouse cells lacking p53, p21, and/or
gadd45 genes. Mol Cell Biol., 20:3705-14.

Smits R, Kielman MF, Breukel C, Zurcher C, Neufeld K, Jagmohan-Changur S,
Hofland N, van Dijk J, White R, Edelmann W, Kucherlapati R, Khan PM,
Fodde R. (1999) Apc1638T: a mouse model delineating critical domains of
the adenomatous polyposis coli protein involved in tumorigenesis and
development. Genes Dev., 13:1309-21.

Solomon E, Voss R, Hall V, Bodmer WF, Jass JR, Jeffreys AJ, Lucibello FC,
Patel l, Rider SH. (1987) Chromosome 5 allele loss in human colorectal
carcinomas. Nature, 328:616-9.

Spence J, Sadis S, Haas AL, Finley D. (1995) A ubiquitin mutant with specific
defects in DNA repair and multiubiquitination. Mol Cell Biol., 15:1265-73.

Srivastava S, Zou ZQ, Pirollo K, Blattner W, Chang EH. (1990) Germ-line
transmission of a mutated p53 gene in a cancer-prone family with Li-
Fraumeni syndrome. Nature, 348:747-9.

Steller H. (1995) Mechanisms and genes of cellular suicide. Science, 26721445-
9.

Stenger JE, Tegtmeyer P, Mayr GA, Reed M, Wang Y, Wang P, Hough PV,
Mastrangelo IA. (1994) p53 oligomerization and DNA looping are linked
with transcriptional activation. EMBO J., 13:6011-20.

Sturzbecher HW, Donzelmann B, Henning W, Knippschild U, Buchhop S. (1996)
p53 is linked directly to homologous recombination processes via
RAD51/RecA protein interaction. EMBO J., 1521992-2002.

88

 

Sugrue MM, Shin DY, Lee SW, Aaronson SA. (1997) Wild-type p53 triggers a
rapid senescence program in human tumor cells lacking functional p53.
Proc Natl Acad Sci U S A,.94:9648-53.

Sung P, Prakash S, Prakash L. (1990) Mutation of cysteine-88 in the
Saccharomyces cerevisiae RADG protein abolishes its ubiquitin-

conjugating activity and its various biological functions. Proc Natl Acad Sci
U S A, 87:2695-9.

Svejstrup JQ, Vichi P, Egly JM. (1996) The multiple roles of transcription/repair
factor TFIIH. Trends Biochem Sci., 212346-50.

Tang M, Shen X, Frank EG, O'Donnell M, Woodgate R, Goodman MF. (1999)
UmuD'(2)C is an error-prone DNA polymerase, Escherichia coli pol V.
Proc Natl Acad Sci U S A, 96:8919-24.

Tateishi S, Sakuraba Y, Masuyama S, Inoue H, Yamaizumi M. (2000)
Dysfunction of human Rad18 results in defective postreplication repair
and hypersensitivity to multiple mutagens. Proc Natl Acad Sci U S A,
97:7927-32.

Thiagalingam S, Lengauer C, Leach FS, Schutte M, Hahn SA, Overhauser J,
Willson JK, Markowitz S, Hamilton SR, Kern SE, Kinzler KW, Vogelstein
B. (1996) Evaluation of candidate tumour suppressor genes on
chromosome 18 in colorectal cancers. Nat Genet, 13:343-6.

Thut CJ, Chen JL, Klemm R, Tjian R. (1995) p53 transcriptional activation
mediated by coactivators TAFII40 and TAFII60. Science, 267:100-4.

Tissier A, McDonald JP, Frank EG, Woodgate R. (2000a) pol iota, a remarkably
error-prone human DNA polymerase. Genes Dev., 14:1642-50.

Tissier A, Frank EG, McDonald JP, lwai S, Hanaoka F, Woodgate R. (2000b)
Misinsertion and bypass of thymine-thymine dimers by human DNA
polymerase iota. EMBO J., 19:5259-66.

Toribara NW, Sleisenger MH. (1995) Screening for colorectal cancer. N Engl J
Med., 332:861-7.

Torpey LE, Gibbs PE, Nelson J, Lawrence CW. (1994) Cloning and sequence of
REV7, a gene whose function is required for DNA damage-induced
mutagenesis in Saccharomyces cerevisiae. Yeast, 10:1503-9.

Torres-Ramos CA, Yoder BL, Burgers PM, Prakash S, Prakash L. (1996)
Requirement of proliferating cell nuclear antigen in RAD6-dependent
postreplicational DNA repair. Proc Natl Acad Sci U S A, 93:9676-81.

89

Torres-Ramos CA, Prakash S, Prakash L. (2002) Requirement of RAD5 and
MMSZ for postreplication repair of UV-damaged DNA in Saccharomyces
cerevisiae. Mol Cell Biol., 22:2419-26.

Trincao J, Johnson RE, Escalante CR, Prakash S, Prakash L, Aggarvval AK.
(2001) Structure of the catalytic core of S. cerevisiae DNA polymerase
eta: implications for translesion DNA synthesis. Mol Cell, 82417-26.

Truant R, Xiao H, Ingles CJ, Greenblatt J. (1993) Direct interaction between the
transcriptional activation domain of human p53 and the TATA box-binding
protein. J Biol Chem., 268:2284-7.

Ulrich HD, Jentsch S. (2000) Two RING finger proteins mediate cooperation
between ubiquitin-conjugating enzymes in DNA repair. EMBO J., 1923388-
97.

Unger T, Nau MM, Segal S, Minna JD. (1992) p53: a transdominant regulator of
transcription whose function is ablated by mutations occurring in human
cancer. EMBO J., 11:1383-90.

VanDemark AP, Hofmann RM, Tsui C, Pickart CM, Wolberger C. (2001)
Molecular insights into polyubiquitin chain assembly: crystal structure of
the Mms2/ch13 heterodimer. Cell, 105:711-20.

Vogelstein B, Fearon ER, Hamilton SR, Kern SE, Preisinger AC, Leppert M,
Nakamura Y, White R, Smits AM, Bos JL. (1988) Genetic alterations
during colorectal-tumor development. N Engl J Med, 319:525-32.

Vogelstein B, Fearon ER, Kern SE, Hamilton SR, Preisinger AC, Nakamura Y,
White R. (1989) Allelotype of colorectal carcinomas. Science, 2442207-11.

Wagner J, Gruz P, Kim SR, Yamada M, Matsui K, Fuchs RP, Nohmi T. (1999)
The dinB gene encodes a novel E. coli DNA polymerase, DNA pol IV,
involved in mutagenesis. Mol Cell, 42281-6.

Wang YC, Maher VM, McCormick JJ. (1991) Xeroderma pigmentosum variant
cells are less likely than normal cells to incorporate dAMP opposite
photoproducts during replication of UV-irradiated plasmids. Proc Natl
Acad Sci U S A, 88:7810-4.

Wang Y, Reed M, Wang P, Stenger JE, Mayr G, Anderson ME, Schwedes JF,
Tegtmeyer P. (1993) p53 domains: identification and characterization of
two autonomous DNA-binding regions. Genes Dev., 722575-86.

Wang XW, Yeh H, Schaeffer L, Roy R, Moncollin V, Egly JM, Wang Z, Freidberg
EC, Evans MK, Taffe BG, Bohr VA, Weeda G, Hoeijmakers JHJ, Forrester
K, Harris CC. (1995) p53 modulation of TFIIH-associated nucleotide
excision repair activity. Nat Genet, 102188-95.

90

Washington MT, Prakash L, Prakash S. (2001) Yeast DNA polymerase eta
utilizes an induced-fit mechanism of nucleotide incorporation. Cell,
107:917-27.

Watkins JF, Sung P, Prakash S, Prakash L. (1993) The extremely conserved
amino terminus of RAD6 ubiquitin-conjugating enzyme is essential for
amino-end rule-dependent protein degradation. Genes Dev., 72250-61.

Weinberg RA. (19950 The retinoblastoma protein and cell cycle control. Cell,
812323-30.

Willers H, McCarthy EE, Wu B, Wunsch H, Tang W, Taghian DG, Xia F, Powell
SN. (2000) Dissociation of p53-mediated suppression of homologous

recombination from G1/S cell cycle checkpoint control. Oncogene, 19:632-
9.

Wilson, D.M., Yang, D.J., Dillberger, J.E., Dietrich, S.E., Maher, V.M.,
McCormick, J.J. (1990) Malignant transformation of human fibroblasts by
a transfected N-ras oncogene. Cancer Res., 50:5587-5593.

Wittschieben J, Shivji MK, Lalani E, Jacobs MA, Marini F, Gearhart PJ, Rosewell
l, Stamp G, Wood RD. (2000) Disruption of the developmentally regulated
Rev3l gene causes embryonic lethality. Curr Biol., 10:1217-20.

Woffendin C, Chen ZY, Staskus K, Retzel EF, Plagemann PG. (1991)
Mammalian mRNAs encoding protein closely related to ubiquitin-
conjugating enzyme encoded by yeast DNA repair gene RAD6. Biochim
Biophys Acta, 1090:81-5.

Woo RA, McLure KG, Lees-Miller SP, Rancourt DE, Lee PW. (1998) DNA-
dependent protein kinase acts upstream of p53 in response to DNA
damage. Nature, 394:700-4.

Woodgate R. (2001) Evolution of the tvvo-step model for UV-mutagenesis. Mutat
Res., 485:83-92.

Xiao W, Lechler T, Chow BL, Fontanie T, Agustus M, Carter KC, Wei YF.
(1998a) Identification, chromosomal mapping and tissue-specific
expression of hREV3 encoding a putative human DNA polymerase zeta.
Carcinogenesis, 19:945-9.

Xiao W, Lin SL, Broomfield S, Chow BL, Wei YF. (1998b) The products of the
yeast MMSZ and two human homologs (hMMSZ and CROC-1) define a

structurally and functionally conserved ch-like protein family. Nucleic
Acids Res., 26:3908-14.

91

Xiao W, Chow BL, Broomfield S, Hanna M. (2000) The Saccharomyces
cerevisiae RAD6 group is composed of an error-prone and two error-free
postreplication repair pathways. Genetics. 155:1633-41.

Xin H, Lin W, Sumanasekera W, Zhang Y, Wu X, Wang Z. (2000) The human
RAD18 gene product interacts with HHR6A and HHR6B. Nucleic Acids
Res., 28:2847-54.

Xiong Y, Hannon GJ, Zhang H, Casso D, Kobayashi R, Beach D. (1993) p21 is a
universal inhibitor of cyclin kinases. Nature, 366:701-4.

Yang D, Louden C, Reinhold DS, Kohler SK, Maher VM, McCormick JJ. (1992)
Malignant transformation of human fibroblast cell strain MSU-1.1 by (i)-7
beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo
[a]pyrene. Proc Natl Acad Sci U S A, 89:2237-41.

Yang, D, Kohler, SK, Maher, VM, McCormick, JJ (1994) v-sis oncogene-induced
transformation of human fibroblasts into cells capable of forming benign
tumors. Carcinogenesis, 15:2167-2175.

Yin Y, Tainsky MA, Bischoff FZ, Strong LC, Wahl GM. (1992) Wild-type p53
restores cell cycle control and inhibits gene amplification in cells with
mutant p53 alleles. Cell, 702937-48.

Yu SL, Johnson RE, Prakash S, Prakash L. (2001) Requirement of DNA
polymerase eta for error-free bypass of UV-induced CC and TC
photoproducts. Mol Cell Biol., 21:185-8.

Zan H, Komori A, Li Z, Cerutti A, Schaffer A, Flajnik MF, Diaz M, Casali P. (2001)
The translesion DNA polymerase zeta plays a major role in lg and bcl-6
somatic hypermutation. Immunity, 142643-53.

Zhan Q, Bae I, Kastan MB, Fornace AJ Jr. (1994) The p53-dependent gamma-
ray response of GADD45. Cancer Res., 54:2755-60.

Zhang H, Xiong Y, Beach D. (1993) Proliferating cell nuclear antigen and p21 are
components of multiple cell cycle kinase complexes. Mol Biol Cell, 4:897-
906.

Zhang Y, Yuan F, Wu X, Wang M, Rechkoblit O, Taylor JS, Geacintov NE, Wang
Z. (2000a) Error-free and error-prone lesion bypass by human DNA
polymerase kappa in vitro. Nucleic Acids Res., 28:4138-46.

Zhang Y, Yuan F, Xin H, Wu X, Rajpal DK, Yang D, Wang Z. (2000b) Human
DNA polymerase kappa synthesizes DNA with extraordinarily Iow fidelity.
Nucleic Acids Res., 28:4147-56.

92

Zhang L, Yu D, Hu M, Xiong S, Lang A, Ellis LM, Pollock RE. (2000c) Wild-type
p53 suppresses angiogenesis in human leiomyosarcoma and synovial
sarcoma by transcriptional suppression of vascular endothelial growth
factor expression. Cancer Res., 60:3655-61.

Zhang Y, Yuan F, Wu X, Taylor JS, Wang Z. (2001) Response of human DNA
polymerase iota to DNA lesions. Nucleic Acids Res., 292928-35.

Zhang Y, Wu X, Rechkoblit O, Geacintov NE, Taylor JS, Wang Z. (2002)
Response of human REV1 to different DNA damage: preferential dCMP
insertion opposite the lesion. Nucleic Acids Res., 30:1630-8.

Zhou J, Ahn J, Wilson SH, Prives C. (2001) A role for p53 in base excision repair.
EMBO J., 202914-23.

93

CHAPTER 2

Re-expression of Wild-type p53 in a Focus-derived Human Fibroblast Strain
that Can Produce Tumors in Athymic Mice Prevents Focus Formation,
Anchorage Independence, and Tumorigenicity, but Does Not Affect Growth

in Culture1

Xi-De Wang, Terrence P. McManus, J. Justin McCormick,

and Veronica M. Maher2

Carcinogenesis Laboratory, Department of Microbiology & Molecular Genetics
and Department of Biochemistry & Molecular Biology. Michigan State University,

Food Safety and Toxicology Building, East Lansing, MI 48824-1302

Running title: INHIBITION OF HUMAN CELL TRANSFORMATION BY p53

Key words: p53, Focus formation, Anchorage-independent growth,

Tumorigenicity, Cell growth

1 Supported by United States Department of Health and Human Services Grants
CA21253 and CA60907 from the National Cancer Institute.
2 To whom requests for reprints should be addressed at: Carcinogenesis

Laboratory, Food Safety and Toxicology Building, Michigan State University,

94

East Lansing, Michigan 48824-1302. Fax: (517)353-9004; E-mail:
maher@msu.edu.

3 The abbreviations used are: FBS, fetal bovine serum; MNU, N-methyI-N-
nitrosourea; PBS, phosphate-buffered saline; SDS-PAGE, sodium dodecyl
sulfate-polyacrylamide gel electrophoresis; TBST, Tris-buffered saline
containing Tween-20; TD, tumor-derived; tTA, tetracycline-controlled

transactivator; TRE, tetracycline-responsive element; UV, ultraviolet radiation.

Abstract

More than half of human tumors lack functional p53. When human
fibroblast cell strain MSU-1.1 was treated with a single dose of carcinogen and
assayed for focus formation, a significant fraction of the focus-derived cell strains
were found to have lost functional p53. When cells from the foci were propagated
and injected into athymic mice, only those lacking p53 function formed tumors.
To determine whether loss of p53 plays a causal role in allowing cells to form
foci, and whether restoration of p53 to these cells would prevent them from
forming tumors, we transfected a wild-type p53 gene into MSU-1.1-y1-2A1, a
focus-derived cell strain that does not contain p53 DNA and produces malignant
tumors in athymic mice (Reinhold et al., Radiat. Res., 692707, 1996). Two
independent transfectants that expressed wild-type p53 protein at a normal level,
i.e., comparable to that of the original, non-transformed parental strain MSU-1.1,
were identified. One of the cell strains replicated at a rate comparable to that of
cell strain MSU-1.1, and the other at a rate 50% lower. However, their frequency

of focus formation and the quality of the foci were very significantly reduced,

95

 

compared to those of the focus-derived strain from which they were derived. The
ability of the two wild-type p53 transfectant cell strains to form colonies in semi-
solid medium (anchorage—independence) or tumors in mice was very significantly
decreased, and the cells derived from the few tumors that did form no longer
expressed p53 or expressed it at very low level. The data indicate that
expression of exogenous wild-type p53 in carcinogen-transfonned, focus-
derived, malignant human fibroblasts can significantly inhibit focus formation,
anchorage-independence, and tumor formation. Expression of functional p53 at a
normal level in these focus-derived cell strains either directly prevented them
from forming tumors, or prevented them from acquiring the genetic change(s)

needed to become tumor-forrning cells.

Introduction

More than half of the human tumors tested have lost functional p53, a
tumor suppressor gene (1) and Li-Fraumeni patients, who inherit one mutant
allele of the p53 gene, are predisposed to soft tissue sarcomas and various other
kinds of cancer (2). In response to DNA damage, p53 protein triggers growth
arrest of cells in the G1 phase of the cell cycle by transactivating p21 (3), a
universal inhibitor of cell-cycle-dependent kinases (4). This arrest allows cells in
G1 additional time to carry out DNA repair before the onset of DNA synthesis. In
badly damaged cells, p53 triggers apoptosis by transactivating Bax, an apoptotic
factor (5). When expression of wild-type p53 is lost, cells are much more likely to
acquire mutations (6), including those involved in malignant transformation.

Reintroduction of wild-type p53 into human tumor-derived cell lines has been

96

shown to cause growth arrest or apoptosis, depending in part on the level of
expression of the transgene (7,8). It can also trigger senescence (9).

To investigate the mechanisms of malignant transformation of human
fibroblasts, McCormick and colleagues (10) developed an infinite life span, near-
diploid, chromosomalIy-stable, nontumorigenic human fibroblast cell strain, MSU-
1.1, from the foreskin of a normal neonate. MSU-1.1 cells can be malignantly
transformed into cells capable of forming tumors in athymic mice by transfection
of an overexpressed activated (mutated) H-ras (11), N-ras (12) or K-ras (13)
oncogene, or by a single exposure to y-radiation (14,15) or a chemical
carcinogen (16,17). Cells from tumors produced by cells overexpressing a
transfected ras oncogene did not exhibit any change in karyotype. The
carcinogen-treated cells were allowed a 7 to 8 day expression period following
exposure to carcinogen before being assayed for ability to form foci. Analysis of
independent focus-derived cell strains for the transactivation function of p53
revealed that 40 to 60% had lost all p53 transactivation function. When clonally
expanded and assayed for the ability to form tumors, a significant fraction (45%
to 70%) of the focus-derived cell strains formed fibrosarcomas after a short
latency (15-17). All of these focus-derived cell strains had lost the transactivating
function of p53. These results strongly suggested that loss of p53 function allows
focus formation by these MSU-1.1 cells and that loss of p53, although not
sufficient in itself to cause such focus-forming cells to form tumors in the athymic
mice, nevertheless plays an essential role in the cells acquiring the additional

changes needed to progress further.

97

 

 

To test the hypothesis that loss of p53 is sufficient to enable human
fibroblasts to form foci and significantly increases their chance of becoming
malignant, we transfected a wild-type copy of p53 into cell strain MSU-1.1y1-2A1,
which was derived from a focus induced by irradiating parental MSU-1.1 cells
with 4.35Gy 5°Co (14). The focus-derived MSU-1.1y1-2A1 cells were shown to
have lost one copy of chromosome 16 and chromosome17. lmmunoprecipitation
studies showed that they do not express any p53 protein. When the cells were
propagated and injected into athymic mice, they formed tumors with a diameter
of 1 cm within 3-7 weeks (14). Therefore, these focus-derived MSU-1.1y1-2A1
cells were used as the target cells to determine whether restoration of wild-type
p53 expression at a normal level could significantly inhibit focus formation,
anchorage-independence, and/or the ability to form malignant tumors. The
results obtained from two independent p53 transfectant strains exhibiting a
normal expression level of p53 protein and retaining a normal growth rate in
culture indicate that loss of p53 plays a causal role in allowing cells to form foci,
to form large-colonies in semi-solid medium (anchorage-independence), and to

form tumors in athymic mice.

Materials and methods
Cell strain and cell culture

The target cell strain used for transfection of wild-type p53 was MSU-
1.1y1-2A1, derived from a focus formed by the original infinite life span parental

human fibroblast cell strain designated MSU-1.1 (10) that had been irradiated

with a single dose of cobalt 60 at 4.35 Gy and assayed for focus formation (14).

98

The focus-derived cell strain was shown in the original study (14) to be capable
of forming tumors in athymic mice. Unless othenivise noted, cells were cultured in
Eagle's minimal medium (Gibco, Gaithersburg, MD), pH 7.2, supplemented with
0.2 mM L-aspartic acid, 0.2 mM L-serine, 1.0 mM sodium pyruvate, and 10%
(v/v) supplemented calf serum (HyCIone, Logan UT). The medium also contained
penicillin (100 Ulml), streptomycin (100 pglml), and hydrocortisone (1 pig/ml).

Cells were cultured in 5% CO; humidified incubators at 37°C.

Plasmid construction

Plasmid pBPSTR1-WTp53.19 was constructed by excising the 1.8 kb
WTp53 DNA fragment from pc53-SN (provided by Dr. B. Vogelstein) by BamH l
digestion and ligating it into pBPSTR1 that had been linearized at the multiple
cloning site by BamH l digestion. pBPSTR1 contains a tetracycline-responsive
promoter upstream of the multiple cloning site, as well as a tTA expression
cassette for enhancement of tetracycline responsiveness, and the gene coding

for resistance to puromycin as the selectable marker.

Transfection

Cells plated at 105 per 60 mm-diameter tissue culture dishes were
transfected ~18 h later with plasmid DNA linearized at the unique Sca I site.
Plasmid DNA, 1.5 pg per dish, was introduced in lipofectamine according to the
manufacturer's instructions (Gibco, Bethesda). Tetracycline (2.0 pg/ml) was
included in the transfection solution. After 48 h, the medium was changed to

medium containing puromycin (1.0 jig/ml) to select for transfectants. Tetracycline

99

(2.0 pg/ml) was added every 2-3 days. The cells were given fresh medium

containing puromycin (1.0 jig/ml) every 5 days.

Growth curves

Cells growing logarithmically were dislodged with trypsin and plated at a
density of 1x 105 cells per 100 mm-diameter dish on day 0. On day 1, 2, 3, 4, 5, 6
and 7, the cells in three dishes were dislodged with trypsin, suspended in isoton
solution and counted by using an electronic counter (Coulter, Hialeah, FL) to

determine the total number of cells per dish.

UV3-irradiation
Cells in exponential growth in 100 mm-diameter dishes at 40-50%
confluence were aspirated, washed with PBS, aspirated again and irradiated with

5 J/m2 UV (254nm) at an incidence dose of ~0.3 Jlmzlsec. Cells were given fresh

medium immediately after irradiation.

Preparation of cell lysates

To assay expression of p53 protein, the protocol described previously by
Qing et al. (18) was used. Briefly, exponentially growing cells were washed with
cold PBS and lysed on ice with lysis buffer composed of 50 mM Tris-HCI, pH7.2,
150 mM NaCl, 1% Triton X-100, 0.1% SDS, 0.5% sodium deoxycholate, 2 mM
phenylmethylsulfonyl fluoride (PMSF), 1 mM EDTA, 0.1 mM sodium vanadate, 2
mg/ml aprotinin. The cell lysates were collected with a rubber scraper and
centrifuged at 14,000 rpm on a microcentrifuge (Eppenforf, Westbury, NY) at

4°C. The supernatant was collected, an aliquot was taken for protein

100

quantification, and the rest was stored at -80°C. The protein concentration of the
lysates was determined using the Coomassie protein assay reagent (Pierce,
Rockford, IL). To assay expression of p21, cells were dislodged with trypsin,
counted with a Coulter counter, centrifuged at 2000 rpm on a microcentrifuge for
10 min, resuspended in PBS and centrifuged again. To lyse the cells, the cells
pellets were lysed with lysis buffer containing 2% SDS, 10% glycerol, 5% 2-

mercaptoethanol, 0.05% bromophenol blue and 62.5 mM Tris, pH 7.2. For 1x 106

cells, 40 pl of lysis buffer was used. The lysates were heated at 95°C for 15

minutes, allowed to cool on ice, briefly centrifuged to collect condensation, and

stored at -80 00.

Western blot analysis

Cell lysates were loaded on a 10% SDS-PAGE gel (40 pg of protein for
p53 and 10 pl Iysate for p21), and the gel was run at constant 50 mA. The
proteins were electrotransfered onto an Immobilon-P membrane (Millipore,
Bedford, MA) for ~18 h at constant 50 mA. The membranes was blocked at 4°C
overnight in 5% (w/v) non-fat dry milk in Tris-buffered saline (20 mM Tris-HCI, pH
7.2, 137 mM NaCl) containing 0.1% (v/v) Tween-20 (TBST), and incubated for 2
h at room temperature with the appropriate mouse monoclonal antibodies, i.e.,
anti-p53 antibody (Clone Pab1801) or anti-p21 antibody (sc-817) (Santa Cruz
Biotechnology, Santa Cruz, CA) at a concentration of 0.5-1.0 pglml. The
membranes were washed and incubated with horseradish peroxidase-conjugated
goat anti-mouse lgG (Sigma, St. Louis, MO) that had been diluted 1220,000 in

TBST solution supplemented with 5% milk. Enhanced chemiluminescence

101

(SuperSignal HRP substrate, Pierce, Rockford, IL) was used to detect the protein
bands, and a Molecular Phosphor Imager system (BioRad, Hercules, CA) was
used to quantitate them. The actin level, assessed with goat polyclonal anti-actin
antibody (sc-1616) (Santa Cruz Biotechnology, Santa Cruz, CA), was used to

normalize the levels of p53 and p21 proteins.

Focus reconstruction assay

Cells in exponential growth, together with 2x105 feeder cells, were
suspended in Eagle's medium supplemented with 20 mM HEPES (Sigma, St.
Louis, MO) and 0.5% FBS (Summit Biotechnology, Fort Collins, CO) and plated
into 100 mm-diameter dishes that had been plated with 5x 10‘ normal, non-
transforrned MSU-1.1 cells in the same medium ~16h earlier. Feeder cells used
were MSU-1.1 cells that had been treated with 30 Gy y-radiation. They could not
divide, but could increase the colony forming ability of the live cells in the first
several days after plating. Sixteen dishes were plated for each cell strain to be
tested. The cells were incubated at 37 °C in the same 5% CO; humidified
incubator, and the medium was replaced every 4 days. At that time, the dishes
were also scanned for foci using a focused beam of light from beneath the
dishes. Some dishes were stained to verify the growth of foci as needed.
Representative foci were photographed before and after fixation with methanol

and staining with methylene blue.

Assay of anchorage-independent growth

102

Cells were assayed for the ability to form colonies in semi-solid medium,
i.e., 0.33% agarose (Seaplaque, FMC, Rockford, ME). 5000 exponentially
growing cells in 1.5 ml of McM medium (19) supplemented with 20 mM HEPES
(Sigma, St. Louis, MO), 0.33% (w/v) agarose and 2% (vlv) FBS (Summit
Biotechnology, Fort Collins, CO) were plated on top of a solidified bottom
agarose layer made up of McM medium supplemented with 2% (w/v) agarose
and 2% (v/v) F38. The top agar was allowed to solidify, and then covered with
3.5 ml of McM medium containing 2% FBS, 20 mM HEPES, penicillin (100 Ulml)
and streptomycin (100 pglml). The cells were incubated at 37 °C in a 3% C02
humidified incubator, and the 3.5 ml of medium was replaced every week for 3
wk. The parental MSU-1.1 cell strain, which does not form colonies in soft agar
and a derivative H-ras-transforrned tumorigenic cell line, MSU-1.1 A210, which
forms large-sized colonies in soft agar, were used as negative and positive
controls, respectively. After 3 wk, the medium overlaying the agar was removed
and glutaraldehyde (2.5%) was added to fix the colonies in the top agar layer.
The dishes were stored at 4 °C until analyzed. The size of colonies was
measured under microscope using a calibrated ruler placed in the eyepiece of

the microscope. Representative fields of colonies were photographed.

Tumorigenicity test
Athymic BALB/c mice of 4-5 weeks of age were injected subcutaneously
in the left front and right hind flank regions with 1x 106 cells suspended in 0.2 ml

of serum-free Eagle's medium. The mice were monitored weekly for tumor

growth, and the size of tumors was measured using a caliper. When a tumor

103

reached 1 cm in diameter, the mice were sacrificed, and the tumor was removed.
Cells from a portion of the tumor were returned to culture, and the rest of the
tumor was fixed with formalin for histological examination. Because MSU-1.1
cells express a transfected neomycin resistance gene (10), making them
resistant to G418, tumor-derived cells were propagated in medium containing
G418 (Gibco, Bethesda, MD) to eliminate any possible contaminating mouse

cells. All mice were sacrificed four months after injection.

Results
Determining whether functional wild-type p53 could be successfully re-

introduced and expressed at a normal level into cell strain MSU-1.1y1-2A1

Exogenous expression of wild-type p53 in cancer cell lines has been
shown to induce growth arrest or apoptosis (7,8), or to promote cellular
senescence (9,20). Such effects indicated that it might be very difficult to obtain
cells that expressed a transfected wild-type p53 gene, but were still capable of
growing normally in cell culture. ln an attempt to control expression of the
transfected wild-type p53 in order to avoid these deleterious effects, we used a
construct that was designed to be tetracycline-regulatable and transfected it into
the focus-derived cell strain MSU-1.1y1-2A1. For the sake of brevity, this strain
will be referred to as 2A1 from now on. The constructed vector, pBPSTR1-
WTp53.19, is designed so that when tetracycline is absent, the tTA binds to the
TRE and activates the expression of the transgene p53. In the presence of
tetracycline, tTA should be unable to bind the TRE, and therefore the expression

of the transgene is shut down. Focus-derived cell strain 2A1 was transfected with

104

the plasmid in medium containing tetracycline, and puromycin-resistant
transfectants were selected in the presence of tetracycline. A total of 55
puromycin-resistant clones were expanded. Lysates for Western blot analysis
were prepared from cells cultured for 7 days in the absence of tetracycline. Of
the 55 cell strains assayed, two, designated MSU-1.1-2A1-88A and MSU-1.1-
2A1-85, were found to express p53 protein. For the sake of brevity, these two
strains will be referred to from now on as cell strains 88A and 85. As shown in
Figure 1, the recipient cell strain 2A1 does not express p53; transfected cell
strain 88A expressed p53 protein at a level comparable to that found in the
parental MSU-1.1 cell strain, and the level of p53 in transfected cell strain 85 was
~2-fold higher than that seen in parental MSU-1.1 cells. Therefore, cell strains
88A and 85 were suitable for determining the role of loss of p53 in carcinogen-
induced human cell transformation. Additional studies with strain 88A revealed
that p53 expression in these cells could not be modulated by tetracycline (data
not shown). Therefore it was not possible to examine the effects of p53
expression on transformation using the same strain cultured in the presence or
absence of tetracycline. Lack of modulation of gene expression using this

expression system has also been reported elsewhere (21).

Evidence that the level of expression of exogenous wild-type p53 did not

greatly affect the rate of growth of the two cell strains

As noted above, expression of exogenous p53 in human cells has been
reported to induce growth arrest (7-9). Therefore, we compared the rate of

growth of strains 88A and 85 with that of their parental cell strain, 2A1, which

105

.«T‘
99° 9.?5 '66 69"“

p53 - - -

 

A

P.-
;.
F.
t

--r-- ' , w. --,.‘ a" _ TVs-i

:l

d

a n a
- 1’

'1'

_. _. u. - m - ._ m «A

Figure 1. Western blot analysis of cell strains 88A and 85 for the expression of
p53 protein. Cell strains MSU-1.1 and 2A1 were used as a positive and a
negative control respectively. For each strain, 100 pg of lysate was loaded. The

p53 protein was probed with monoclonal anti-p53 antibody (Pab1801). Actin was

used as a loading control.

106

does not express p53. As shown in Figure 2, the population doubling time of
strain 88A was 24 h, which is very similar to that of the non-transfected strain
2A1, i.e., 22 h, and the population doubling time for strain 85, which expresses
p53 at a slightly higher level, i.e., 35 h. These results, indicating that the level of
p53 expression in these two strains in the absence of tetracycline did not have a
significantly deleterious effect on their rate of cell growth, allowed us to use them

to examine the effects of p53 expression on other properties of the cells.

Evidence that the exogenous p53 in the transfected strains was functional

To determine whether the transfected wild-type p53 in strains 88A and 85
was functional, we tested using Western blotting whether UV radiation increased
the level of expression of p53 in the cells and also that of p21 protein, the product
of a gene for which p53 is a transcription factor (22). Such a test of the
functionality or activation of p53 has been commonly used (23,24). Parental
strain MSU-1.1, which expresses wild-type p53, was used as a positive control,
and focus-derived cell strain 2A1 was used as a negative control. As shown in
Figure 3, the relative basal level of p21 protein in each of the four strains
corresponded to the basal level of p53 protein in the same cell strains. For
example, 2A1 cells did not express either p53 or p21; strain 88A had a lower
basal level of p53 and of p21 than parental cell strain MSU-1.1; and strain 85 had
a higher basal level of p53 and of p21 than strain MSU-1.1. In response to UV,
strain 2A1, which lacks expression of p53, showed no upregulation of expression

of p21, whereas strain 88A, as well as MSU-1.1 cells, exhibited increased

107

Figure 2. Expression of exogenous p53 in cell strains 88A and 85 does not
significantly affect the rate of cell growth. Cell strains 88A (0), 85 (I), and cell
strain 2A1 used as the recipient for transfection of p53 (A) were plated at 1x 105
per 100 mm-diameter plates on day 0. On each of the following seven days, cells
on 3 p100's of each strain were dislodged with trypsin, combined, and counted
with an electronic counter. The average number of cells on each dish for each
strain was calculated. The data point of 2A1 (A) on day 1 has been offset slightly
for clarity. The population doubling time of strains 2A1, 88A, and 85 were ~22h,

~24h, and ~35h, respectively.

108

 

 

 

 

 

 

E: x. in .8 e__8 .o .8532

Day

109

MSU-1 .1 88A 85 2A1

 

 

0924092409240924h

 

 

 

 

 

anwm amen? 'P‘fl? .W-‘V hi'l‘ Jim-3"... '11“

~ ° (I
“1

p21

 

 

 

Figure 3. Expression of p53 and p21 proteins in cell strains 88A and 85 in
response to UV irradiation. Cell lysates were prepared from exponentially
growing cells prior to UV treatment (labeled as 0) or 9 or 24 hours (labeled as 9
or 24) after UV treatment at 5Jlm2. Western blot analysis was performed using
anti-p53 (Pab1801) or anti-p21 (sc-817) mouse monoclonal antibodies. Cell
strain MSU-1.1, which contains wild-type p53, and cell strain 2A1, which lacks
p53 expression, were included as a positive control and a negative control,

respectively. Actin was the loading control.

110

expression of both p53 and p21. The level of both p53 and p21 protein in strain
85 did not change significantly as a result of UV treatment. These data, showing
the p53-dependent expression of p21 protein in cell strains 88A and 85, and the
lack of expression of p53 and p21 in the non- transfected strain 2A1, strongly

suggest that the function of p53 had been restored in strains 88A and 85.

Effect of expression of exogenous p53 on focus formation by the focus-

derived cell strain 2A1

Focus reconstruction assays are used to evaluate the ability of cells to
proliferate, form a multilayer, and pile up on top of confluent monolayer of lawn
cells. The 2A1 cell strain was derived from a focus formed by the parental strain
MSU-1.1 after y-irradiation (14). To test whether p53 expression in the p53
transfectant cell strains 88A and 85 inhibited focus formation, focus
reconstruction assays were carried out, plating the same number of clonable
cells on the lawn of cells in each dish for all strains tested. Pilot studies showed
that plating 80 cells of strains 88A and 2A1 and 500 cells of strain 85 per 100
mm-diameter tissue culture dish, in presence of 2x105 feeder cells per dish gave
a plating efficiency of 60-70 clones on each dish. To minimize possible variation
in focus formation by cells to be tested that might reflect the status of the lawn
cells used, we assayed the various cell strains plated onto two different
populations of MSU-1.1 cells, i.e., SB62-7 cells, a non-cloned population, and
8362-79 cells, a clonal population derived from it. The former cells grow a little
faster than the latter, but otherwise the lawn cells grow in an identical manner.

After 2 to 3 wk, foci formed by the parental focus-derived 2A1 cells on top of the

111

monolayer of lawn cells could easily be seen using a focussed beam of light. In
contrast, with p53-expressing cell strains 88A and 85, foci could only be
differentiated from the background lawn cells after 4 wk. Strain 2A1 formed
distinct foci, while strains 88A and 85 formed only weak foci. Compared to the
foci formed by 2A1 cells, those formed by cell strains 88A and 85 contained
fewer crossing layers in a much more limited area (see Figure 4). As shown in
Table 1, which presents the numbers of foci formed by 88A, 85 and 2A1 cells on
the two separate lawns, the frequency of focus formation of strain 88A was five
times lower than that of the parental 2A1 cells, and that of strain 85 was ten
times lower. The reduction in both the frequency of focus formation and the
quality of foci in two independent strains, 88A and 85, clearly indicates that
expression of wild-type p53 at a normal level inhibits focus formation by these

cells.

Expression of p53 also reduced the anchorage-independence

To obtain additional evidence of the inhibiting effect of p53 on human cell
transformation, we tested the anchorage independence of cell strains 2A1, and
its two derivative strains, 88A and 85, i.e., their ability to form colonies in semi-
solid agarose. Anchorage-independence, another important characteristic of
malignantly-transformed cells, has been widely used to determine whether a
particular cell strain has been transformed into malignant cells (15,25). For this
assay, MSU-1.1, the infinite life span, non-tumorigenic parental strain from which
the 2A1 cells arose, and cell strain A210, an H-ras-transformed, tumorigenic

derivative of MSU-1.1 cells, were included as the negative and positive controls,

112

 

Figure 4. A representative set of foci formed by cell strains 2A1, 88A and 85.
Eighty 2A1 and 88A cells or 500 85 cells, together with 2x105 feeder cells, were
plated into p100 tissue culture dishes that had been plated with 50,000 normal
cells ~16h earlier: Cells were refed and checked every 4 days. Dishes were

stained with methylene blue.

113

Table 1. Frequency of focus formation by cell strains 88A, 85, and the parent

 

 

 

strain 2A1
Cell Average Number of distinct foci Percentage of cells able
strain number of per dishb to form focib
clonable
lawn 1° lawn 2° lawn 1° lawn 2°
cells plated
per dish"
2A1 67 3114 321:6 461:6 47:1:10
88A 59 7:2 612 1214 1014
85 67 3 :t 1 2 :l: 1 4 :I: 1 3 :t 2

 

a Determined from assaying colony forming ability of cells plated with y-irradiated

(30Gy) cells plated at 4000 cells/cmz.

b Data presented as mean :t standard deviation. Six dishes were counted for

each cell strain.

° Lawn 1: 8362-7; lawn 2: 8862-79.

114

respectively. As shown in Figure 5, 2A1 cells formed large-sized colonies, with
size distribution similar to that of the positive control cell strain A210, whereas
88A and 85 cells, with the function of wild-type p53 restored, showed greatly
reduced ability to form colonies in soft agar. As shown in Table 2, the 2A1 cells
formed colonies with a diameter greater than 70pm at a frequency of >20%. In
contrast, only ~1% of 88A cells and ~0.5% of 85 cells formed colonies with a
diameter of 70pm or more. Unlike 2A1 cells, which could produce colonies with a
diameter of 2105pm with a frequency of ~10%, the 88A and 85 cells never did
so. The number and size of colonies formed in soft agar by 88A and 85 cells did
not differ from those formed by non-transformed parental MSU-1.1 cells. These
data indicate that induced expression of exogenous p53 in the focus-derived
strain 2A1 strongly suppressed its anchorage-independence. The decrease in
anchorage-independence of these p53-transfected cell strains correlated well

with the decrease in their focus forming ability.

Effect of expression of exogenous p53 on the tumorigenicity of cell strains

88A and 85

A significant fraction of the cell strains derived from foci induced by
treating MSU-1.1 cells with benzo(a)pyrene diol epoxide (16) or ionizing radiation
(15) can form tumors in athymic mice. This is true for cell strain 2A1 generated
by selection for focus formation. When the cells derived from the focus were
propagated and injected into athymic mice, they proved to be highly tumorigenic
(14). Because expression of p53 so greatly reduced the focus-forming ability of

cell strains 88A and 85, we determined whether p53 expression could also

115

 

Figure 5. Photos of representative field of colony formed by cell strains 88A, 85
and 2A1 in soft agar. MSU-1.1, the immortal and non-tumorigenic original
parental strain,. and cell strain A210, an H-ras-transforrned, tumorigenic
derivative of MSU-1.1 cells, were included as the negative and positive controls,

respectively. The photos were taken under microscope with 6x magnification.

116

Table 2. Size distribution of colonies formed by cell strains 88A, 85, the parent

strain 2A1, and two control strains

 

Cell strains Number of colonies of a given diameter per 5000 cells assayed:

 

 

35-70 pm 70-105 pm 2105 pm
A210 572 91 1 445
2A1 525 599 476
88A 21 1 57 0
85 144 20 O
MSU-1.1 23 0 0

 

117

reduce the tumorigenicity of the cells. Cell strains 88A and 85 were injected
subcutaneously into athymic mice. As a control, 2A1 cells were also injected into
mice at the same time. As expected, cell strain 2A1 proved to be highly
tumorigenic, forming tumors in all 18 sites of injection. In contrast, the incidence
of tumor formation by cell strains 88A and 85 was greatly reduced. Cell strain 85
did not form tumor at any of the 18 injection sites; strain 88A formed tumors at
only 6 out of 18 injection sites. The frequency of tumor formation by 88A cells
was consistent in three independent injection experiments, with 1-3 tumors out of
6 injection sites. What is more, the length of the time required for the 88A cells to
form tumors that reached 0.5 cm in diameter was ~6wk, i.e., 1 wk longer than
required for 2A1 cells. These data indicate that expression of exogenous wild-
type p53 protein eliminated or markedly reduced the tumorigenicity of the cells.
Histological examinations of tumor slides showed that all the tumors
formed by 2A1 cells and 88A cells were high grade round spindle cell sarcomas.
Cells from the largest tumors formed were propagated and designated 88A-
Tumor Derived (TD)1, -TD2, -TDB, -TD4 and 2A1-TD1, -TDZ, and analyzed for
the expression of p53 protein by Western blotting. As expected, the 2A1-TD1 and
2A1-TD2 did not express any p53 protein. What is more significant, the cells
derived from the tumors that had not been expected, i.e., 88A-TD1, 88A-TD2 and
88A—TD4 had lost expression of p53 protein, and those from strain 88A-TD3
showed a greatly reduced level of p53 protein. These data strongly suggest that
the tumors formed following injection of 88A cells that had been expressing a

transfected p53 gene resulted from the growth in the animals of cells that had

118

Table 3. Tumorigenicity of cell strains 88A, 85, and the parental strain 2A1

 

 

Cell strains Tumor incidencea Tumor latencyb p53 expression in
tumor-derived
(wk)
cells°
2A1 1 8118 ~ 5 no
88A 6/18 ~ 6 no or markedly
reduced
85 0/18 N/A N/A

 

a Number of sites with tumor per number of sites injected.

b The length of the time required for cells to form tumors of 0.5 cm in diameter.

° As determined by Western blotting shown in Figure 6.

119

«0 ‘0'» «9 «0% «db «0..
65? '3? “:41“? 15%? we. if” ”M '1, 8‘3,» %$?‘

‘.

V A

. l .

.’ I'll .
f.

- - - M— ---.. . ~f ‘.. .e. -. _. .22.: . .

 

Mm

p53 P,

Hm a. .m --- ”a: .numed‘a‘n Fr...— _.__.._._._ -..~__. . . -...‘_--.—_-

 

 

aetin m w “2%"

.

.
P i
._'._

£54.12... H--.4 -M..-___..._H_.x_4_fi1._x;e ---_--..-'_.a---.l.

 

Figure 6. Lack of or markedly reduced expression of p53 protein in cells derived
from tumors formed by 88A cells. Cells from four largest tumors formed by
injected 88A cells, designated as 88A-Tumor Derived (T D)1, -TD2, -TD3 and -
TD4, were propagated and evaluated for the expression of p53 protein by
Western blot analysis. Lysates of 88A and 85 cells, of 2A1 and 2A1-TD1, -TD2
cells, were used as positive controls and negative controls, respectively. p53
protein was detected using an anti-p53 monoclonal antibody (Pab1801), and

actin was used as the loading control.

120

lost expression of wild-type p53 or in which expression of p53 had been greatly
reduced. Because all the injections were made using a common population of
cells that had been propagated as a pool, such loss or reduction of expression of
the transfected p53 is not likely to have occurred before the cells were injected
into the athymic mice, but rather reflects selection for a growth advantage in the
mice of cells with reduced , levels of p53. Transgene (p53 cDNA)-specific
amplification by polymerase chain reaction in 88A-T01 cells and 88A-T02 cells
showed that these two tumor-derived strains had lost their transfected DNA (data
not shown). The absence or reduced frequency of tumor formation by cell strains
85 and 88A, and the lack of or low level of p53 expression in tumors that formed
following injection of cell strain 88A, strongly suggest that p53 suppresses tumor
formation and that loss of p53 expression plays a causal role in human cells

becoming malignant.

Discussion

In the studies of the malignant transformation of MSU-1.1 by y-irradiation
(15) or by MNU (17), a significant fraction (>40%) of the focus-derived cells were
found to have lost the function of p53. Of the 13 independent focus-derived
strains assayed that had been induced by a single exposure of MSU-1.1 cells to
60Co irradiation, 11 showed loss of p53 transactivation activity. Assays of 40
independent focus-derived cell strains induced by MNU showed that 15 (~40%)
lacked functional p53. These data suggest that p53 plays an important role
relative to focus formation. In these transformation studies referred to above, it

was also found that a high percentage (62% and 29%, respectively). but not all,

121

of the focus-derived cell strains assayed that had lost p53 function were capable
of forming tumors in athymic mice. However, all the focus-derived strains that did
form tumors lacked p53. This led to the hypothesis that loss of p53 enables
human fibroblast MSU-1.1 cells to form distinct foci on a monolayer of normal
cells, and that, although loss of p53 is not sufficient to convert the focus forming
cells into malignant cells, it enables or predisposes the fibroblasts to acquire an
additional change(s) necessary for them to become malignant, i.e., able to form
malignant tumors in athymic mice. If so, restoration of wild-type p53 expression
in a focus-derived cell strain should inhibit focus formation and tumor formation
by the cells. It was to test these hypotheses that we transfected wild-type p53
cDNA into a focus-derived cell strain, 2A1, that had been shown to lack p53. Our
study with cell strains 88A and 85 show that restoration of wild-type p53 inhibited
the focus formation, anchorage-independence and tumorigenicity of these cell
strains.

p53 suppresses tumor by a number of mechanisms, including growth
arrest (7), apoptosis (8) and senescence (9). These mechanisms were
discovered when wild-type p53 protein was overexpressed in cancer cell lines,
and a common feature of these mechanisms is the inability of the p53
transfectants to replicate. In our study, out of the 55 strains tested, we identified
two independent strains that express p53 protein, and these two strains express
p53 at a level comparable to the nontumorigenic parental strain MSU-1.1. Our
failure to identify strains that overexpress the exogenous protein probably reflects

the deleterious effect on cell growth of overexpression of exogenous wild-type

122

p53 protein. The two strains, 88A and 85, expressing a normal level of p53, grew
normally. Strain 88A replicated as fast as the parental strain 2A1 in culture; strain
85 had 50% longer doubling time. The fact that the growth rate of cell strains 88A
and 85 was virtually normal facilitated our study of their transformed phenotypes.
The effect of overexpression of wild-type p53 protein on cell growth appears to
vary with cancer cell type. For example, the human glioma cell line U87MG has
wild-type p53 function. The growth of this cell line, as measured by clonogenic
survival, is only reduced by 25% even when 2025 times more exogenous wild-
type p53 protein is expressed (26). In an independent study, the growth of this
same cell line did not change when exogenous p53 protein was introduced (27).
Our data showing that expression of wild-type p53 inhibited focus
formation strongly suggest that loss of p53 function results in focus formation.
This result agrees with a previous study reported by Maclean et al. (28) that
inactivation of the wild-type p53 by SV40 large T antigen causes focus formation
by fibroblasts derived from a patient with Li-Fraumeni syndrome. Wild-type p53
also inhibits focus formation induced by transfected oncogenes. In the study of
transformation of primary rat embryo fibroblasts, Eliyahu et al. (29) found that
wild-type p53, but not mutant p53, greatly reduces the number of foci induced by
combinations of bona fide oncogenes, such as myc plus ras or adenovirus E 1A
plus ras. p53 also suppresses focus formation induced in primary rodent
epithelial cells by EM and E18 oncogenes. Interruption of p53 function by
adenovirus E4orf6 protein promotes the transformation of these cells by E 1A and

E 1B oncogenes (30). Studies carried out in this laboratory, including the present

123

study and those by O'Reilly et al. (15) and Boley et al. (17), identify the inhibitory
role of p53 on focus formation of human fibroblasts induced by various
carcinogens. This information provides insight into the tumor suppressor function
of p53 from a new perspective. It should be noted that factors other than loss of
p53 also induce focus formation. For example, MSU-1.1 cells possessing wild-
type p53 genes can still be induced to form foci by transfection of activated
(mutated) oncogenes including ras (11-13). It is important to note that in
transformation studies of MSU-1.1 cells induced by y—radiation (15) or by MNU
(17), focus-derived cell strains that retained their wild-type p53 function were also
identified. Furthermore, a substantial percentage of human cancers have been
determined not to have a mutation in the p53 gene (11).

Both of the cell strains expressing transfected wild-type p53 showed
reduced anchorage-independent growth potential and the ability to form tumors
in athymic mice. This is not surprising because the ability of cells to grow in
semisolid medium has been closely correlated with tumorigenicity in animal
models (31). The correlation between anchorage-independence or tumorigenicity
and loss of wild-type p53 function has been reported as well. For example, as an
animal model for human urothelial carcinomas, an immortalized nontumorigenic
rat urothelial cell line, MYP3, expressing wild-type p53, was used for studying the
effect of loss of p53 function on induction of malignant phenotypes. The
investigators found that expression of p53 antisense RNA resulted in a significant
reduction in p53 protein level, a stimulation of anchorage-independent growth,

and an acquired ability to form malignant tumors in athymic mice (32). In the

124

present study, cell strain 88A formed tumors in several sites of injection, but in
the four tumor-derived cell strains that we tested for the expression of the
transgene, its expression level very significantly reduced. A similar loss of an
exogenous p53 transgene has been reported in human colorectal carcinoma cell
lines (33). The loss of p53 expression or greatly reduced level of p53 expression
in tumors formed by cell strain 88A strongly suggests that loss of p53 function is
essential for the tumorigenicity of these cells.

The p53 protein, composed of 393 amino acid residues, can be divided
roughly into three functional domains, i.e., the amino-terminal activation domain,
the central core sequence-specific DNA binding domain and the multifunctional
carboxy-terminal domain (34). The tumor suppressor functions of p53 such as
growth arrest, apoptosis and senescence require the transactivation function of
p53. It has also been reported that p53 can suppress transformation using a
function other than transactivation, as p53 mutants lacking domains required for
transactivation are still capable of suppressing transformation, albeit at lower
efficiency than the wild-type protein (reviewed in 34). In addition, p53 can prevent
tumor formation by interfering with angiogenesis of human fibroblasts by
upregulating expression level of thrombospondin-1, a potent inhibitor of
angiogenesis (35) or down-regulating vascular endothelial growth factor (VEGF)
expression (36,37). At present, we do not know for certain how p53 suppresses
foci and tumors in our system. It could be a combination of more than one of the

mechanisms mentioned above, or a novel mechanism;

125

In summary, our data show that wild-type p53 inhibits focus formation as
well as tumor formation in athymic mice by carcinogen-treated, focus-derived
MSU-1.1 fibroblasts, and that p53 suppresses the transformation without altering

the rate of cell growth in culture.

Acknowledgements

We thank Dr. Bert Vogelstein at Johns Hopkins University School of
Medicine for kindly providing us with the wild-type p53 cDNA plasmid. We also
appreciate helpful discussions with Dr. Sandra O’Reilly in this laboratory and the

excellent technical assistance of Clarissa Dallas.

References

1. Greenblatt, M.S., Bennett, W.P., Hollstein, M., and Harris, C.C. Mutations
in the p53 tumor suppressor gene: clues to cancer etiology and molecular
pathogenesis. Cancer Res., 54: 4855-4878, 1994.

2. Srivastava, S., Zou, Z.Q., Pirollo, K., Blattner, W., Chang, E.H. Germ-line
transmission of a mutated p53 gene in a cancer-prone family with Li-
Fraumeni syndrome. Nature, 348: 747-749, 1990.

3. el-Deiry, W.S., l—larper, J.W., O'Connor, P.M., Velculescu, V.E., Canman,
C.E., Jackman, J., Pietenpol, J.A., Burrell, M., Hill, D.E., Wang, Y.,
Wilman, K.G., Mercer, W.E., Kastan, M.B., Kohn, K.W., Elledge, S.J.,
Kinzler, K.W., and Vogelstein, B. WAF1/CIP1 is induced in p53-mediated
G1 arrest and apoptosis. Cancer Res., 54: 1169-1174, 1994.

4. Xiong, Y., Hannon, G.J., Zhang, H., Casso, D., Kobayashi, R., Beach, D.
p21 is a universal inhibitor of cyclin kinases. Nature, 366: 701-704, 1993.

5. White, E. Life, death, and the pursuit of apoptosis. Genes Dev. 10: 1-15,
1996.

6. Smith ML, Fornace AJ Jr. Genomic instability and the role of p53
mutations in cancer cells. Curr Opin Oncol. 7:69-75, 1995.

126

10.

11.

12.

13.

14.

15.

16.

17.

Polyak, K., Waldman, T., He, T.C., Kinzler, K.W. and Vogelstein, B.
Genetic determinants of p53-induced apoptosis and growth arrest. Genes
Dev., 10: 1945-1952, 1996.

Chen, X., Ko, L.J., Jayaraman, L. and Prives, C. p53 levels, functional
domains, and DNA damage determine the extent of the apoptotic
response of tumor cells. Genes Dev., 10: 2438-2451, 1996.

Sugrue, M.M., Shin, D.Y., Lee, W.L., and Aaronson, S.A. Wild-type p53
triggers a rapid senescence program in human tumor cells lacking
functional p53. Proc. Natl. Acad. Sci. USA, 94: 9648-9653, 1997.

Morgan, T.L., Yang, D.J., Fry, D.G., Hurlin, P.J., Kohler, S.K., Maher,
V.M., McCormick, J.J. Characteristics of an infinite life span diploid human
fibroblast cell strain and a near-diploid strain arising from a clone of cells
expressing a transfected v-myc oncogene. Exp. Cell Res., 197: 125-136,
1991.

Hurlin, P.J., Maher, V.M., McCormick, J.J. Malignant transformation of
human fibroblasts caused by expression of a transfected T24 HRAS
oncogene. Proc Natl Acad Sci USA, 86: 187-191, 1989.

Wilson, D.M., Yang, D.J., Dillberger, J.E., Dietrich, S.E., Maher, V.M.,
McCormick, J.J. Malignant transformation of human fibroblasts by a
transfected N-ras oncogene. Cancer Res., 50: 5587-5593, 1990.

Fry, D.G., Milam, L.D., Dillberger, J.E., Maher, V.M., McCormick, J.J.
Malignant transformation of an infinite life span human fibroblast cell strain
by transfection with v-Ki-ras. Oncogene, 5: 1415-1418, 1990.

Reinhold, D.S., Walicka, M., Elkassaby, M., Milam, L.D., Kohler, S.K.,
Dunstan, R.W., McCormick, J.J. Malignant transformation of human
fibroblasts by ionizing radiation. Int J Radiat Biol, 69: 707-715, 1996.

O'Reilly, 8., Walicka, M., Kohler, S.K., Dunstan, R., Maher, V.M.,
McCormick, J.J. Dose-dependent transformation of cells of human
fibroblast cell strain MSU-1.1 by cobalt-60 gamma radiation and
characterization of the transformed cells. Radiat Res., 150: 577-584,
1998.

Yang, 0., Louden, C., Reinhold, D.S., Kohler, S.K., Maher, V.M., and
McCormick, J.J. Malignant transformation of human fibroblast cell strain
MSU-1.1 by (i)-7 beta,8 alpha-dihydroxy-9 alpha,10alpha-epoxy-7,8,9,10-
tetrahydrobenzo [a]pyrene. Proc. Natl. Acad. Sci. USA, 89: 2237-2241,
1992.

Boley, S.E., McManus, T.P., Maher, V.M., McCormick, J.J. Malignant
transformation of human fibroblast cell strain MSU-1.1 by N-methyl-N-

127

18.

19.

20.

21.

22.

23.

24.

25.

26.

27.

28.

nitrosourea: evidence of elimination of p53 by homologous recombination.
Cancer Res., 60: 4105-4111, 2000.

Qing, J., Maher, V.M., Tran, H., Argraves, W.S., Dunstan, R.W.,
McCormick, J.J. Suppression of anchorage-independent growth and
matrigel invasion and delayed tumor formation by elevated expression of
fibulin-1D in human fibrosarcoma-derived cell lines. Oncogene, 15: 2159-
2168, 1997.

Ryan PA, Maher VM, McCormick JJ. Modification of MCDB 110 medium
to support prolonged growth and consistent high cloning efficiency of
diploid human fibroblasts. Exp Cell Res., 172:318-28, 1987.

Wang, Y., Blandino, G., Oren, M., and Givol, D. Induced p53 expression in
lung cancer cell line promotes cell senescence and differentially modifies
the cytotoxicity of anti-cancer drugs. Oncogene, 17: 1923-1930, 1998.

Shockett, PE, and Schatz, D.G. Diverse strategies for tetracycline-
regulated inducible gene expression. Proc Natl Acad Sci U S A, 93: 5173-
5176, 1996.

el-Deiry, W.S., Tokino, T., Velculescu, V.E., Levy, D.B., Parsons, R.,
Trent, J.M., Lin, D., Mercer, W.E., Kinzler, K.W., Vogelstein, B. WAF1, a
potential mediator of p53 tumor suppression. Cell, 75: 817-825, 1993.

Ford, J.M., Hanawalt, P.C. Expression of wild-type p53 is required for
efficient global genomic nucleotide excision repair in UV-irradiated human
fibroblasts. J Biol. Chem., 272: 28073-28080, 1997.

Ljungman, M., Zhang, F., Chen, F., Rainbow, A.J., McKay, B.C. Inhibition
of RNA polymerase II as a trigger for the p53 response. Oncogene, 18:
583-592, 1999.

Schwartz MA. Integrins, oncogenes, and anchorage independence. J Cell
Biol., 139:575-8, 1997.

Geng L, Walter S, Melian E, Vaughan AT. Transfection of a vector
expressing wild-type p53 into cells of two human glioma cell lines
enhances radiation toxicity. Radiat Res., 150:31-7, 1998.

Lang FF, Yung WK, Raju U, Libunao F, Terry NH, Tofilon PJ.
Enhancement of radiosensitivity of wild-type p53 human glioma cells by
adenovirus-mediated delivery of the p53 gene. J Neurosurg., 892125-32,
1998.

Maclean, K., Rogan, E.M., Whitaker, N.J., Chang, A.C., Rowe, P.B., Dalla-
Pozza, L., Symonds, G., Reddel, R.R. In vitro transformation of Li-

128

29.

30.

31.

32.

33.

34.

35.

36.

37.

Fraumeni syndrome fibroblasts by SV40 large T antigen mutants.
Oncogene, 9: 719-25, 1994.

Eliyahu, D., Michalovitz, D., Eliyahu, S., Pinhasi-Kimhi, O., Oren, M. Wild-
type p53 can inhibit oncogene-mediated focus formation. Proc. Natl. Acad.
Sci. USA, 86: 8763-8767, 1989.

Nevels, M., Rubenwolf, S., Spruss, T., Wolf, H., Dobner, T. The
adenovirus E4orf6 protein can promote E1A/E1B-induced focus formation

by interfering with p53 tumor suppressor function. Proc. Natl. Acad. Sci.
USA, 94: 1206-1211, 1997.

Freedman VH, Shin SI. Cellular tumorigenicity in nude mice: correlation
with cell growth in semi-solid medium. Cell, 3:355-9, 1974.

Okamoto M, Hattori K, Fujimoto K, Tanaka Y, Gloosby CL, Oyasu R.
Antisense RNA-mediated reduction of p53 induces malignant phenotype
innontumorigenic rat urothelial cells. Carcinogenesis, 19:73-9, 1998.

Baker, S.J., Markowitz, S., Fearon, E.R., Willson, J.K., Vogelstein, B.
Suppression of human colorectal carcinoma cell growth by wild-type p53.
Science, 249: 912-915, 1990.

K0, LJ., and Prives, C. p53: puzzle and paradigm. Genes Dev., 10: 1054-
1072, 1996.

Dameron, K.M., Volpert, O.V., Tainsky, M.A., Bouck, N. Control of
angiogenesis in fibroblasts by p53 regulation of thrombospondin-1.
Science, 265: 1582-1584, 1994.

Bouvet, M., Ellis, L.M., Nishizaki, M., Fujiwara, T., Liu, W., Bucana, C.D.,
Fang, B., Lee, J.J., and Roth, JA. Adenovirus-mediated wild-type p53
gene transfer down-regulates vascular endothelial growth factor

expression and inhibits angiogenesis in human colon cancer. Cancer
Res., 58: 2288-2292, 1998.

Zhang, L., Yu, 0., Hu, M., Xiong, S., Lang, A., Ellis, L.M., Pollock, R.E.
Wild-type p53 suppresses angiogenesis in human leiomyosarcoma and
synovial sarcoma by transcriptional suppression of vascular endothelial
growth factor expression. Cancer Res., 60: 3655-3661, 2000.

129

CHAPTER 3

The Function of the Human Homolog of Saccharomyces cerevisiae REV1 Is

Required for Mutagenesis Induced by UV Light

Peter E. M. Gibbs’, Xi-De Wang“, Ziqiang Li“, Terrence P. McManus',

W. Glenn McGregor“, Christopher W. Lawrence”, and Veronica M. Maher?

* Department of Biochemistry and Biophysics, University of Rochester School of
Medicine and Dentistry, Rochester, NY 14642; and " Carcinogenesis Laboratory,
Department of Microbiology and Department of Biochemistry, Michigan State

University, East Lansing, MI 48824

Communicated by James E. Cleaver, University of California, San Francisco, CA,

January 2, 2000 (received for review November 18, 1999)
*To whom reprint requests should be addressed. E-mail:

christopher_lawrence@urmc.rochester.edu.

Abbreviations used are: RACE, rapid amplification of cDNA ends; Pol, DNA
polymerase; EST, expressed sequence tag; HPRT, hypoxanthine

phosphoribosyltransferase.

Data deposition: The sequence reported in this paper has been deposited in the

GenBank database (accession no. AF206019).

130

Abstract

In Saccharomyces cerevisiae, most mutations induced by a wide range of
mutagens arise during translesion replication employing the REV1 gene product
and DNA polymerase Q. As part of an effort to investigate mammalian mutagenic
mechanisms, we have identified cDNA clones of the human homologs of the
yeast REV genes and examined their function in UV mutagenesis. Previously, we
described the isolation of a human homolog of yeast REV3, the catalytic subunit
of Pol g, and here report the identification and sequence of a human homolog of
yeast REV1. This gene was isolated by identifying an expressed sequence tag
encoding a peptide with similarity to the C terminus of yeast Rev1p, followed by
sequencing of the clone and retrieval of the remaining cDNA by 5' rapid
amplification of cDNA ends. The human gene encodes an expected protein of
1,251 residues, compared with 985 residues in the yeast protein. The proteins
share two amino-terminal regions of ~100 residues with 41% and 20% identity, a
region of z320 residues with 31% identity, and a central motif in which 11 of
13 residues are identical. Human cells expressing high levels of an hREV1
antisense RNA grew normally, and were not more sensitive to the cytotoxic effect
of 254 nm UV radiation than cells lacking antisense RNA. However, the
frequencies of 6-thioguanine resistance mutants induced by UV in the cells
expressing antisense hREV1 RNA were significantly lower than in the control
(P = 0.01), suggesting that the human gene has a function similar to that of the

yeast homolog.

131

Introduction

Information about the mechanisms that generate mutations in eukaryotes
is likely to be useful for understanding human health concerns, such as
genotoxicity and cancer (1). At the present, these mechanisms have been
investigated most intensively in budding yeast, Saccharomyces cerevisiae, and in
this organism almost all induced mutations arise during translesion replication, a
process that promotes elongation past sites of unrepaired lesions that might
otherwise block this event (1, 2). The major pathway for translesion replication in
yeast employs DNA polymerase (Pol) Q (3) together with Rev1 protein (4), an
enzyme that has two functions. These include a deoxycytidyl transferase activity
that incorporates dCMP opposite abasic sites in the template and a second, as
yet poorly defined, activity that is required for replication past a wide variety of
lesions as well as abasic sites (J. R. Nelson, P.E.M.G., A. M. Nowicka,
D. C. Hinkle and C.W.L., unpublished observations). In addition to this general
pathway, yeast also possesses a specialized pathway for translesion replication
employing Pol n (5), a distant homolog of Rev1 protein that is encoded by RAD30
(6, 7). Although the substrate range of this enzyme has not yet been fully defined,
it is likely that it entails only a few types of lesion, which Pol 11 appears to bypass
with relatively high accuracy. Pol 11 appears to contribute very little to overall
mutagenesis compared with Pol {/Rev1 both because RAD30 mutants have little
effect on mutagenesis and because mutants lacking the Pol ; pathway are

substantially deficient in mutagenesis induced by almost all mutagens.

132

DNA repair and damage tolerance mechanisms in yeast often provide
good models for these processes in mammals, and evidence of several kinds
suggests that this is likely to be the case with translesion replication. We (8) and
others (9, 10) have identified and sequenced cDNA clones of a human homolog
of the yeast REV3 gene, which encodes the catalytic subunit of Pol 4', and a very
similar gene has been described in the mouse (11). More particularly, we have
further shown that UV-induced mutagenesis is markedly reduced in human cells
expressing high levels of a REV3 antisense RNA, suggesting that the human
gene is also likely to be used in translesion replication (8). In addition, human
cells have been shown to possess two homologs of the yeast RAD30 gene (12-
15). As further evidence for a yeast-like mechanism for translesion replication in
humans, we report here the isolation and sequence of a cDNA clone of a human
homolog of yeast REV1, together with evidence that it too is used in UV
mutagenesis. A recent report also describes this gene, and provides evidence

indicating that it possesses a deoxycytidyl transferase activity (16).

Materials and methods

cDNA synthesis, 5' Rapid Amplification of cDNA Ends (RACE), and cloning

Total RNA or polyadenylated RNA from human brain (Stratagene) was
used as a template for cDNA synthesis, using Superscript II Reverse
Transcriptase (Life Technologies, Gaithersburg, MD) and primers specific for the
candidate hREV1 cDNA clone (leBAC55; American Type Culture Collection).
After removal of residual primer, an oligo(dC) tail was added to the cDNA with

terminal transferase, and the cDNA selectively amplified by PCR, using elongase

133

(Life Technologies) or Taq polymerase, a nested hREV1-specific primer, and a
commercial primer with 3' complementarity to the oligo(dC) tail. The PCR
products were separated on agarose gels, and material from the trailing edge of
the DNA smear was purified for use as a substrate for a second amplification with
Taq polymerase; in this instance, both primers had 5' sequences containing
multiple dU residues. The products were again separated on agarose gels, and
the largest visible DNA species were purified and treated with uracil N-
glycosylase in the presence of the pAMP-1 vector (Life Technologies). The
resulting annealed DNAs were used to transform Escherichia coli DH5a cells. For
assembling clones with a nearly complete insert sequence, PCR used two
hREV1-specific primers, each of which contained a restriction enzyme
recognition site in the 5' sequence. Subsequent digestion with the appropriate
restriction nucleases enabled ligation with similarly digested vector DNA. A
plasmid (leNo-10) containing the entire ORF and 3'-untranslated region, but

with a truncated 5' end, was constructed in pSPORT1 (Life Technologies).

DNA purification and sequence analysis

Plasmid DNAs were isolated by alkaline lysis, and in many cases further
purified on midi columns (Qiagen, Chatsworth, CA) for sequence analysis. The
initial clone, pHIBAC55, and clones from the first two rounds of 5' RACE were
sequenced entirely by the dideoxy method with Sequenase ll. Later clones were
sequenced by using dye terminator chemistries (Perkin-Elmer Applied
Biosystems). Primers for sequencing and for PCR were developed as required

from the known hREV1 sequence as it became available. Both strands of each

134

clone were sequenced, and all regions of the cDNA were sequenced in at least
three clones (except for the 5' 26 nucleotides, where only one clone was
available) to establish a consensus sequence for hREV1 free of reverse
transcription or PCR-generated errors. Attempts to confirm the sequence at the
extreme 5' end of the mRNA with data from human expressed sequence tag
(EST) sequences were unsuccessful, because none carried sequence at the 5'

end of the cDNA.

Preparation of cells that express hREV1 antisense RNA

A plasmid designed to express antisense hREV1 RNA was constructed by
inserting a 4,117-bp REV1 fragment into pTet—Puro in the antisense orientation.
The pTet-Puro plasmid, described previously (8), contains a puromycin-
selectable marker and places the gene of interest under the control of the TetP
promoter (17, 18), with the gene being transcribed in the absence of tetracycline
(Tet OFF system). The 4,117-bp sequence contained the complete hREV1 ORF,
the entire 3' untranslated region, and a short oligo(A) sequence. Initial
experiments, using a 972-bp fragment containing 75 bp of the hREV1 5'
untranslated sequence cloned in the antisense orientation into pTet-Puro, proved
incapable of suppressing hREV1 function. This was probably caused by an
interaction between the antisense RNA and 288 rRNA, and indeed the hREV1 5'
untranslated sequence has regions of strong similarity to part of the rRNA
sequence. The Xhol and Sail fragments from pEcNo-4 containing the biologically
effective 4,117-bp sequence were cloned into the Sall site of the vector pTet-

Puro, to give the hREV1 anti-sense expressing plasmid, pR1P27-AS. The

135

orientation of the insert in this clone was determined by restriction enzyme

analysis and by sequencing across the ligation boundaries.

Plasmid pR1P27-AS was transfected into 7AGM cells, which were derived
from MSU-1.2, a near diploid, nontumorigenic, karyotypically stable human
fibroblast cell line obtained originally from foreskin material of a normal neonate
(19). Strain 7AGM was engineered to contain the tetracycline-controlled
transactivator (tTA), which can activate transcription of genes of interest
controlled by the Tet-responsive element. The pTet-tTAk plasmid used to
introduce the tTA element carried the gene for histidinol resistance (20). pR1P27-
AS transfectants were selected for resistance to puromycin and screened for the

level of expression of hREV1 antisense by Northern blotanalysis.

Northern blot analysis

The conditions used for comparing the level of expression of hREV1
antisense in the transfectant cell strains were described previously (21). Briefly,
15 pg total RNA was electrophoresed on a denaturing formaldehyde gel,
transferred to a Hybond-N membrane by a downward capillary transfer
technique, and fixed by UV crosslinking. The template DNA for preparing the
hREV1 antisense DNA probe was excised from the pR1P27-AS plasmid using
EcoRl restriction. A z2,900-bp fragment from the 5' end of the insert hREV1
cDNA was radiolabeled by random-primer labeling (21). The probe to be used for
the loading control was similarly prepared from PCR-amplified cDNA of the
hypoxanthine phosphoribosyltransferase (HPR'I') gene of human cells. Northern

hybridization was performed at 42°C overnight in 50% formamide, containing

136

SSPE [NaCl (0.75 M)/NaH2PO4-H20 (0.05 M)/EDTA (pH 8.0) (0.5 mM)] and the
other components listed in ref. 22, and the blot was washed as described (21).

Variation in RNA loading per lane was evaluated by probing with HPRTcDNA.

Determination of the cytotoxic and mutagenic effects of UV radiation

Cells were routinely cultured in medium containing 10% supplemented calf
serum (HyCIone), hydrocortisone (1 uglml), penicillin (100 units/ml), and
streptomycin (100 pglml). The medium used was McM medium (22) or Eagle's
MEM, modified by addition of L-aspartic acid (0.2 mM), L-serine (0.2 mM), and
pyruvate (1 mM). The cytotoxic effect of the 254-nm UV radiation was determined
from the survival of colony-forming ability, as described (23). Briefly, cells were
plated into a series of dishes at cloning densities (100-1,000 cells per 100-mm-
diameter dish, depending on the cloning efficiency of the strain and the expected
survival). When the cells had attached and flattened out, the medium was
removed, and the cells were rinsed and irradiated as described (24), using the
designated doses. The mutagenic effect of UV was determined from the
frequency of HPRT-defective, 6-thioguanine-resistant cells. Briefly, for each
experiment, cells in exponential growth were plated into a series of 150-mm-
diameter dishes at a density of 0.5-2 x 106 cells per dish. For each dose of UV,
sufficient dishes were used to ensure at least 1 x 106 surviving cells. At the same
time, cells were plated at cloning density into a series of 100-mm-diameter dishes
to be used for determining the cytotoxic effect of each dose. Again, when the
cells had attached and flattened out, the medium was removed, and the cells

were rinsed and irradiated. For each experiment, 1 x 106 cells were mock-treated

137

as a control. Fresh medium containing serum was returned to the cells
immediately after irradiation, and the cells were refed with culture medium after
24 h. The cells plated at cloning densities were allowed 14 days to form colonies,
with one additional refeeding after 7 days. The cells irradiated at high density for
mutation induction were allowed to replicate for 4-5 days. They were then
detached from the dishes, pooled, and 1-2 x 106 plated again and allowed to
continue replicating for 4 to 5 additional days to allow depletion of preexisting
HPRT. The unused population for each dose was stored in liquid N2 for future
use. After an 8- to 9-day expression period, at least 1 x 106 cells from each
population were selected for resistance to 6-thioguanine at a density of
500 cells/cmz, as described (23). A portion of the cells from each population was
also plated in nonselective medium at a density of 100 cells per 100-mm-
diameter dishes to assay the colony-forming ability of the cells at the time of
selection. This value was used to correct the observed frequency of mutants for

the cloning efficiency of the cells at the time of selection (23).

Results and discussion

Human cells possess a homolog of yeast REV1

A candidate human REV1 cDNA clone was identified by screening the
dbEST database of the National Center for Biotechnology Information, using the
BLAST algorithm. This screen yielded a single EST (GenBank T08134) among
the approximately 52,000 entries listed at that time, and the corresponding clone
leBAC55 was obtained from the American Type Culture Collection. The 2.13-kb

human DNA insert in this plasmid was fully sequenced and found to consist of

138

1,813 bp of the 3' end of an ORF, 296 bp of 3' untranslated DNA, and 20 bp of
poly(A). The remainder of the ORF, together with at least some of the 5'
untranslated region, was obtained by using five rounds of 5' RACE, with the fifth
round extending the sequence to a point beyond which no further elongation was
obtained. To eliminate possible errors of reverse transcription and amplification, a
consensus sequence was established from at least three independent clones at

each round of 5' RACE.

The hREV1 ORF (GenBank accession no. AF206019) encodes an
expected protein of 1,251 residues (Fig. 1A) with significant identity to yeast
REV1 (Fig. 2) and to candidate REV1 genes from other species, including
Caenorhabditis elegans, Arabidopsis thaliana, Schizosaccharomyces pombe,
and Drosophila melanogaster (data not shown). Significant identity exists in two
amino-terminal regions, each z100 residues in length, that exhibit 41% and 20%
identity with their corresponding yeast sequences, a region of 2830 residues with
31% identity, and a centrally located motif that is highly conserved in Rev1
proteins and also found in E. coli dinB. A total of perhaps nine sequence motifs
are suggested by alignments with the Rev1 proteins from S. cerevisiae,
S. pombe, C. elegans, D. melanogaster, and A. thaliana (Fig.1B). Motif l lies
within a region that shows weak homology to the terminal region of the BRCA1
gene (BRCT), although the similarity is greater with the Rev1 protein from
budding yeast than with the human protein and the possible functional
significance is unclear. However, of greater functional significance, motif l in the

human protein contains G76 (highlighted in Fig. 1A), homologous to G193 in

139

A.

I
Knaccwnkkarnrsaa:AGGYHAAKVWKIyrgrasneamcxcarasrtFSGVA:rvnsy?orsgggcnkpfixgfiggotnvrrsrSKTTHIIATNLPNAKI 130

KELKGEKVIRPEW:VPSIKAGRLLSYIPYQLYTKQSSVOKGLSFNPVCRPEDPLPGFSNIAKQLNNRVHHIVKKIETENEVKVNSHNSWEEEDENNDFSF 203

VDLEOTSPGPKONGIPHPRUSTAItNGHTFSSNGALKTQDCLVFHVNSVASPLSPAFSQEEDKAEKSSTDFRDCTLQQLOQSTRNTDALRNPHRTNSFSL 300
n
SPLHSNTKINCAFHSTVQGPSSTKSTSSVSTFSKAAPSVPSKPSDCNFISNPYSB§§gfifllSHWKCELTEFVNTLORQSNGIFPCREKLKKMKTSRSALV 400
Ill
VTDTCDHSVLNSPRHOSCIMHVDMD:FFVSVGIRNHPULKCKPVAVTSNRSTCRAFLRPGANPQLSWQYYQKKILKGKAADIPDSSLHENPDSAQRNGID 500
IV V VI
SVLSRAQ1asfgngBQLQlfifiggFFGHAKQLCPKLQAUPYDFHRYKEVAOTLYETLASYTHNEEAVCCDEALVDITEILAfiTKLTPDEFANAVRMEIKD 500
VII
crkcnasvcIGsn:gzagggggknkyocotHLkpaevccrIRGQLVTNLPGVGHSMESKLASLG:KrgcULQYMTMAKLQKEVGPKTQQMLrnrcucnoo 700
VIII IX
FPVRTLKLRKSVSAEINYC}FFTQPKEAEAFLLSLSEEICRRLEATGMHGKRLTBKIHVEKPGAPVETAKFCCHS CDNIARTvTLchTcNAKIICKAM 800

 

 

 

LNHFHTMKLNISUMRGVGIHVNOLVPTNLNFSTCPSRFSVQSSHFPSSSYSVRDVFQVQKAKKSTEHEHKEVFRAAVDLEiSSRSHTCTFLPPFPAHLPT 900
SPDTNKAHRSGKWNGLHTPVSVOSPLNLSIEVPSFSOL398VLERLPPDLREQVEQVCAVQOAFSHGDKKKEPVNGCNTGILPQPVCTVLLOIPEPOESN 1300
SUBSENLIALPAFSQVDPSVFARLFABLQRELKAAYDCROROGENSTEOOSASASVPKNFLLHLKAAVHEKFRNKKKKTTGSPKRICSFLNNKLLNSPAK 1100
1LPGACGSPQKLIDGFLKHEGPPEEKPLEELSASTSGVPGMSSLQSDFAGCVRPPAPNLAGRVEFNDVKTLLREWZTTISOPHLEDILOVVKYCTDLIEE 1200

KULEKLDLVI KYMKRLMQQSVESVWNMAFD? I LDNVQVVL'QQTYGSTLKVT ‘ 12 51

 

 

 

 

 

 

 

 

 

 

 

 

THIII

I II III IV V VI VII VIII IX

 

 

 

 

 

 

 

 

 

 

Figure 1. (A) Sequence of the translation product of hREV1 mRNA. The
underlined sequences numbered l-IX are possible sequence motifs, and the
highlighted residues in motifs I, III, and VI are known to be important for function.
(B) Depiction of regions of similarity to C. elegans and D. melanogaster
sequences (open boxes) together with the location of the predicted motifs. The

darker filled boxes indicate the location of motifs Ill and VI.

140

4 9
370 432
17 126

277 \ K 506 825
I I I | I

1251

 

: It. v.5" -' ’ , l. J * 9 n. ‘_._."-.T> -’ “Ni“.
‘ . z‘ a ‘ ‘ -' 1 ,‘ . ‘ .. , '. ‘ "L ‘ - .
_"I-."-g ; . .$_ :. ...- .- ‘ .; ..:_‘.{‘

57°/

410/0 ZOO/o 31%

J human Rev1p

 

I lit‘:— ;;{:I I JELZ‘LSIL' .2‘" 3. A" 17:13.13: $33
I I J K |
131 244 735
337 395

358 371

J yeast Rev1p

I
985

Figure 2. Schematic representation of the alignment of hREV1 and yeast REV1

protein sequences. Regions with significant identity are shaded, and the percent

identity is indicated.

141

yeast and the site of the G193R mutation found in the yeast rev1-1 mutant (25);
the Rev1-1 protein retains a significant fraction of its deoxycytidyl transferase
activity, but has lost almost all of its general bypass function (J. R. Nelson,
P.E.M.G., A. M. Nowicka, D. C. Hinkle and C.W.L., unpublished observations).
Conversely, the highly conserved DXD and DE sequences found in motifs Ill and
VI, respectively, appear to be concerned with catalysis (5, 26), and a Pol
77 D156A, E157A tandem double mutation lacks polymerase activity (5).

The sequence 5' to the ATG codon at the start of the main ORF contains
an out-of-frame ATG sequence at nucleotide -35, initiating a small reading frame
that terminates at a TGA stop codon overlapping the main ORF ATG. Further, the
sequence context of the out-of frame ATG is almost as good as that of the ORF
ATG. This feature suggests that the hREV1 message is translated very
inefficiently, and predicts that hRev1 protein levels are likely to be low (27). A
number of the clones analyzed encoded a protein of 1,250, rather than
1,251, residues, in which the sequence CAGCAG at nucleotides 1041-1046 was
replaced by CAG. A similar pair of sequences was also found in the homologous
region of the mouse REV1 sequence (data not shown); in both cases, they may
result from slippage at the 3' splice site of an intron. The protein sequence given
in Fig. 1A is identical with that of ref. 16, but differs by one synonymous mutation
(Ile1150, ATT vs. ATC), that may represent a polymorphism. Finally, a sequence
yielding an exact match to the 3'-tenninal 60% of the ORF of hREV1 is described
in GenBank as encoding a protein that interacts with a3A integrin (28); the

significance of this observation is unclear.

142

Although determining the sequence of the ORF presented few problems,
establishing the sequence of the 5' untranslated region of the mRNA was less
straightforward, and it is unlikely that it is complete. Because the 5' sequence is
GC-rich, attempts to extend primers into this region resulted in multiple premature
termination events in clones with intron sequences, as identified both by the
presence of canonical 3' splice junction sequences and by the loss of the ORF.
Other attempts recovered clones showing rearrangements of the cDNA sequence
that presumably occurred during either synthesis or amplification. To confirm that
the GC-rich sequence indeed constituted the 5' untranslated sequence, sense-
strand primers were designed specific to each of the possible upstream
sequences, together with a common antisense primer. Only the GC-rich specific
primer yielded an appropriate amplification product (data not shown). Additional
evidence supporting this conclusion is provided by the sequences of three mouse
ESTs (accession nos. AA4202230, Al019222 and Al481088), which show strong
similarity to the region of hREV1 flanking the putative initiation codon. In contrast,
sequence identified by an alternative upstream primer was clearly an artifact,
because it matched sequence of an mRNA from human cortex (HUMMRNAC,

accession no. L10374).

UV-induced mutagenesis is much reduced in human cells expressing

hREV1 antisense RNA

In yeast, the REV1 gene is required for 395% of the base pair
substitutions induced by UV (19), and rev1 mutants are more readily killed by this

radiation, although this hypersensitivity is relatively modest (30). Unlike REV3

143

however, the REV1 gene appears to be required to a much smaller extent for the
production of frame-shift mutations induced by UV. The extent varies among the
different genetic sites investigated, however, and at three of them >80% of the
UV-induced frameshifts depended on REV1 function (29, 31). Results with other
mutagens, although less extensive, appear to be similar to those with UV, both
with respect to base pair substitutions and frameshifts (32). To examine whether
human REV1 is also required for UV mutagenesis, we have used the antisense
method used previously with hREV3 (8), in which high levels of an antisense
RNA are expressed under the control of the TetP promoter. To this end, a series
of independent puromycin-resistant colonies obtained by transfection of parental
7AGM cells with hREV1 antisense-expressing plasmid, pR1P27-AS, were
isolated and expanded to 2.40 x 106 cells. A portion was used for Northern blot
analysis for antisense expression, and the rest were cryopreserved. Two
unequivocally independent clones, designated 7AGM-1ZB-R1 and 7AGM-17C-
R1, found to express antisense RNA at a level much higher than the level of
natural transcript from the endogenous hREV1 gene (Fig. 3), were chosen for
study. The level of expression of antisense REV1 RNA in these two strains,

corrected for slight differences in RNA loading per lane, was very similar (Fig. 3).

The frequency of 254-nm UV-induced mutants and the sensitivity to UV
cell killing in these two cell strains was compared with that in their nontransfected
parental cell strain, 7AGM. In all but one experiment, the parental cell strain and
one or other or both of the derivative strains 7AGM-12B-R1 and 7AGM-17C-R1

were compared simultaneously. As shown in Fig. 4A, these two cell strains

144

17C 128 7AGM

hRev1 mRNA —
hRev1 AS RNA _

   

  
 

HPRT mRNA -;,

 

Figure 3. Northern blot analysis of the level of expression of hREV1 antisense
RNA in the cell strains. RNA extracted from the two derivative cell strains, 7AGM-
17C and 7AGM-123, which had been transfected with plasmid pR1P27-AS
containing the 4,117-bp sequence of the hREV1 RNA in an antisense orientation,
and from their nontransfected parental cell strain, MSU-1.2-7AGM, and analyzed
for expression of hREV1 antisense RNA (hREV1 AS RNA) and/or the
endogenous hREV1 sense RNA. The latter mRNA, which is 24.4 kbp in length,
can be seen as a faint band just above the antisense band in the first two lanes,
and in the third lane. The lower band, approximately 1.4 kbp in length, is the
endogenous HPRT mRNA, which was used to normalize the amount of RNA
loaded per lane. There was no significant difference between the two derivative

cell strains in the level of expression of the antisense RNA.

145

expressing hREV1 antisense RNA were not more sensitive than their parental
Strain to the cytotoxic effect of UV. The line with the slightly steeper slope is a
least squares fit to the data from the parent strain 7AGM and also from strain
7AGM-17C-R1; the other line represents the least squares fit to data from strain
7AGM-12B-R1. Although the survival curves did not show a significant difference
between these two strains and their parental strain, the frequency of 6-
thioguanine-resistant mutants induced in the two strains expressing hREV1
antisense RNA was significantly lower (P= 0.01) than that seen with their
parental cell strain (Fig. 4B). The 7AGM-17C-R1 cells showed a 264% decrease;
the 7AGM-1ZB-R1 cells showed a $4% decrease in frequency.

The results shown in Fig. 4B for the latter strain were obtained from two
independent experiments, using 0, 11, 13, and 15J/m2 of UV in one experiment,
and 0, 12, and 14 J/m2 of UV in the other. No mutants were seen in the
unirradiated control population. The slope of the line for this strain in Fig. 4B was
fitted to the five data points from these two experiments. Although the slope is
virtually parallel with the x axis, the line cannot, in reality, intersect the y axis
above zero because the data represent the increase in frequency of mutants
above the background frequency in the population. Cell strain 7AGM-17C-R1 was
tested in three experiments. In each, the UV fluences used were 0, 11, 13, and
15 J/mz. The values shown for each UV fluence represent the average of the
three determinations. Nevertheless, the slope of the line shown in Fig. 4B for this
strain was fitted to the nine individual data points. The background frequencies of

mutants per 106 cells in the unirradiated population for these three experiments,

146

i.e., 0, 0, and 5, were almost as low as those observed with cell strain 7AGM-
1ZB-R1. The values shown in Fig. 4B for parental cell strain 7AGM were taken
from four experiments. Except for a single determination at 10 J/mz, the data are
the average from two or three independent determinations in which the same UV
fluence was used. Again, the values shown for each fluence, except 10 Jim2 UV,
represent the average of the multiple determinations. Nevertheless, the slope of
the line was fitted to the ten individual determinations obtained. The background
frequency of mutants per 106 cells in the experiments with this strain were
4, 10, 13, and 17. To allow a comparison of the surviving fractions, the number of
mutants seen, the total cells assayed for mutants, and their corresponding
cloning efficiency at the time of selection for 6-thioguanine resistance, an
example of the data obtained from an experiment for each strain is shown in
Table 1.

The data in Fig. 48 indicate that, in cells expressing of hREV1 antisense,
the frequency of UV-induced mutants was significantly reduced. Unlike what is
found with yeast, however, there was no evidence for increased sensitivity to the
cytotoxic effect of UV radiation in either of the two cell strains expressing the
hREV1 antisense, and in which, presumably, the level of Rev1 protein is reduced
by virtue of the antisense RNA. Such a lack of hypersensitivity may well result
from the redirection of DNA damage into another pathway, one in which
mutations are less likely to occur, or it may reflect a decreased importance of
translesion replication for survival in these human cells, compared with yeast

cells.

147

Figure 4. Percent survival (A) and frequencies of UV-induced 6-thioguanine
resistant mutants (B) in the parent cell strain 7AGM and the hREV1 antisense
RNA-expressing cell strains 7AGM-17C-R1 and 7AGM-1ZB-R1, plotted against
fluence of 254 nm UV. The data are the average of results from four experiments
with the parent strain, 7AGM; three experiments with 7AGM-17C-R1 cells; and
two experiments with 7AGM-1ZB-R1cells. The background frequencies of
mutants, which have been subtracted to obtain the frequencies induced above
the background frequency by UV (23), are cited in the text. The mutant
frequencies observed for untreated control and each UV fluence were corrected
for the cloning efficiency of the cells determined after an 8-day expression period,
i.e., at the time they were plated into selection medium containing 6-thioguanine.
The average cloning efficiencies for control and UV-irradiated cells were: 7AGM
cells, 333%: 1.4% (SEM for four independent experiments); 7AGM-17C-R1
cells, 26.7 :t 1.3% (SEM for three independent experiments); and 7AGM-1ZB-R1

cells, 31.8% :t 0.9% (SEM for two independent experiments).

148

0 3 6 9 12 15 18

 

 

 

 

 

 

 

 

100 rIrlirlrrrirrrrr'
PA 1
'6
.2 " ‘
2
3
‘0 - -
H
C
Q)
0
L-
d)
a _ d
A 7AGM
O 7AGM-17C-R1
I 7AGM-128-R1
1o llllllllJLIlLlliL
7o II'IIIIIIWI'IT1I
BA 7AGM
O 7AGM-17C-R1
:1”- 60 - I 7AGM-123m -
0
U
2
£50- -
C
.2
0
«040- A "
O
‘-
$-
8
£30”
'5
o 9.
(920’ "
j.
'8
$310— . "
s I'
011114111m

0 3 6 9 12 15 18
UV (Jlmz)

149

Table 1. Example of UV cytotoxicity and mutagenicity in parental cell strain

MSU-1.2-7AGM and two derivative cell strains expressing hREV1 antisense

 

RNA
Cell UV Percent Cells Mutants Cloning Mutant Induced
strain (J/mz) survival selected observed efficiency frequency frequency
(X106) 0%) (x106) (x106)
7AGM 0 1 00 1 4 40 10 0
1 0 62 1 1 8 36 50 40
1 2 52 1 22 40 55 45
7AGM- 0 1 00 1 .8 2 24 5 0
17C-R1 11 47 1.8 6 24 14 9
1 3 33 1 .8 1 2 21 32 27
1 5 20 1 .8 7 21 1 9 14
7AGM- 0 1 00 1 0 33 0 0
128-R1 1 1 57 1 O 32 O O
1 3 35 1 .5 1 32 2 2
1 5 26 1 .5 0 30 0 0

 

150

Acknowledgements

We thank Dr. Dennis Gilliland of Michigan State University, Department of

Statistics & Probability, for statistical analysis of the mutagenicity data, and

Beatrice Tung for advice and technical assistance with the human cell studies.

This work was supported by US. Public Health Service Grants GM21858 (to

OWL), E809822, and CA56796 (to V.M.M.), and CA73984 (to W.G.M.) from the

National Institutes of Health.

References

10.

Lawrence, C. W. & Hinkle, D. C. (1996) Cancer SUN. 28, 21-31.

Morrison, A., Christensen, R. B., Alley, J. A., Beck, A. K., Bernstine, E. G.,
Lemontt, J. F. & Lawrence, C. W. (1989) J. Bacteriol. 171, 5659-5667.

Nelson, J. R., Lawrence, C. W. & Hinkle, D. C. (1996) Science 272, 1646-
1649.

Nelson, J. R., Lawrence, C. W. & Hinkle, D. C. (1996) Nature (London)
382, 729-731.

Johnson, R. E., Prakash, S. & Prakash, L. (1999) Science 283, 1001-
1004.

McDonald, J. R, Levine, A. S. & Woodgate, R. (1997) Genetics 147,
1557-1568.

Roush, A. A., Suarez, M., Friedberg, E. C., Radman, M. & Siede, W.
(1998) Mol. Gen. Genet. 257, 686-692.

Gibbs, P. E. M., McGregor, W. G., Maher, V. M., Nisson, P. & Lawrence,
C. W. (1998) Proc. Natl. Acad. Sci. USA 95, 6876-6881.

Morelli, C., Mungall, A. J., Negrini, M., Barbanti-Brodano, G. & Croce, C.
M. (1998) Cytogenet. Cell Genet. 83, 18-20.

Lin, W., Wu, X. & Wang, Z. (1999) Mutat. Res. 433, 89-98.

151

11.

12.

13.

14.

15.

16.

17.

18.

19.

20.

21.

22.

23.

24.

25.

Van Sloun, P. P. H., Romeijn, R. J. & Eeken, J. C. J. (1999) Mutat. Res.
433, 109-116.

Matsutani, C., Araki, M., Yamada, A., Kusumoto, R., Nogimori, T.,
Maekawa, T., lwai, S. & Hanaoka, F. (1999) EMBO J. 18, 3491-3501.

Matsutani, C., Kusumoto, R., Yamada, A., Dohmae, N., Yokoi, M., Yuasa,
M., Araki, M., lwai, S., Takio, K. & Hanaoka, F. (1999) Nature (London)
399, 700-704.

Johnson, R. E., Kondratick, C. M., Prakash, S. & Prakash, L. (1999)
Science 285, 263-265.

McDonald, J. P., Rapic—Otrin, V., Epstein, J. A., Broughton, B. C., Wang,
X., Lehman, A. R., Wolgemuth, D. J. & Woodgate, R. (1999) Genomics
60, 20-30.

Lin, W., Xin, H., Zhang, Y., Wu, X., Yuan, F. & Wang, Z. (1999) Nucleic
Acids Res. 27, 4468-4475.

Gossen, M. & Bujard, H. (1992) Proc. Natl. Acad. Sci. USA 89, 5547-
5551.

Shockett, P., Difilippantonio, M., Hellman, N. & Schatz, D. G. (1995) Proc.
Natl. Acad. Sci. USA 92, 6522-6526.

Lin, C. C., Wang, Q., Maher, V. M. & McCormick, J. J. (1994) Cell Growth
Differ. 5, 1381-1387.

Hartmann, S. C. & Mulligan, R. C. (1988) Proc. Natl. Acad. Sci. USA 85,
8047-8051.

Qing, J., Maher, V. M., Tran, H., Argraves, W. S., Dunstan, R. W. &
McCormick, J. J. (1997) Oncogene 15, 2159-2168.

Ryan, P. A., Maher, V. M. & McCormick, J. J. (1987) Exp. Cell Res. 172,
318-328.

Maher, V. M. & McCormick, J. J. (1996) in Technologies for Detection of
DNA Damage and Mutations, ed. Pfeifer, G. P. (Plenum, New York), pp.
381-390.

Patton, J. D., Rowan, L. A., Mendrala, A. L., Howell, J. N., Maher, V. M. &
McCormick, J. J. (1984) Photochem. Photobiol. 39, 37-42.

Larimer, F. W., Perry, J. R. & Hardigree, A. A. (1989) J. Bacteriol. 171,
230-237.

152

26.

27.
28.

29.
30.
31.

32.

Gerlach, V. L., Aravind, L., Gotway, G., Schultz, R. A., Koonin, E. V. &
Friedberg, E. C. (1999) Proc. Natl. Acad. Sci. USA 96, 11922-11927.

Kozak, M. (1986) Gel/44, 283-292.

Wixler, V., Laplantine, E., Geerts, D., Sonnenberg, A., Petersohn, D.,
Eckes, B., Paulsen, M. & Aumailley, M. (1999) FEBS Lett. 445, 351-355.

Lawrence, C. W. & Christensen, R. B. (1978) J. Mol. Biol. 122, 1-21.
Lemontt, J. F. (1971) Genetics 68, 21-33.

Lawrence, C. W., O'Brien, T. & Bond, J. (1984) Mol. Gen. Genet. 195,
487-490.

McKee, R. H. & Lawrence, C. W. (1979) Genetics 93, 375-381.

153

CHAPTER 4

Expression of Exogenous hRev1 in Human Cells Devoid of Mutations as a
Result of Antisense hREV1 Restores their Ability to Be Mutated by UV or

Benzo[a]pyrene Diol Epoxide

Xi-De Wang‘, Christopher W. Lawrencez, Yoshiki Murakumo3

J. Justin McCormick‘, and Veronica M. Maher"

1Carcinogenesis Laboratory, Department of Microbiology and Molecular Genetics
and Department of Biochemistry and Molecular Biology, Food Safety and
Toxicology Building, Michigan State University, East Lansing, MI 48824-1302.
2Department of Biochemistry and Biophysics, University of Rochester School of
Medicine and Dentistry, Rochester, NY 14642. 3Department of Pathology,
Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku,

Nagoya 466-8550, Japan

Runnng title: ROLE of hRev1 IN UV- AND BPDE- INDUCED MUTAGENESIS
* Corresponding author. Mailing address: Carcinogenesis Laboratory, Food
Safety and Toxicology Building, Michigan State University, East Lansing, MI

48824-1302. Phone: (517)-353-7785. Fax: (517)-353-9004. E-mail:

maher@msu.edu.

154

Abstract

Expression of antisense RNA against hREV1 gene in human fibroblasts in
culture reduced the frequency of 6-thioguanine-resistant mutants induced by
ultraviolet (UV)(254 r.m) radiation up to 20-fold, i.e., to 5% of that induced in the
parental cell strain (Gibbs et al., Proceedings of the Natl. Acad. Sci. USA,
9724186, 2000). To determine whether the decrease in mutagenicity in the
antisense RNA-expressing cell strains resulted from decreased levels of hRev1
protein, we prepared a series of rabbit polyclonal antibodies against hRev1-
specific peptides. Western blotting with these antibodies indicated that the level
of expression of hRev1 in the parental cell strain used in the above mutation
studies, i.e., cells not transfected with antisense, was very low, too low to
quantify a decrease in protein expression caused by hREV1 antisense. As an
alternative approach to see if the level of hRev1 protein determines the
frequency of UV-induced mutants, we transfected an hREV1 gene coding for a
Flag-tagged protein into the antisense-expressing cell strain that exhibited a UV-
induced mutant frequency only ~ 5% of its normal control parental cell strain.
Two transfectant strains expressing the exogenous Flag-hRev1 protein at a
detectable level were assayed for the frequency of UV-induced mutants. For
each dose of UV in both recipients, the frequency of mutants increased ~ 8-fold
over that of the recipient cell strain, i.e., from 5% of that induced in the normal
parent to 40%. This series of cell strains was also used for a similar investigation
of the role of hRev1 in mutagenesis caused by (i)-7j3,8a-dihydroxy—9a,10a-

epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE). The frequency of mutants

155

induced by BPDE in the recipient cell strain expressing antisense RNA was ~ 4%
of that induced in the normal parental strain. The frequency of mutants induced
by BPDE in one of the two transfectant cell strains expressing Flag-tagged
hRev1 protein increased ~ 73-fold, i.e., to 29% of that found in the normal
parental strain; in the other strain, it increased ~3.3-fold, to 13% of the parent.
Sequencing of the HPRT gene of independent mutants induced by BPDE in
these cell strains showed that the majority were base substitutions, and that the
kinds of base substitutions did not differ from those induced by BPDE in the
parental control cell strains or those found previously in this laboratory to be

induced by BPDE in foreskin-derived finite life span human fibroblasts.

Introduction

In Saccharomyces cerevisiae yeast exposed to a variety of DNA
damaging agents, the products of the REV3, REV7 and REV1 genes contribute
only minimally to cell survival, but are absolutely required for damage-induced
mutagenesis (Lawrence and Hinkle, 1996). The Rev3 and Rev7 proteins
constitute DNA polymerase zeta, with Rev3 being the catalytic subunit, and Rev7
the non-catalytic regulatory subunit (Nelson et al., 1996a). Pol zeta is capable of
replicating past UV-induced thymine-thymine cyclobutane dimers, the major
photoproduct of UV radiation (Nelson et al., 1996a). In cell-free assays, Rev1
protein has been shown to exhibit a DNA template-dependent deoxycytidyl
transferase activity, by which it transfers a dCMP opposite an undamaged G
template, but it does not insert other deoxynucleotides opposite their respective

template deoxynucleotides. S. cerevisiae Rev1 can also efficiently incorporate a

156

dCMP opposite an abasic site in vitro, and this can subsequently be extended
efficiently by DNA polymerase zeta (Nelson et al., 1996b). Lawrence and
colleagues (Nelson et al., 2000) also showed that in S. cerevisiae, REV1 is
required for replicating past (6-4) T-T UV photoproducts, a process that usually
does not require dCMP insertions, suggesting that Rev1 has a second function in
participating in translesion synthesis.

The potential relevance of the error-prone translesion synthesis
mechanism involving REV3, REV7 and REV1 to human carcinogenesis
stimulated a successful search for human homologs of these yeast genes (Xiao
et al., 1998a; Gibbs et al., 1998, 2000; Lin et al., 1999b; Murakumo et al., 2000).
Studies designed to eliminate, or greatly decrease, the level of hRev3 and hRev1
protein in human cells by use of antisense showed that the frequency of 6-
thioguanine resistant mutants induced by UV in human fibroblasts expressing
high levels of hREV3 or hREV1 antisense RNA was reduced very significantly
(Gibbs et al., 1998; 2000). It has not been possible to demonstrate directly the
relationship between reduced UV-mutagenicity and reduced hRev3 or hRev1
proteins in cell strains expressing antisense RNA using Western blotting. As an
alternative approach, we transfected a cell strain that expressed hREV1
antisense RNA at a high level and exhibited 95% reduction in the frequency of
UV-induced mutants (Gibbs et al., 2000) with an exogenous hREV1 gene coding
for hRev1 protein Flag-tagged at the amino-terminus (Flag-hRev1). Transfectants
expressing the transgene were identified by Western blotting using an antibody

against the tag. Two of these latter cell strains, along with several control strains

157

transfected with an empty vector, as well as the original parental human
fibroblast strain and its hREV1 antisense-expressing derivative strain, i.e., the
one used as the recipient for the Flag-tagged hRev1, were compared for the
frequency of mutants induced by UV radiation and also BPDE, a reactive
metabolite of benzopyrene. The results show that expression of hRev1 protein in
the transfectant strains significantly increased the frequency of UV or BPDE-
induced mutants. DNA sequencing of the cDNA of the HPRT gene in the BPDE-
induced mutants revealed that the kinds of mutations induced were virtually

identical to those found previously in this laboratory (Yang et al., 1989).

Materials and methods

Cell strains and cell culture

The cells strains were derived from MSU-1.2, a derivative of MSU-1.1 cell
strain, an infinite life span, near-diploid, karyotypically-stable cell strain
established in this laboratory from the foreskin of a normal neonate (Morgan et
al., 1991). Unless othenrvise noted, the cell strains were cultured in Eagle's
minimal medium (Gibco, Gaithersburg, MD), pH 7.2, supplemented with 0.2 mM
L-aspartic acid, 0.2 mM L-serine, 1.0 mM sodium pyruvate, and 10% (v/v)
supplemented calf serum (HyCIone, Logan UT). The culture medium also

contained penicillin (100 Ulml), streptomycin (100 uglml), and hydrocortisone (1

pglml). Cells were cultured in 5% C02 humidified incubators at 37°C.

Plasmids and DNA transfection

158

The plasmid used for expression of the Flag-hRev1 protein,
pcDNA3.1(+)lFLAG-hREV1 was described previously (Murakumo et al., 2000).
The recipient cells were plated at 105 per 60 mm-diameter culture dish and
transfected ~18 h later. Because pcDNA3.1(+)lFLAG-hREV1 contains the gene
for neomycin resistance as its selectable marker, and the recipient MSU-1.2 cells
already express this marker (Morgan et al., 1991), a co-transfection with a
plasmid pcDNA6N5-HisA which carries the gene for resistance to blasticidin was
performed, using 5 pg pcDNA3.1(+)IFLAG-hREV1 and 0.5 pg pcDNA6N5-HisA
per 60 mm-diameter culture dish. The plasmid DNA was introduced in
lipofectamine following the manufacturer's instructions (Gibco, Bethesda). After
48 h, the medium was changed to medium containing blasticidin (5 jig/ml) to
select for transfectants. The cells were given fresh culture medium containing

blasticidin (5 uglml) every 5 days.

Analysis for the expression of hRev1 protein

Cells growing exponentially were washed with cold phosphate-buffered
saline (PBS) and then lysed on ice with lysis buffer composed of 50 mM Tris-HCI,
pH 7.2, 150 mM NaCl, 0.5% NP-40, 50 mM NaF, 1 mM sodium vanadate, 1 mM
DTT, 1 mM benzamidine, 1 mM phenylmethylsulfonyl fluoride (PMSF), 25 pig/ml
aprotinin and 25 jig/ml leupeptin. The cell lysates were collected with a rubber
scraper and centrifuged at 14,000 rpm on a microcentrifuge (Eppendorf,
Westbury, NY) at 4°C. The supernatant was collected, an aliquot was taken for
protein quantification using the Coomassie protein assay reagent (Pierce,

Rockford, IL), and the rest was stored at -80°C. Unless otherwise noted, lysates

159

(100 pg of protein) were loaded on a 6% SDS-PAGE gel, and the gel was run at
constant 35 mA. The proteins were electrotransfered onto an lmmobilon-P
membrane (Millipore, Bedford, MA) for ~18 h at constant 35 mA. The membranes
were blocked at room temperature for 2-3 h in 5% (w/v) non-fat dry milk in Tris-
buffered saline (20 mM Tris-HCI, pH 7.2, 137 mM NaCl) containing 0.1% (vlv)
Tween-20 (T 831'), and incubated with the mouse monoclonal anti-Flag antibody
(Clone M1, Sigma, St. Louis, MO) at the recommended concentration of 10 pg/ml
or the rabbit anti-hRev1 polyclonal antibody (antibody 468) at 1:600 dilution for 2
h at room temperature. The membranes were washed and incubated with
horseradish peroxidase-conjugated goat anti-mouse or anti-rabbit lgG (Sigma,
St. Louis, MO) that had been diluted 1:20.000 in TBST solution supplemented
with 5% milk. Enhanced chemiluminescence substrate (SuperSignal Pico,
Pierce, Rockford, IL) was used for blots using anti-Flag antibody, and Maximum
Sensitivity Substrate (Supersignal Femto, Pierce, Rockford, IL) was used for
blots using anti-hRev1 antibody. The amount of Ku-80 protein, assessed with
rabbit polyclonal anti-Ku80 antibody (Serotec, Raleigh, NC) and same
chemiluminescent substrates as those for detection of hRev1 protein on the
same blot, was used as the loading control. For the Western blotting experiments
designed to explore the detection limit of protein using a Femto sensitivity
substrate, a Dot-Blot anaysis was first performed to optimize the concentrations
of primary and secondary antibodies, and SuperBIock blocking reagent (Pierce,

Rockford, IL) and nitrocellulose membrane (Amersham Pharrnacia, Piscataway,

160

NJ) were used as recommended by the manufacturer of the chemiluminescent

substrate.

Determination of the cytotoxic and mutagenic effects of UV radiation

The cytotoxicity of UV was determined from the number of colonies
formed at each dose, and the mutagenic effect of UV was determined from the
frequency of UV-induced HPRT mutants, as selected by resistance to 40 pM 6-
thioguanine (T6). The procedures used have been described previously (Gibbs
et al., 2000; Li et al., 2002). Briefly, to determine the cytotoxic effect of UV,
sufficient cells were plated per 100-mm-diameter dish to obtain ~50 colonies per
dish. The number plated depended on the expected survival and the cloning
efficiency of the strain. To determine the frequency of induced mutants, cells
were plated into a series of 150-mm-diameter dishes (P150's) at a density of 0.5x
106 to 2 x 106 cells per dish, depending on the expected survival at each dose
and allowing cells enough room to grow for 4 days. For each dose, sufficient
dishes were used to ensure at least 1 x 106 surviving target cells. Approximately
12-16 h after plating, the medium was removed, and the cells were rinsed with
PBS and irradiated, with the dishes revolving on a turntable, at an incidence dose
of ~0.3 Jlmzlsec. Cells were given fresh medium immediately after irradiation and
refed again after 24 h. For determining cytotoxicity, cells were allowed 14 days to
form colonies, with one additional feeding. To determine the frequency of
mutants, cells were allowed 8-10 days of an expression period to allow depletion
of preexisting Hprt protein, with one harvesting, pooling of cells of the same

dose, and plating of 1-2 x 10‘5 cells at the 4"‘-5th day post irradiation. A total of 1 x

161

106 to 2x 106 cells from each population were selected for resistance to 40 pM 6-
TG at a density of ~500 cells/cmz. To assay their colony-forming ability at the
time of selection, cells from each population were plated in nonselective medium
at cloning density. This value was used to correct the observed frequency of TG-
resistant (TG') mutants. Parental MSU-1.2 cells have a cloning efficiency of
~50%. To increase the cloning efficiency of any derivative strain that cloned at a
lower frequency, and to increase the number of cells per clone, feeder cells
composed of TGr clonal populations derived from the same cell strain were y-
irradiated with 30Gy to prevent them from replicating and were plated at a
density of 4x 103 cells/cm2 at the end of the expression period, i.e., when the
target cells were selected for frequency of mutants and cloning efficiency. The
cells used for cloning efficiency were stained with crystal violet and the colonies
were counted. After ~14 days of selection, with one feeding of the cells after 7
days, the dishes containing cells assayed for mutant frequency were searched
for colonies using a focused beam of light from below. The TGr colonies were
isolated and stored at -80°C for later sequencing of their HPRT gene. The dishes
were then stained and any remaining colonies observed were added to the total

number.

Determination of the cytotoxic and mutagenic effects of BPDE

The procedures for determining the cytotoxic and mutageneic effects
BPDE have been described previously (Yang et al., 1991). Briefly, cells in
exponential growth were plated into a series of p100’s at a density of 5.5x

105cells/dish. When cells had attached and flattened out, at ~12-16h post plating,

162

the medium was removed, the cells were rinsed with PBS, and fresh serum-free
medium was added. The BPDE, dissolved in anhydrous dimethylsulfoxide, was
delivered into each culture dish with micropipette and mixed immediately. After
1h, the medium was removed and replaced with fresh culture medium. The cells
in two dishes for each dose, and for the mock-treated control, were immediately
harvested, pooled and plated at cloning density to determine the cytotoxicity of
BPDE. The rest of cells, were allowed an expression period of 8-10 days before
being selected with 40 pM 6-TG. The procedure for expression and selection was

the same as that used for determining the mutagenic effects of UV.

Sequencing of mutant HPRT cDNA

Cell pellets of TGr colonies (~500-2,000 cells/pellet) derived from the
mutagenesis assays were lysed and the HPRT cDNA was amplified using
procedures and reagents essentially described by Yang et al. (1989). The
amplified HPRT cDNA was purified with QlAquick PCR purification kit (Qiagen,
Valencia, CA) and sequenced using Therrno Sequenase Primer Cycle
Sequencing Kit (Amersham Pharmacia, Piscataway, NJ) with primer C
(AAACTCAACTTGAACTCTCA) labeled with Cy5.5 and primer 7
(GACATTCTTICCAGTI’AAAGT) labeled with Cy5.0. The sequencing was
analyzed using Microgene Clipper System (Visible Genetics, Toronto, Ontario,

Canada).

163

Results

Efforts to determine the cellular level of hRev1 protein

In the previous study to determine if hREV1 plays a role in UV-induced
mutagenesis (Gibbs et al., 2000), we showed that two human fibroblast strains
expressing antisense hREV1 RNA at high levels, i.e., 7AGM-17C-R1 and 7AGM-
12B-R1, exhibited a significant reduction in the frequency of UV-induced
mutants, compared to that of their parental strain 7AGM. For each dose, the
mutant frequencies observed were 40% and 5% that of the parental cell strain,
respectively. Subsequently, a third cell strain expressing a high level of antisense
RNA was tested. It gave a frequency ~18% that of the parent (data not shown).
Additional studies showed that the low mutant frequency in these three strains
was not the result of such artifacts as loss of HPRT mutant cells by metabolic
cooperation from neighboring HPRT wild-type cells; a slower rate of growth of the
cells, allowing longer time for excision repair prior to DNA replication past the
photoproducts; or an increase in the number of X chromosomes, on which the
HPRT gene is located (data not shown). Taken together, these data strongly
suggest that the function of hREV1 is required for mutagenesis induced by UV
light in human cells.

The more direct way to test the hypothesis that the reduction of UV
mutability of these cells strains resulted from impaired function of hREV1 was to
determine that antisense reduced or eliminated expression of the target protein,
as was seen for hMms2 in studies from this laboratory (Li et al., 2002). We

independently prepared rabbit polyclonal antisera against three discrete hRev1

164

peptides of 18, 19 and 21 amino acid residues, each of which was predicted to
have high antigenicity. To facilitate the identification of hRev1 protein on Western
blots, we prepared a positive control hRev1 protein by overexpressing a Flag-
tagged hRev1 protein in kidney 293 cells, using a transient expression protocol.
The overexpressed Flag-hRev1 protein could be easily detected by Western
blotting using anti-Flag antibody (data not shown). Using this positive control
lysate, we tested the potential of the rabbit anti-hRev1 peptide antisera for use in
Western blotting. Our pilot studies indicated that two of the polyclonal antisera
failed to recognize even the hRev1 protein overexpressed in 293 cells, and the
third, designated 468, recognized the overexpressed Flag-hRev1 protein, as
shown in Figure 1 (Lane 1). However, this best antibody, i.e., antibody 468, could
not detect an endogenous hRev1 band at that position in the control 293 cells or
in the parental cell strain 7AGM used in our study. We determined that the 293
cells transiently overexpressing Flag-hRev1 protein had at least 20-fold more
hRev1 protein than the control non-transfected 293 cells or 7AGM parental cell
strain. Western blot analyses using carefully optimized blotting conditions,
including optimum primary antibody concentration, secondary antibody
concentrations, blocking reagent, and maximum sensitivity fluorescent substrate,
revealed that the lowest amount of protein that could be detected was about 6.3

pg (Lane 7), 16-fold less the amount of protein displayed in Lane 1, and linear

range fell between 50 pg and 12.5 pg of protein (Lane 4, 5 and 6). Under the

165

1234567891011
hRev1 ‘ 1‘...”

KU’BO *.~‘ P- g... .2... . I a

Figure 1. Evidence that endogenous hRev1 protein in the parental strain 7AGM
was below the detection limit of Western blotting. Lane 1, positive control human
kidney 293 cells, transiently expressing Flag-hRev1; lane 2, control kidney 293
cells; lanes 3 and 11, original parental cell strain 7AGM. 100 pg of protein was
loaded in these lanes. Lanes 4 through 10, kidney 293 cells transiently
expressing Flag-hRev1 protein, but with only 50, 25, 12.5, 6.3, 3.1, 1.5, 0.8 pg of
protein loaded, respectively. The hRev1 protein was probed with rabbit polyclonal
antibody 468. Ku-80 protein was used as the loading control. These blots were

developed using maximum sensitivity chemiluminescence substrate.

166

same conditions, 100 pg of protein prepared from the parental 7AGM cells
yielded no obvious band (Lane 11). Tests showed that use of lysates prepared
from nuclei rather than from whole cells did not produce any advantage (data not

shown).

Cell strains expressing exogenous Flag-hRev1 protein

To circumvent the difficulty of not being able to detect endogenous hRev1
protein on Western blots as a means of determining that the decreased
frequency of mutants induced by UV resulted from a decrease in expression of
hRev1 as a result of antisense RNA-expression, we employed an alternative
strategy. We introduced an exogenous hREV1 gene into the 7AGM-1ZB-R1
antisense RNA-expressing cell strain that showed the greatest reduction in UV
mutagenesis, i.e., 20-fold (Gibbs et al., 2000). If the reduction in mutagenesis in
this antisense RNA-expressing cell strain was caused by lower level of hRev1
protein, expression of exogenous hRev1 protein should increase the UV-
mutability of this cell strain. To facilitate the detection of exogenous protein
expressed in the transfectant clones, we transfected this recipient strain with a
plasmid expressing a hRev1 protein with a Flag-tag fused in-frame at the amino-
terminus. The expression of Flag-hRev1 could be detected by Western blot
analysis using an anti-Flag mouse monoclonal antibody. Following transfection,
~90 blasticidin-resistant transfectant clones were expanded and screened for
expression of Flag-hRev1 protein. A total of 14 clones showed expression of the
exogenous Flag-hRev1 protein. As shown in Figure 2, two cell strains,

designated 7AGM-12B-R1-FR1C1 and 7AGM-12B-R1-FR1CZ, expressed Flag-

167

1 2 3 4 5 6

1.....- """" - my - w

r I "a 9" "'" Flag-hRev1

Figure 2. Western blot analysis of transfected cell strains for the expression of
exogenous Flag-hRev1 protein. Lanes 1 and 2, cell strains transfected with the
vector alone, as a control; lanes 3 and 5, transfectant strains, C1 and CZ,
expressing Flag-hRev1 protein; lane 4, a transfectant that failed to express Flag-
hRev1 protein; lane 6, the positive control, i.e., 293 cells transiently transfected
with FLAG-hREV1 plasmid. The Flag-hRev1 protein was detected using mouse

monoclonal anti-Flag antibody. Ku-80 was used as a loading control.

168

hRev1 at a higher level than other transfectants. These were chosen for further

studies.

Effects of Flag-hRev1 expression on UV-induced mutagenesis

These two transfectant cell strains expressing exogenous Flag-hRev1
protein, hereafter designated C1 and CZ for brevity, were used as target cell
strains in UV-induced mutagenesis assays to determine whether UV-mutability of
the antisense-expressing recipient cell strain could be restored as a result of the
exogenous hRev1 expression. That latter strain, 7AGM-1ZB-R1, hereafter
referred to as 123-R1 for brevity, which expressed high level of antisense hRE V1
RNA, had shown an approximately 95% reduction in frequency of UV-induced
mutants compared to the normal parental strain (Gibbs et al., 2000), a level of
reduction comparable to that seen in a yeast rev1 mutant strain (Lawrence and
Hinkle, 1996). Three independent experiments were carried out to determine the
cytotoxic and mutagenic effects of UV (254nm) in the two transfectant cell
strains. As shown in Figure 3A, the cell strains did not exhibit a significant
change in sensitivity to the cytotoxic effect of UV. This result was not unexpected
because even the strains expressing high levels of antisense hREV1 RNA
showed a survival comparable to the parental 7AGM cell strain (Gibbs et al.,
2000). However, as shown in Figure 3B, the two transfectan strains, C1 and C2,
showed a significantly increased frequency of UV-induced TGr mutants,
compared to their recipient strain, 123-R1. Both of them exhibited approximately
8-fold higher frequency than the parental strain 7AGM-12B-R1, to a level of

~40% of the frequency of the original normal fibroblasts 7AGM. In contrast, cell

169

 

Figure 3. Evidence that expression of Flag-hRev1 protein does not greatly affect
cell survival, but significantly increases the frequency of UV-induced mutants. A)
UV cytotoxicity. B) Frequencies of UV-induced TGr mutants. The data for the
original parental strain 7AGM (A) and the antisense RNA-expressing strain 12B-
R1 (I) from Gibbs et al. (2000) are shown for comparison. (0), cell strain C1; (:1),
cell strain C2. The data shown by open symbols represent three independent
experiments. For the mutagenicity data, the latter are shown as the mean at
standard deviation. The data shown by (0) have been offset slightly from the
dose used, i.e., 13 and 15 J/m2 for clarity. To obtain UV-induced frequencies, the
background frequencies of mutants, which ranged from 0 to 9 per 106 clonable
cells, have been subtracted. The mutant frequencies were corrected for the
cloning efficiency of the cells at the time they were plated into selective medium.
These ranged from ~30% to 90%. The survival curves for strains C1 and CZ are
the same. They are virtually superimposed on the lower curve and have been

omitted for clarity.

170

 

 

0
100 T I l l r I r l I r r I r r I r 1

   

 

 

 

 

 

 

 

 

 

. A ‘
b 1
:alij‘ai;
1.13131 3
.2 * -+ '
alaforf': g
m h -r
straini’é- ‘E
0
2
. 1‘. 0
mo. IL _ -
lasers
' A 7AGM
0 C1
””3“ D 02
I 1ZB-R1
110.11: 101L111lrrlrrl11|l,
IICIeSIE 7oil'j'l'IlIIIIIT1r
B
Claret: m A
g 60 "O 7AGM .q
‘ - C1
Home 2 El 02
"' 1 -R1
~Z~r .3 5° _' 23 _
me... :
.0.
'13,? o
as. o 40 P _
S!
we??? 5
8.
1’ 30 - _
3
0
[—
3 1-
:3 1o - ._
U
s I I
o 1 1 L1 I l j 1 [W

0 3 6 9 12 15 18
uv (Jlmz)

171

strains transfected with an empty vector, or that were transfected with the FLAG-
hREV1 plasmid, but did not express the exogenous protein, remained virtually
immutable (data not shown). These data indicate that the reduced capability of
the antisense RNA-expressing strain in UV-induced mutation can be restored by
exogenous expression of hRev1, strongly suggesting that the function of hREV1

is required for UV-induced mutagenesis.

Effects of Flag-hRev1 expression on BPDE-induced mutagenesis

Both the photoproducts induced by UV light and the bulky adducts formed
by benzo[a]pyrene, including BPDE, cause DNA helix distortion, and these DNA
lesions are repaired by the same repair mechanism, i.e., nucleotide excision
repair (Friedberg et al., 1995). To explore the possibility that the mutagenic effect
of these two kinds of lesions requires the function of the same pathway, i.e., the
pathway involving hREV1, we determined the frequency of BPDE-induced
mutants in the original parental strain 7AGM, strain 1ZB-R1 that expresses a high
level of antisense RNA, and the two transfectant strains that express exogenous
hRev1 protein. As shown in Figure 4A, the cytotoxic effect of BPDE as a function
of dose in the parental strain 7AGM and the antisense RNA-expressing strain,
1ZB-R1, were very similar; that in the two exogenous hRev1 expressing strains,
C1 and C2, was slightly less. However, as shown in Figure 4B, in contrast to the
high frequency of mutants induced by BPDE in the parental fibroblasts, cell strain
7AGM, values similar to those found previously in this laboratory for finite life
span human fibroblasts (Yang, et al., 1982; Aust et al., 1984), the frequency of

induced mutants in 1ZB-R1 was very low, suggesting that the disruption of

172

Figure 4. Evidence that expression of Flag-hRev1 protein does not greatly affect
cell survival but significantly increases the frequency of BPDE-induced mutants.
A) BPDE cytotoxicity. B) Frequencies of BPDE-induced TGr mutants. (A), the
original parental strain 7AGM; (I), the antisense RNA-expressing strain 1ZB-R1;
(0), C1; (a), C2. The data shown by open symbols represent three independent
experiments. For the mutagenicity data, the latter are shown as the mean i
standard deviation. Four data points shown by (I) have been offset slightly from
the dose used, i.e., 0.075, 0.10, and 0.15 pM for clarity. To obtain BPDE-induced
frequencies, the background frequencies of mutants, which ranged from 0 to 4
per 106 clonable cells, have been subtracted. The mutant frequencies were
corrected for the cloning efficiency of the cells at the time they were plated into
selective medium. These ranged from ~30% to 90%. The survival curves for
strains C1 and CZ, and the survival curves for 7AGM and 1ZB-R1 are virtually

the same. Only two lines are drawn for clarity.

173

 

0.00 0.05 0.10 0.15 0.20
100 . r r r r r
_ A
E - -
3
m
‘E " I ‘
d}
2
§ _ A _
A 7AGM
0 c1
1:] c2
I 128-R1
1o 1 l 1 I l 1
150 ' I I l I I 1
- ‘ 7AGM B 1
C1 A
m ~ El cz ~
= I 128-R1 A
o L _
o
2 . _
é ‘ -
2 100 -
o I -
“’2
'5
O. -
fl
3 .
o
('2 50 - -
'5 _ _
0
O
3 _ s
"O
E
o 1 j ' 1
0.00 0.05 0.10 0.15 0.20
BPDEluMI

 

 

 

 

 

 

 

174

hREV1 function by expression of antisense RNA greatly abolished the mutations
induced by BPDE. Conversely, as also shown in Figure 48, with expression of
the exogenous Flag-hRev1 protein, both cell strains C1 and C2 showed a
restoration of BPDE-induced mutagenesis, to a level significantly higher than
their parental strain 1ZB-R1, 5-fold and 3-fold respectively. All the data were
results from at least two independent mutagenesis assays, with selection of 1X
106 cells for each dose. Figure 5 showes the relationship between frequency of
induced mutants and cytotoxicity of BPDE, using the same set of data as used
for Figure 4. The dependence of mutagenesis induced by BPDE on the cells’
functional status of hREV1 strongly supports the indispensable role of hREV1 in

mutagenesis induced by BPDE.

hREV1 is required for the majority of UV- and BPDE-induced base

substitutions

The restoration of UV and BPDE mutability of the antisense RNA-
expressing strain by exogenous expression of Flag-hRev1 protein strongly
indicated that the function of hREV1 had been markedly impaired in this cell
strain, and that the greatly reduced mutation was caused by decreased function
of hREV1. The fact that cell strain 1ZB-R1 exhibited an ~ 94% reduction in
frequency of UV-induced mutants indicated that the function of hREV1 is
required for the vast majority of mutations induced by UV. In finite life span
foreskin-derived human fibroblast cells, the vast majority of mutations induced by
UV are base substitutions, with about 50%-60% being C->T transitions

(McGregor et al., 1991; Wang et al., 1993). These data, along with the data in

175

160

 

 

 

 

 

A7AGM
001
1:02
I1ZB-R1 100%
A
:13 1
8 120 -
2
1%
1: A
2
0 A
"*3
h
a 80 +
=;."-’ A
d)
O
“o A
I-
8 29%
3 4o -
1:
E
13%
4%
10
Percent Survival

Figure 5. Frequency of mutants induced by BPDE expressed as a function of
percent survival of the target cells. This figure shows all the data points used for
preparing Figure 4. They demonstrate the degree of increase in mutagenesis
observed when exogenous hRev1 protein was expressed in the hREV1
antisense-expressing recipient cell strain, 12B-R1. (A), 7AGM; (I), 1ZB-R1; (0),
C1; (1:1), CZ.

176

Figure 3B, predict that hRev1 is involved in generation of C->T transitions.
Similarly, the fact that the cell strain 1ZB-R1 also showed a ~96% reduction in
the frequency of BPDE-induced mutants predicts that hREV1 is involved in the
vast majority of mutations induced by BPDE.

To test this prediction for BPDE, we sequenced the HPRTcDNA of BPDE-
induced HPRT mutant clones derived from the cell strain C1, which expressed
exogenous Flag-hRev1 protein. If hRev1 is required for nearly all BPDE-induced
mutagenesis, the kinds of mutations generated in cell strains expressing
exogenous Flag-hRev1 should resemble those of the original parental strain
7AGM. We first obtained the spectrum of BPDE-induced HPRT mutants derived
from asynchronously growing cells of the original parental strain 7AGM, an
immortal, near-diploid fibroblast strain, and compared with that of a normal
human fibroblast strain previously obtained in this laboratory (Yang et al., 1991).
In asynchronously growing normal diploid human fibroblasts, the majority of
mutations induced by BPDE are base substitutions, with approximately 94% of
them involving guanine, primarily G-)T transveslons and ~5% involving adenine
(Yang et al., 1991). As summarized in Table 1, the mutation spectrum induced by
BPDE in 7AGM resembled that of the normal fibroblast strain (Yang et al., 1991).
Approximately 80% of these base substitutions are transversions and the most
predominant transversion is G-)T transversion. As expected, sequencing of the
BPDE-induced HPRT mutants derived from the Flag-hRev1 expressing strain C1
revealed a very similar spectrum. The similarity of mutation spectrum from C1

and that of its parental strain 7AGM, along with the data shown in Figure 4B and

177

5, clearly demonstrates that the function of hREV1 is required for virtually all

BPDE-induced mutageneis.

Discussion

The difficulty of detecting hRev1 protein might reflect the low expression
level of hRev1 protein in human cells. Indeed, the 5' untranslated region of
hREV1 mRNA contains an out-of-frame start codon, which would initiate the
translation of an unrelated small peptide and would severely reduce the
translation efficiency of full-length hRev1 protein (Gibbs et al., 2000; Lin et al.,
1999b). hREV3, believed to function in the same pathway as hREV1, also has
properties in its mRNA that would greatly reduce the translation efficiency of the
hRev3 protein. Such properties are the presence of an out-of-frame start codon
(Gibbs et al., 1998), and the propensity of forming a stem-loop structure at the 5’
untranslated region (Lin et al., 1999a). As a consequence, hRev3 protein might
also be very low. The low expression level of Rev1 and Rev3 proteins appears to
be evolutionarily conserved, because the yeast REV3 gene also has an out-of-
frame ATG start codon (Morrison et al., 1989), and efforts to detect endogenous
yeast Rev3 or Rev1 proteins using antibody-based techniques were not
successful either (Lawrence, unpublished studies).

The biochemical studies of many proteins involved in mutagenesis have
been greatly facilitated by the use of protein tags and their corresponding
commercially available ligands that the tags bind to and/or high-quality
monoclonal antibodies. Fusion of a glutathione S-transferase (GST) tag to the N-

terminus of yeast Rev3 protein allowed affinity purification of sufficient amount of

178

~.- $81.43 ..~ _.

.comtmano .3 2o; 633.05 2m 65 .28: ._m up 95>

.3 b05593 £5 E co>_..ou 903 6:: :8 3385.... SEE .mcto: cmam or. 9E: usEonéEmQQ m .8 .53 owes... ..

 

 

 

 

 

NN 2 «N .93
3.3 P 3;: N 3.9 N o t <
35: o 3;: N 35V N < A. o
3.NNV N 3N8 v 3.5 4 22.23
3.9 F 3.8 F 33 F e A: <

o 3.3 N 3.9 N o ¢<
3.5 m. 3.5 N 3.9 N o t o
3&9 2 3.3 a 3:9 3 e A. o
3.3 N 3.5 3 3.8V 2 2229,23

.2: :8 9:5 35.02 .29: ass... was... BEHEANFéofi 82.3383...

Us uoaomno Amcozazmnzm __m E 3.9 mcozazmnam mama so .3832 ommm

 

 

.mammfiofic 5:5; 2036 cam 95 2E: new 59%

REES N. Tam—2 9: E coo-65 muss. 05 53> £905 gem: ommmfléml maocomoxo om_m Sn .<zm omcomzcm {Ema

mmoaxo “m5 m__oo so ocom HQQI 05 E wodm .3 6835 20:32.33 owns .6 mac: 9: ho comtmanO ._. oEmb

179

Rev3 protein, and helped elucidate the interaction of Rev7 to Rev3 and the
formation of the Pol zeta entity by this interaction (Nelson et al., 1996a). The
deoxycytidyl transferase activity of yeast Rev1 protein was also originally
identified using recombinant Rev1 protein fused in-frame with GST tag at the N-
terminus and purified with glutathione-sepharose columns (Nelson et al., 1996b).
In the characterization of human Rev1 protein (hRev1), Lin et al. (1999b)
expressed and purified a recombinant hRev1 protein fused with six histidine
residues at its N-terminus as well. The data indicate that these proteins, tagged
at the N-terminus, retained their functions. The present study was designed to
determine whether the reduced mutagenesis observed with cell strains
expressing antisense hREV1 RNA was a result of a loss of hREV1 function. We
introduced expression of an exogenous hRev1 protein in one of these strains. To
facilitate the detection of the exogenous protein, we expressed hRev1 protein
tagged with the Flag tag at the N-terrninus. Evidence that this fusion protein
retains its wild-type conformation comes from its ability to bind hRev7 protein
shown by co-immunoprecipitation using anti-Flag monoclonal antibody
(Murakumo et al., 2001).

The present studies with two derivative cell strains, showing that
expression of this exogenous tagged hRev1 protein partially restored the
mutability of the recipient cell strain expressing antisense RNA, strongly support
a role for hREV1 in mutagenesis induced by UV and BPDE. The fact that
expression of hRev1 did not increase the frequency of UV-induced mutants to

the level seen in the parental cell strain 7AGM, but only to 40% of the value,

180

probably reflects the continued expression of hREV1 antisense in these two cell
strains. Alternatively, the transfectants might simply not have produced as much
protein, or activity of the recombinant fusion protein in participating in translesion
synthesis is less than that of endogenous hRev1 protein. The observation that
the increase in the frequency of mutants induced by BPDE in the same
exogenous hRev1-expressing strains is not quite as high as that induced by UV
might indicate that the tagged hRev1 protein participates better in bypassing UV
photoproducts than BPDE adducts. Nevertheless, the reduction of mutation
frequency in cell strains expressing antisense RNA and the reversal of this
reduction by exogenous hRev1 protein clearly indicate the requirement for
hRE V1 in damage-induced mutagenesis.

The human homolog of yeast REV1 (hREV1) appeared to have functions
similar to the yeast gene. The human Rev1 protein (hRev1) has a specific G
template-dependent transferase activity (Lin et al., 1999b), and the ability to
incorporate dCMP opposite abasic sites (Lin et al., 1999b). Our previous studies
showed that human fibroblasts expressing high level of antisense RNA of hRE V1
(Gibbs et al., 2000) and hREV3 (Gibbs et al., 1998), a gene believed to function
in the same pathway as hREV1, exhibited greatly reduced mutation frequency
induced by UV. Very recently, hRev1 protein has been shown to incorporate
dCMP opposite damaged guanines, including 8-oxoguanine and the bulky
guanine adduct formed by benzo[a]pyrene in in vitro primer extension assays
(Zhang et al., 2002). If, in human cells, hRev1 protein inserts dCMP opposite

template G damaged by benzo[a]pyrene, and DNA polymerase Pol kappa

181

extends from the 3' -OH, as demonstrated by in vitro primer extension assay
(Zhang et al., 2002), this bypassing will be error-free, as C is the correct base
pair for G. Then, reduction in the function of hREV1 will cause an increase in
BPDE-induced mutagenesis. However, we did not obtain such a result. Our data
showed that expression of antisense RNA greatly reduced the frequency of
BPDE-induced mutants, and expression of exogenous hRev1 protein in this
antisense-expressing strain restored the frequency of mutants, strongly
suggesting that hRev1 is involved in an error-prone mechanism. Sequencing of
BPDE-induced mutants also showed that hRev1 is required for the vast majority
of base substitutions induced by BPDE. The error-free bypassing of BPDE
adduct by hRev1 protein might play a minor role in human cells.

In summary, by introducing expression of exogenous hRev1 protein in the
cell strain expressing antisense hREV1 RNA and seeing that this increased the
frequency of mutants 8-fold, we confirmed our previous conclusion that the
function of hREV1 is required for UV-induced mutagenesis. In the present study,
our data with the cell strain expressing high level of antisense (123-R1)
exhibiting a 96% reduction in the frequency of mutants induced by BPDE and the
reversal of this reduction by exogenous hRev1 protein also indicated that hREV1
is involved in BPDE-induced mutagenesis. Furthermore, we provided evidence
that hREV1 is involved in generating nearly all kinds of base substitutions

induced by BPDE.

Acknowledgements

182

We thank Dr. Yun Wang for helpful discussion. This research was
supported by US Public Health Service grants GM21858 (to C.W.L.) and
Department of Health and Human Services and National Institutes of Health

Grants ES-09822 and CA 91490 (to V.M.M.).

References

Aust AE, Drinkwater NR, Debien K, Maher VM, McCormick JJ. (1984)
Comparison of the frequency of diphtheria toxin and thioguanine
resistance induced by a series of carcinogens to analyze their mutational
specificities in diploid human fibroblasts. Mutat Res., 125295-104.

Friedberg, E.C., Walker,G.C. and Siede,W. (1995) DNA Repair and
Mutagenesis, American Society of Microbiology Press, Washington, DC.

Gibbs PE, McGregor WG, Maher VM, Nisson P, Lawrence CW. (1998) A human
homolog of the Saccharomyces cerevisiae REV3 gene, which encodes
the catalytic subunit of DNA polymerase zeta. Proc Natl Acad Sci U S A.
95:6876-80.

Gibbs PE, Wang XD, Li Z, McManus TP, McGregor WG, Lawrence CW, Maher
VM. (2000) The function of the human homolog of Saccharomyces
cerevisiae REV1 is required for mutagenesis induced by UV light. Proc
Natl Acad Sci U S A. 97:4186-91.

Lawrence CW, Hinkle DC. (1996) DNA polymerase zeta and the control of DNA
damage induced mutagenesis in eukaryotes. Cancer Surv. 28:21-31.

Li Z, Xiao W, McCormick JJ, Maher VM. (2002) Identification of a protein
essential for a major pathway used by human cells to avoid UV- induced
DNA damage. Proc Natl Acad Sci U S A. 99:4459-64.

Lin W, Wu X, Wang Z. (1999a) A full-length cDNA of hREV3 is predicted to
encode DNA polymerase zeta for damage-induced mutagenesis in
humans. Mutat Res. 433:89-98.

Lin W, Xin H, Zhang Y, Wu X, Yuan F, Wang Z. (1999b) The human REV1 gene
codes for a DNA template-dependent dCMP transferase. Nucleic Acids
Res. 27:4468-75.

McGregor WG, Chen RH, Lukash L, Maher VM, McCormick JJ. (1991) Cell
cycle-dependent strand bias for UV-induced mutations in the transcribed

183

strand of excision repair-proficient human fibroblasts but not in repair-
deficient cells. Mol Cell Biol 11:1927-34.

Montgomery RA, Dietz HC. (1997) Inhibition of fibrillin 1 expression using U1
snRNA as a vehicle for the presentation of antisense targeting sequence.
Hum Mol Genet. 6:519-25.

Morgan TL, Yang DJ, Fry DG, Hurlin PJ, Kohler SK, Maher VM, McCormick JJ.
(1991) Characteristics of an infinite life span diploid human fibroblast cell
strain and a near-diploid strain arising from a clone of cells expressing a
transfected v-myc oncogene. Exp Cell Res. 197:125-36.

Morrison A, Christensen RB, Alley J, Beck AK, Bernstine EG, Lemontt JF,
Lawrence CW. (1989) REV3, a Saccharomyces cerevisiae gene whose
function is required for induced mutagenesis, is predicted to encode a
nonessential DNA polymerase. J Bacteriol 171:5659-67.

Murakumo Y, Roth T, Ishii H, Rasio D, Numata S, Croce CM, Fishel R. (2000) A
human REV7 homolog that interacts with the polymerase zeta catalytic
subunit hREV3 and the spindle assembly checkpoint protein hMADZ. J
Biol Chem. 275:4391-7.

Murakumo Y, Ogura Y, Ishii H, Numata S, lchihara M, Croce CM, Fishel R,
Takahashi M. (2001) Interactions in the error-prone postreplication repair
proteins hREV1, hREV3, and hREV7. J Biol Chem 276:35644-51.

Nelson JR, Lawrence CW, Hinkle DC. (1996a) Thymine-thymine dimer bypass
by yeast DNA polymerase zeta. Science. 272:1646-9.

Nelson JR, Lawrence CW, Hinkle DC. (1996b) Deoxycytidyl transferase activity
of yeast REV1 protein. Nature. 382:729-31.

Nelson JR, Gibbs PE, Nowicka AM, Hinkle DC, Lawrence CW. (2000) Evidence
for a second function for Saccharomyces cerevisiae Rev1 p. Mol Microbiol.
37:549-54.

Qing, J., Maher, V.M., Tran, H., Argraves, W.S., Dunstan, R.W., McCormick, J.J.
Suppression of anchorage-independent growth and matrigel invasion and
delayed tumor formation by elevated expression of fibulin-1D in human
fibrosarcoma-derived cell lines. Oncogene, 15: 2159-2168, 1997.

Wang YC, Maher VM, Mitchell DL, McCormick JJ. (1993) Evidence from
mutation spectra that the UV hyperrnutability of xeroderma pigmentosum
variant cells reflects abnormal, error-prone replication on a template
containing photoproducts. Mol Cell Biol., 13:4276-83.

Xiao W, Lechler T, Chow BL, Fontanie T, Agustus M, Carter KC, Wei YF. (1998)
Identification, chromosomal mapping and tissue-specific expression of

184

Fr“.

 

hREV3 encoding a putative human DNA polymerase zeta.
Carcinogenesis, 19:945-9.

Yang LL, Maher VM, McCormick JJ. (1982) Relationship between excision repair
and the cytotoxic and mutagenic effect of the 'anti' 7,8-diol-9,10-epoxide of
benzo[a]pyrene in human cells. Mutat Res., 942435-47.

Yang JL, Maher VM, McCormick JJ. (1989) Amplification and direct nucleotide
sequencing of cDNA from the lysate of low numbers of diploid human
cells. Gene. 832347-54.

Yang JL, Chen RH, Maher VM, McCormick JJ. (1991) Kinds and location of
mutations induced by (i)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-
epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene in the coding region of the
hypoxanthine (guanine) phosphoribosyltransferase gene in diploid human
fibroblasts. Carcinogenesis. 12271-5.

Zhang Y, Wu X, Rechkoblit O, Geacintov NE, Taylor JS, Wang Z. (2002)
Response of human REV1 to different DNA damage: preferential dCMP
insertion opposite the lesion. Nucleic Acids Res 30:1630-8.

185

APPENDIX

Roles of DNA Polymerase zeta and Rev1 Protein in Eukaryotic Mutagenesis

and Translesion Replication

C.W. LAWRENCE: P.E.M. GIBBS,* R.S. MURANTE,* X.-D. WANG} 2. LI,‘ T.P.
MCMANUS,T W.G. MCGREGORF J.R.NELSON,* o.c. HINKLE,‘ V.M. MAHER"
*Department of Biochemistry and Biophysics, University of Rochester School of
Medicine and Dentistry, Rochester, NY 14642; 7Carcinogenesis Laboratory,
Department of Microbiology and Department of Biochemistry, Michigan State
University, East Lansing, MI 48824; ‘Department of Biology, College of Arts and

Sciences, University of Rochester, Rochester NY 14627

Running Head: DNA polymerase zeta and Rev1 protein
Corresponding author: Christopher W Lawrence

Department of Biochemistry and Biophysics

University of Rochester Medical Center

601 Elmwood Avenue

Rochester, NY 14642

Phone: (716) 275-2948

Fax: (716) 275—6007

email: christopher_lawrence@urmc.rochester.edu

186

In addition to mechanisms that specifically repair DNA, such as those that
excise damaged nucleotides or bases, eukaryotes possess several processes
that promote the tolerance of unrepaired DNA damage. These processes are
concerned with overcoming a potentially lethal property of unrepaired template
damage, namely its propensity to block or severely impede DNA replication. One
of the strategies used to overcome this inhibition is translesion replication, in
which extension of the nascent strand at blocked forks is promoted by means of
specialized DNA polymerases that are presumably more tolerant of alterations in
primer/ template structure than the normal replicases. In budding yeast, the
eukaryote in which tolerance mechanisms have been most intensively studied,
the major process for translesion replication employs products encoded by the
REV1, REV3, and REV7 genes (Lemontt 1971; Lawrence et al. 1985a,b). Rev3p
is the catalytic subunit of DNA polymerase zeta (Morrison et al. 1989; Nelson et
al. 1996a), a distant relative of DNA polymerase delta (Braithwaite and lto 1993),
and Rev7p is another subunit of pol zeta (Torpey et al. 1994; Nelson et al.
1996a). Rev1p contains domains found in a superfamily that includes the E. coli
UmuC and DinB gene products, the DinB homolog encoded by yeast RAD30,
and the products of several mammalian RAD30 homologs (reviewed in
Woodgate 1999; Gerlach et al 1999). Although the process employing the pol
zeta and Rev1p enzymes provides only a modest fraction of the capability of
yeast to tolerate unrepaired damage, the activities of these enzymes are a major
source of both spontaneous and induced mutations. For the most part, pol zeta

and Rev1p are responsible for the production of basepair substitutions and single

187

nucleotide frameshifts, rather than larger additions or deletions, and their
activities are usually the principal source of single nucleotide alterations. Greater
than 98% of the basepair substitutions induced by UV irradiation, and > 90% of
the UV-induced frameshift mutations depend on the function of the pol zeta, for
example (Lawrence and Christensen 1979; Lawrence et al. 1984). Rev1p

function is responsible for > 94% of UV-induced basepair substitutions, but

appears to have less involvement in the induction of frameshift mutations, at

least at some genetic sites (Lawrence and Christensen 1978; Lawrence et al.
1984). Although less well investigated, the mutagenic effectiveness of other
mutagens such as ionizing radiation (McKee and Lawrence 1979) and chemical
agents (Lawrence et al 1985b) also depend substantially on pol zeta and Rev1p
function. In addition, pol zeta is responsible for between 50% and 75% of
spontaneous mutations (Quah et al. 1980), for the increased spontaneous
mutation resulting from transcription (Datta and Jinks-Robertson 1995),
recombination (Holbeck and Strathern 1995), and the overexpression of 3-methyl
adenine glycosylase (Glassner et al. 1998), and for a substantial fraction of the
enhanced spontaneous mutagenesis in an excision-defective strain (Roche et al
1994). Genetic evidence suggests that, aside from translesion replication, pol
zeta and Rev1p play no part in any other replication, recombination, or repair
activity. Recent evidence suggests that yeast possesses a second translesion
pathway, which employs DNA polymerase eta (Johnson et al. 1999), encoded by

RADSO (McDonald et al. 1997). A comparison between the phenotypes of

188

T"

RAD30 and REV mutants indicates that pol eta has a relatively restricted range
of substrates.

Translesion replication is one of several DNA damage tolerance activities
carried out by the yeast RAD6 pathway. The activities of this pathway are often
referred to as post replication repair, a double misnomer because they do not
repair DNA and they do not necessarily occur after replication in excision repair
proficient cells; they are more accurately called mechanisms for the restoration of
replication competence (Lawrence 1994). The mutant phenotypes of the RADG
and RAD18 genes, which encompass those of all other genes in the RADG
epistasis group and are the most extreme (Lawrence et al. 1974, Cassier-
Chauvat and Fabre 1991), suggesting that they regulate the pathway (McKee
and Lawrence 1979). Rad6p, which is an E2 ubiquitin conjugase (Jentsch et al.
1987) and Rad18p, which binds single-stranded DNA (Jones et al 1988), form a
heterodimer that perhaps targets the conjugase activity to sites of unrepaired
damage (Bailly et al. 1994). The DNA polymerase zeta l Rev1p translesion
replication subpathway also appears to be regulated by the checkpoint genes
RADQ, RAD17, RAD24, and MEC3 because, like the REV loci, deletion mutants
of these genes are deficient in UV-induced mutagenesis (Paulovich et al. 1998).
A plausible, but as yet untested, hypothesis is that the checkpoint gene products
are part of a system that monitors the status of replication forks, and activates
the pol zeta / Rad1p subpathway when forks are blocked. If this is correct,
ubiquitination of some target protein is presumably part of the monitoring

process. Unlike the bacterial SOS system, there is little evidence for

189

 

transcriptional control of this subpathway in yeast (Larimer et al. 1989; Singhal et

al. 1992).

PROPERTIES OF YEAST POL zeta AND Rev1p

The properties of pol zeta have been investigated by co-expressing a
glutathione-S—transferase-Rev3 fusion protein (Gst-Rev3p) and normal Rev7p in
yeast, and purifying the complex by affinity chromatography on glutathione-
Sepharose (Nelson et al. 1996a). Although Gst-Rev3p alone shows a low level of
DNA polymerase activity, the Gst-Rev3p/Rev7p complex is 20- to 30-fold more
active. A plasmid expressing Gst-Rev3p fully complements the mutant phenotype
of a rev3 deletion strain, indicating that the fusion protein retains a substantial
level of activity in vivo, and cleavage of the Gst moiety from the fusion protein
only increased its activity by about two-fold. Since pol zeta has not yet been
purified from cells without overexpression of Rev3p and Rev7p, we cannot rule
out the possibility that the native enzyme contains additional subunits.

Pol zeta is a poorly processive enzyme, dissociating from half of the
template molecules after adding only 5 3 nucleotides under conditions in which
extension mostly resulted from a single polymerase binding event, though in
some instances up to ~ 200 nucleotides were added. Pol zeta does not possess
a 3' to 5' exonuclease proofreading activity, and is relatively insensitive to
aphidicolin (> 90% activity at 200 mM) and dideoxynucleotide triphosphates (>
90% activity at 100 pM), and only moderately sensitive to butylphenyl-guanosine
triphosphate (70% activity at 10 pM). Of particular relevance to its role in

translesion replication, pol zeta appeares to be much more tolerant of abnormal

190

Faun..-

structure in the template and at the primer terminus than the major replicases.
For example, under conditions apparently involving only a single binding event,
pol zeta extended the primer past a thymine-thymine cis-syn cyclobutane dimer
with ~ 10% efficiency but with yeast DNA polymerase alpha the efficiency was <
1% (Nelson et al. 1996a). Although, as described below, it has subsequently
been discovered that pol zeta is unlikely to be primarily responsible for dimer
bypass in vivo, such a result nevertheless suggest that pol zeta possesses a
superior capability for tolerating template distortion. In addition, pol zeta extends
a terminally mismatched primer more efficiently than pol alpha on dimer-free
templates, again indicating a reduced stringency for normal duplex structure at
the primer terminus (T able1). Apparent extension efficiencies were determined
for all combinations of terminal nucleotide in the primer paired or mispaired with
each nucleotide in the template, using templates in which the next nucleotide
beyond the primer terminus was either dA or dG, and extension driven by
increasing concentrations of d‘lTP or dCTP respectively. Extension efficiencies
with pol zeta were from two-fold to more than 1000-fold higher than for pol alpha
for any given mismatch. For pol zeta the actual efficiencies ranged from 0.3% to
54%, whereas for pol alpha the range was 0.002% to 3%. It appears that pol zeta
is less accurate than the major replicases not only by virtue of a lack of
proofreading, but also because it lacks some of the stringent requirements for
correct primer/template structure.

As shown by the REV1 mutant phenotype, lesion bypass by pol zeta also

requires the function of Rev1p. To date, the clearest enzymatic function

191

 

.oFmEEoEoEta .._..m ..

 

 

192

 

 

 

 

ONN.F N 99 x m0 NbF x o.m N-oF x 0m N-oF x Em ._..._.
moF Fm 99 x N.N NbF x NF F-oF x md 1: x in ._..0
m N 99 x Qm NbF x o.N NbF x N.N NbF x N6 ._..0
F F F F ._..<
FN o 93 x Om L: x m.F NbF x cs 1: x F.N 0;.
m? mmN.F .19 x 0v 9? x m.» F-oF x oN NbF x md 0.0
F» mmN 99 x N.N 19 x mi F.oF x oN :9 x m.F 0.<
F F F F 0.0
mm mm F.oF x o.N :9 x ed F.oF x m.F NbF x «.0 09
8F N 93 x 0F FroF x N.N n.oF x F.N n.oF x N.N 0.0
mm 0 ..OF x Om NbF x o.F F.oF x N.N NbF x Em 0.<
F F F F 0.0
NFm mam 99 x F.N 93 x 0+ N-oF x m.F N-oF x F.v <.0
NF m n.oF x mN 99 x 0m NbF x F.F. NbF x N.N <.0
mum mm #9me 0.9an P.oFx F.F F.oFxo.F <.<
F F F F <.._.
n_._.0FU nth 3.06 nth 3.06 Fr?
22.... .0933 .8 2E. 6565.0 3:905... c2228 Eofiaam 55656 53:38 E283 bun: Ema
29m ommLoEzoa <20 93 omEoEboa <20 ommn _mc_FEo._.
3230 so. \ v.25
“5:20.96 c2238 Eofiaa<
22.350 ex :25

 

mocoFmEmE gmaémmn .mEEoF 29.6 Eo: 22m .3 .0 ES .8 .3 c2238 .8 365850 E9maa< .F oEmP

associated with this protein is a deoxycytidyl transferase activity which inserts
dCMP opposite an abasic site in the template and, with a much reduced
efficiency, opposite template guanine and adenine nucleotides (Nelson et al.
1996b). No evidence was found to indicate utilization of any nucleotide
triphosphate other than dCTP. As shown below, this activity is responsible for the
preferential insertion of dCMP opposite abasic sites in vivo that had been
observed previously (Gibbs and Lawrence 1995); unlike E. coli, in which an “A-
rule” for such insertion is observed (Lawrence et al. 1990), yeast exhibits a “C-
rule”. Replication past an abasic site in vitro by pol zeta is strongly stimulated by
Rev1p (Nelson et al. 1996b), and the same is probably true in vivo. The Rev1p
dC transferase activity has received the greatest attention, in large part perhaps
because other members of the Rev1/UmuC/DinD/Rad30 superfamily have
subsequently been found to be DNA polymerases (reviewed in Woodgate 1999;
Gerlach et al. 1999). Nevertheless, the transferase activity is unlikely to
constitute the Rev1p function required for the bypass of most lesions other than
abasic sites, and the protein appears to possess a second activity whose
enzymatic basis is at present poorly understood.

Evidence pointing to the existence of this second function includes the
observations that Rev1p-dependent bypass of a thymine-thymine pyrimidine (6-
4) pyrimidinone UV photoproduct in vivo does not entail the incorporation of dC,
and that bypass of an abasic site in a rev1-1 mutant is much reduced, even
though Rev1-1p retains a substantial fraction of the normal level of (10

transferase activity, which bypass of an abasic site is expected to require (Nelson

193

 

 

et al. 2000). In these experiments, the frequencies of lesion bypass were
estimated by transforming REV" or rev mutant yeast strains with duplex DNA
plasmids that carried either a T-T (6-4) photoadduct, a T-T cyclobutane dimer, or
abasic site centrally located in a 28 nucleotide single-stranded region. The
number of these transforrnant colonies, normalized to the number obtained with a
lesion-free but otherwise identical sample of control plasmid, provided an
estimate of bypass frequency, since replication past the lesion and gap-filling is a
necessary precursor to the creation of transformants. Replicated plasmid
products were then analyzed to identify the nucleotides inserted opposite the
lesion.

As shown in Table 2, the presence of a T-T (6-4) photoproduct in the
plasmid strongly inhibits gap filling, and only 17% of the plasmids were
completely replicated in a REV” strain. Lesion bypass in the successfully
replicated plasmids was largely dependent on Rev1 p, since the fraction of fully
replicated plasmids fell to 1% in the rev1 deletion strain. Incorporation of dCMP
opposite one or the other of the thymine nucleotides occurred in only 1 of 72
REV+ transforrnants analyzed, however, suggesting that the transferase activity
was at best rarely employed during bypass of the T-T (6-4) lesion. A low
frequency of dCMP insertion was also seen in a previous study (Gibbs et al.
1995). Bypass of the T-T (6-4) photoproduct was also much reduced in the rev1-
1 (G193R) and rev3 deletion strains. Interestingly, neither Rev1p nor Rev3p

function appeared to be required for the bypass of a T-T cyclobutane dimer,

194

 

 

 

m o 8 F 0 ad NF Yo c0328 «38
v o mm mm flu om md TEE
mF o 2: o vd mu o.F cozmfiv 39
w F o F F mm mm mm F F 5mm
8%.... .26 $3. mzoo $83 as
59:32 $.33 metau Brows 02820:: $393 .5 Amman? $9 $96
93 282 .055 F.F :19 F-F .6 3288

 

63> Fe 235 53.3 m>$ cam .F-F>9 603200 32 .3mm E 8:26

ocoEEEEQ :18 mEEEEE ._...._. new .886 0:32.228 Finds W... 6% 223m an F0 amoeba—um: mmmgm .N 038.

195

indicating that bypass of this lesion is carried out by another DNA polymerase,
most probably by pol eta (Johnson et al. 1999).

As expected, since dCMP is incorporated preferentially in vivo (Gibbs and
Lawrence 1995), bypass of an abasic site during gap-filling was also found to be
Rev1p-dependent. Twenty three percent of plasmid molecules were fully
replicated in the REV strain, but only 0.4% in a rev1 deletion strain. In keeping
with the earlier study, dCMP was incorporated opposite the abasic site in 89% of
the bypass events in the REV strain (Table 2), but such insertion was not
observed in the few bypass events occurring in the rev1 deletion strain, indicating
that this event is Rev1p dependent. Significantly, however, reduced bypass and
gap filling (2.3%) also occurs in the rev1-1 mutant strain. The G193R rev1-1
mutant protein retains about 60% of the wildtype Rev1p dC transferase activity
(Figure 1), and bypass events in the rev1-1 mutant entailed the incorporporation
of dCMP in roughly the same proportion as seen in the REV strain (Table 2). It
therefore appears that bypass of an abasic site requires both the transferase and
the second Rev1 p function.

The enzymatic basis of this second Rev1p function is not known.
Simultaneous overproduction of Rev1p, Gst-Rev3p, and Rev7p in yeast, followed
by glutathione sepharose chromatography of cleared cell extracts, provides no
evidence for the association of Rev1p with pol zeta. However, as noted above,
the native enzyme may embody other subunits in addition to Rev3p and Rev7p,
and one of these may mediate a Rev1p association. Alternatively, pol zeta and

Rev1p may be independently recruited to a stalled replication fork, resulting in a

196

TC C
AGGOTGAAC

30
1 50

rev1-1mg) o —> o to

 

50

REV1(“Q) o to 8 ‘- o—b

21nt—>

20 nt —>

 

rF o co co_ co
Product <- o to Q 0: Is
F a) a) O) IN 0')
‘- F 1- 6 (O 1- ‘-

(fmoles) 0

Figure 1. Assay of dCMP transferase activity for Gst-Rev1p and Gst-Rev1-1p
fusion proteins. These activities insert dCMP opposite the abasic site in the
template, designated 0, extending the 20 nucleotide 32F end labelled primer by
one nucleotide. Amounts of the 21 nucleotide product were estimated by

phosphorimaging.

197

transient association. Whatever the nature of the association, Rev1p is likely to
facilitate nucleotide insertion and primer extension by pol zeta, since genetic
evidence suggests that the second function is more concerned with base

substitution mutagenesis than with misalignment and frameshift mutagenesis.

FUNCTIONS OF POL zeta AND REV1p IN HUMANS

Saccharomyces cerevisiae has proved to be a good model for mammalian
repair functions, and repair proteins are often conserved from budding yeast to
humans. We have therefore used the yeast Rev1 and Rev3 protein sequences
to isolate cDNA clones that encode their human counterparts (Gibbs et al. 1998;
2000). The human REV3 gene encodes an expected protein of 353kD, about
twice the mass of yeast REV3 (173kD), with regions of significant identity
principally at the amino terminus and at the carboxy terminus, which is the
polymerase domain. Similar regions of homology are found in REV3 genes from
other organisms (Figure 2). The polymerase domain is more closely related to
that of yeast than to those in the human replicases, and is clearly of the pol zeta
type. About 40% of human REV3 mRNA contains a 128 nucleotide insertion,
probably the result of alternate splicing, at position +139/140 of the open reading
frame which introduces a stop codon, and upstream out-of-frame ATG codons,
features indicating that, as with yeast, cellular levels of Rev3p are probably quite
low. The human REV1 gene encodes a 138kD protein with significant identity to
the 112kD yeast protein over the amino two-thirds of the protein (Gibbs et al.
2000). Alignments with the Rev1p from other species (Figure 3) identify 9

sequence motifs, the first of which lies in a region with weak homology to the

198

Figure 2. Sequence comparisons of conserved regions in the amino- and
carboxy-terminus of Rev3p from various species. Sc, Saccharomyces cerevisiae
(Morrison et al. 1989); Sp, Schizosaccharomyces pombe (accession
No.AL355632); Hs, Homo sapiens (accession No. AF058701); Mm, Mus
musculus (accession No. AF083465); Dm, Drosophila melanogaster (J. Eeken,
P. van Sloun, and R. Romeijn, unpubl.); At, Arabidopsis thaliana (accession No.
AC011020); Lm, Leishmania major (accession No. AL122012). Residues

conserved in at least 5 of the sequences are highlighted.

199

 

ScREV3
SpREV3
HsREV3
MmREV3
DmREV3
IHREV3
LmREV3

ScREV3
SpREV3
HsREV3
MmREV3
DmREV3
AtREV3
LmREV3

ScREV3
SpREV3
HsREV3
MmREV3
DmREV3
AlREV3
LmREV3

ScREV3
SpREV3
HsREV3
MmREV3
DmREV3
AlREV3
LmREV3

ScREV3
SpREV3
HsREV3
MmREV3
DmREV3
AtREV3
LmREV3

ScREV3
SpREV3
HsREV3
MmREV3
DmREV3
AtREV3
LmREV3

ScREV3
SpREV3
HsREV3
MmREV3
DmREV3
AtREV3
LmREV3

SCREW
SpREV3
HsREV3
MmREV3
DmREV3
AlREV3
LmREV3

ScREV3
SpREV3
HsREV3
MmREV3
DmREV3
AtREV3
LmREV3

ScREV3
SpREV3
HsREV3
MmREV3
DmREV3
AtREV3
LmREV3

YFRIOLNNQDYYHSKPTFLDPSHGESLPLNOFSQVPNIRVFGALPTGHQVLCHVHGILPYMFIKYDGQITDTSTLRHORCAOVHKTLEVKIRASFKRKKDDKH
2 RF5IEYIDWELSPCDPAYDFVGKDLPTRNEELTTVPVIRVFGLNEEAETVCCFIRNVFPYIYVEYSSFABTLDLEVPDFLSOLOTSINYALALAAR .......
1 MFSVRIVTRDYYHASPLOGLDTCOSPLTOAPVKKVPVVRVFGATPAGOKTCLHLHGIFPYLYVPYDGYGQOPESYLSOMAFSIDRALN..VAL ..........
1 MFSVRIVTRDYYMASPLPGLDTCQSPLTOLPVKKVPVYRVFGATPRGQKTCLHLHGIFPYLYYPYIGYGQQPESYLSOHAFSIDRALN..
VYSVRLVIRDFYMEKPQFGMDPCLFGAARKE1KRVPVIRVFGGNSRGQKTCHHTHGVFPYLYIPYDKKDLSPGAGHPADGRAPDKAIN..ISL ..........
VFSLRIVSIDYYMASPIPGYNICYSSFQGSEVNEVPVIRIYGSTPAGOKTCLHIHRALPYLYIPCSEIPLEHHKGVDGSTLALSLELE..KALKLK .......
SSSFPPPCSNLCLATSAIGSDGDPLILTGGSLGSSAANR..GGYTQ.RRACLHVHGVYPSLLLPQYDRNVSAD ................ QLAAQLEAVALRVL

219
191
183
183
195
200
342

DLAGDKLGNLNFVADVSVVKGIPFYGYHVGWNLFYKISLL ..... NPSCLSRISELIRDGK.
.ANPETYKPAVOSVOLVKG:PFYGYSFCFOKFLKICL ..... FSPKNRDRLVDLFROGA.
.GNPSSTAQHVFKVSLVSGMPFYGYHEKERHFMKIYL ..... YNPTMVKRICELLQSGA.
.GNPSSTAQHVFKVSLVSGMPFYGYHEKERHFHKIYL ..... YNPAMVKRICELLOSGA.
.GOGSSNAQHVFKIOLVKGIPFYGYHRVEHOFLKIYM ..... FNPRFVRRAANLLOSGA.ILSKNFSPHESHVPYILQFHIDYNLYGMSYVHVPLEVLKFR
.GNAASKROHIHDCEIVRAKKFYGYHSTEEAFVKIYLYPYSSYHPPDVARAASLLLAGA.VLGKSLQPYESHIPFILOFLVDYNLYGMGHVH..ISKMKFR

ARQGTFVPTQQLVHNVHIVHRFNVYGYRPHAHAFYEVELI ..... DPDLLPRVVDVLQNSTEVGGRRWQLYDAHYRYHTQFMVRWRVSGIAPFPLPAGRCHVR

IFGKKFEIYESHIPYLLQWTADFNLFGCSNINVD..RCYFR
ILNKVIQVYESHLPYLLOFMVDHNLYGCAPIDLDDSIIKRD
IMNKFYOPHEAHIPYLLQLFIDYNLYGMNLIN..LAAVKFR
IMNKCYQPHEAHIPYLLQLFIDYNLYGMNLIN..LRAVKFR

656
640
2286
2278

QKRKKSSVHDSLTHLTLEIHANTRSDKIPDPAIDEVSMIIWCLEEETFP' DLDIAYE. .GIMIVHKASE ...... DSTFPTKIQHCINE. IPVMFYESEFEMFEALTDL
SKVFRKDP{SCVRILALELFCCSHGGLTPDPTKDSIECCFWAYQEDVNSSMIDRV.. .GEIVVDKSAS ...... NSSFGRSFPSC ..... TVLVVNS ELELINEVIGL
OEAKALHEIQNLTLISVELHARTRRDLEPDPEFDPICALFYCISSDTPLPDTE.KTELTGVIVIDKDKTVFSODIRYOTPLLIRSGI.TGLEVTYAAI‘EKALFHEIANI
QERKALHEIQNLTLISVELHARTRRDLQPDPEFDPICALFYCISSDTPLPDTE.KTELTGVIVIDKDKTVTHQDIRSOTPLLIRSGI.TGLEVTYARPEKALFQEITNI

1288 QQAKADIDCNHLTIITLEVFVSTRGDLQPDPHHDEIRCLFYAI..EHSLPDEKLPSKACGYIMVN...TV..QDLLSEGPF...HGIDRDIEVQVVTSEAEAFEALLAL
1026 RDPASMGAGOOLTILSIEVHAESRGDLRPDPRFDSVNVIALVVON ..... DDSFVAEV..FVLLFSPDSIDQRNV ......... DGL.SGCKLSVFLEEROLFRYFIET
1932 AAASASGGDAGVDSGAAERAFPSVAEEGPTKS.GRGGAEAGELAAARYRSRSLASHAARPFTVRHGSSGGGGEPLPSQESVOSATAMAARSSVLSSAASSASWTAPLRD
VLLLDPDILSGFEIHNFSWGYIIERCOKIHOFDIVR ....... ELARV ................... ......KCOIK...TKLSDTWGYRHSSGIHITGRHMINTWRALRSDV
NROLDPTIVCGYEVHNSSWGYLIERASYRFNYDLPE ....... QLSRL ......................... KCTSKANFAKKENANKYTTTSSINIVGRHVLNIWRILRGEV
IKRYDPDILLGYEIQMHSWGYLLQRAAAL.SIDLCR ....... MISRVPDDKIENRFRRER ........................ DEYGSYTMSEINIVGRITLNLWRIMRSEV
IKRYDPDILLGTEIQHHSWGYLLQRAAAL.SVDLCO ....... MISRVPDDKIENRFRAER ........................ DDYGSDTHSEINIVGRITLNLWRIMRKEV
CERWDADIYRGYEIEMSSWGYVIDRAKHL.CFNIAP ....... LLSRVPTQKVRDFVDEDR ........................ EQF.TDLDVEMKLCGRILLDVNRLMRSEI
LCKWDPDVLLGWDIQGGSIGFLAERAROL.GIRFLN ....... NISRTPSPTTTNNSDNKRKLGNNLLPDPLVANPAQVEEVVIEDEHGRTHASGVHVGGRIVLNAHRLIRGEV
LDEGDDGVDGGGTDDVGTKGGACGRRGGALAGOTVEYREGGTMRHAHTGLORNGQSPLLARPRAAVDLRAAASASALPRPAAAAADHYAKRFGTNHHVVGRICTSLGRDLRKEI
NLTOYTlBSAAFNILHKRLPHFSFESLTNMWNAKKST ..... TELKTVLNYWLSRAQINIQLLRKODYIARNIBOARLIGIDFHSVYYRGSOFKVESFLIRICKSESFILLSPG
NLLNYSLENVVLNIFKKOTPYYNOADKVHLHOSSR.F ..... HEKQILLNYMLNRTRYCLEILSACAIVTKIREQARIIGICfMSVISRGSQFKVESIMFRIAKPENYIFPSPS
RLTNYTFENVSFHVLHQRFPLFTFRVLSD.NFDNKTD ..... LYRWKMVDHYVSRVRGNLQMLEQLDLIGKTSEMARLFGIQFLHVLTRGSQYRVESMMLRIRKPMNYIPVTPS
GLTNYTFENVSFHVLHQRF LFTFRVLSD.HFDNKTD ..... LYRWKMVDHYVSRVRGNLQMLEQLDLIGKTSEMARLFGIQFLHVLTRGSOTRVESMMLRIAKPHNYIPVTPS
ALTSYTFENVMYHILHKRCPWHTAKSLTE.NFGSPCT ....... RNIVMEYYLERVRGTLTLLEQLDLLGRTSEMAKLHWHSVLRGLSRGSQFRVESMMLRIRKPKNLVPLSPS
KLNHYTIEAVSEAVLRQKVPSIPYKVLTE.WFSSGPA ..... GRRYRCIEYVIRRANLNLEIMSOLDHINRTSELARVFGIDFFSVLSRGSQTRVESMLLRLAHTONYLAISP3

KMPSYSLSHVHVOLLGOPLPYFTDSYLSELFLTPQCADALGGGERHTALRYLASRVAAPHRIACKLRWFTRLLEFSRMYGILTKEVITRGSQFRVBATLLHFAHPLRYRMLSPS

.EI.NLTENNLGVSKFSLPRNILAL..LKNDV.TIRPNGVVYRKTSVRKSTLSKML
.IV.N.GKVKLGFMFHSSNPNIVNL..IKNDV. YISFNGYAYVKRNVRKSILAKML
.NLGKYDEFKFGCTSLRVPPDLLYO..VRHDI. TVSPN- SVAFVKPSVRKG‘LPRML
.NLGKYDEFKFGCTSLRVPPDLLYQ..IRHUV.TVSPNGVAFVKPSVRKGVLPRML

KRDVRKOKALECVPLVMEPFSRFYK.SPLIVLDFQSLYPSIMICYNYCYSTMIGRVR.
RKQVREONALEALPLVMEPKSLLYN.NPVVVLDFQSLYPSIIIAYNLCTSTCLGPVK.
VOQRSQMRAPOCVPLIMEPRSRFYS.NSVLVLDFQSLYPSIVIAYNYCFSTCLGHVE.
IOORSQMRAPOCVPLIMEPRSRFYS.NSVLVLDFOSLYPSIVIAYNYCFSTCLGHVE.
VQARAHMRAPEYLALIHEPZSPFYA.DPLIVLDFOSLHPSMIIAYNYCFSTCLGRVE..HLGEVRPLNLARRSSEFRGRCCRS..CWSTIWLLFACGVVFVKREVREGILPRHL
NOQVASQPRMEC‘HPL'MEPESAFYD.DPVIVLDFQSL“1PSMIIAXNLCFSTCLGKLA..HL. KMNTLGVSSYSLDL. .DVLOD. .LNQILOT. .PNF VM”1VPPEVRRGILPRLL
LSQVHRQPRIECIPLJHQPKSGLYRHDPV”VLDFRSL1PSLAIAYNLC{STCLGIVQPHSHGRLGVLPRERQSNAALAELLPDDGTQHDGVVFoPNGAMEVPPSIRVGLLPQHV

EIGDDNTTLFRLLNNVQL!LKLLRNVTYGYTSR"FSGRHPCSDLADSIVDTGRETLEKRIDIIEKDETN"RFVVY"DTDSIFVYLPGYTRIRRFCIG
EELIETRNHVKRGMK..DC. .DSDW{'NK”LNS ROLLLKLIRNVTWIGYT ASFSGRMPC? EIADT IVETGREI LSXSLEYINTLDFCHAKVV:uDTDSlFVPLFGATKF"RF”IG
EEILFTREHVKQSMK..AYKQDRA.LSRMLLRROLJLKL.ANVT.GITbALFbCFMPCIEvQDSIVHRARETLERAIKLVNDTKKWUARVVXGDTDSHF"LLRGATKECSFHIG
EEILFTRLHVKQSMK..SYKQDRA.LSRMLNRRQLSLKLIRNVTFGYTAANFSGRMPCIEVGDSIVHKRRETLEPAIKLVNDTKHHGRPVVYGDTDSMFVLLKGATKEQSPKIG
TEIL“TR;MVKOSMK..LHKDSSA. LORILHSRQLTLKLMANVTYGYT.KAHFSGRMPSV VGDS”VSFFRETLERRIKLVENNEFWFVRVVYFDTDSVFVL?FGRNRFFRFRIG
EEIL? TRI MVKKAHK.. KLTPSEAVLHRIFNRRQLALKL RNVTYG1TRAGFSGRMPCAELADSIVQCGRFTLEKRISFVNANDNWNARVVYFDTDQHFVIIRFRTVKFAFVVG
QR’LETRFEVOAALKHIAVPSGDITMOQRLOEQQLALKMLANVT{GYTRASYTGRMPCVDLAEAIVSLGRQTLERTIELIHNTPAWRAEVVYGDTDSLFVRLAGRTKMDAFRIG

TDILDVRVHIKKTHN..

HRMAERVTONNPPWPIFLKFEKVYHPSILI KKRYVGFSYESPSQTLPIFDRKGIETVRRDGIPAQQKIIEKCIRLLFOTKDLSKIKKYLONEFFKIQIGKVSAODFCFRKEVKL
QOLANNITERPPSPIRLKFEKI YFPCFLLAKRRYVGEKEESVSQRAPIFEARGIETVRRDGTP.QQQLLRRCLEILFRTRDLSTVKRLFQNVCYOIMSGNVPVMDFCFLXE{RL
OEIRERVTATNPFPVKLFFEKVYLPCVLQTKKRYVGYVYETLLQKDP”F’AKFIET"PRD”CPA”§KILERSLFL FRTRDISLIKQYVQRQCMKLLEGKASIQDFIFAKEYR.
QEIAEA HVTA NPR F’K.LKFEK‘HYIPC LQTKKP”IVGYHYETLE QREPVFDRKGIETVRRDE CPA“"KILERSLFLLFLTRDISLIRQYVHRQCHKLVEGRASIQDFIFMhExR
EEIARA”TEMNP¢PJKLKLEYV}-PC}L KKPYVGYlYETADQRQP‘{EAKJIETLRRDJCPA AKMDEKVLRILFuTQD‘SDIKLYVCRQSPSCCR.QANLOD.IFAhErR.
OEIA A TFVNFHPVTLKYEKVYHPC"LL'KFRYVGY”YEQP'QRRPTFTRFGIETVRFDMCFAVRKTHEDSLFLFFEQKN'SK”KSYLYRQWKRILSGRVSLQDFIFAKEVRL
QEAADAVTRSSPAPIRLVFEKVLLPCLLLXKRRXAGYNWSSP QEAPT FLAkGIEJVRRDQCPAT AQLTDRLLRLLLDDASATVLRQSYYAAVERLOSGAANPLQCIFRRAVKL

LLRERCVSPEEFLEGE.NLELDSRYYINKILIPPLDRLFNLIGINVGNWAOEIVK.SKRASTTTTK
E..KYKELSTAPPGAVMARRLMTKDFRPEPOYGERYPVLIIAAAPGT.TLANRSVAPEEFLSSS.FSOLDINYYINNSLIPPLDRFLNLLGASAQSNYHEMPK.PRTSLKLTET
GSFSYKPGACVP.ALELTRKMLTYDRRSEPOVGERYPYVIIYGTPGV.PLIOLVRRPVEVLQ.DPTLRLNATYYITKOILPPLARIFSLIGIDVFSNYHELFRIHKATSSSRSE
GSFSYRPG ACVP ALELTRKMLAYDRRSEPP”GER”PYVIIYGTPGL.PLIQLIRRPAEVLQ.DPTLRLNRTYYITKOILPPLARIFSLIGIDVFSHYQELPRIQKATSSSRSE
GINLEYKPTAC’P ALELTRKWMORDPRH‘JPRRGGR. PLHNSQRFPGM.OLIRLVRSPHDILA.NEGHKINAIYYITKAIIPPLHRCLLLIGANVHDWFASLPRKLLMTPAVGTO
GT'fSTRDSSLLPPAAIVATKSMKADERTEPRYMERVPY’VIHGEPGA.R LVENVVDPLVLLDVDTPYRLNDLYYINKOIIPALQRVFGLVGADLNQWFLEMPRLTRSSLGQRPL
GRYKDADDTHLPLARRLAFQQMEKDATQTPCWGERLPYVVVRSTTATNKLTDKVLHPERLLQVHDTHSLDATYYIVRHVNRTLDRMFYLVGISFGRWYQAMPRRRTRHAALLNL

G..AYKSEKTRPAGAVVVKRRINEDHRAEPOYKERIPYLVVKGKQGQ.

VENITRVGTSAT ...... CCNCGEELTKICS...LQ..LCDDCLEKRSTTTLSFLIKKLKROKEYOTLKTVCPTCSYRYTSDRGIENDHIASKCNSYDCPVFYSRVKAER 1484
VKGGIQKKTLDTFLMEKLCSSCLKNNIEIIPDKINS..LCSECLKNPCATISKAVTOHNAYNKKLSLLFDICRGCSKLSSSDPVL ........ CKSNSCKVYYDRAKTEN 1462
PEG..RKGTISQYFTTLHCPVCD....DL .TOHG..ICSKCRSOPQHVAVILNQEIRELERQQEQLVKICKNC ....... TGCFDR..HIPCVSLNCPVLFKLSRVNR 3115
LEG..RKGTISOYFTTLHCPVCD....DL .TQHG..ICSKCRSOPQHVAIILNQEIRELERKQEQLIKICRNC ....... TGSFDR..HIPCVSLNCPVLFKLSRVNR 3107
RIGGSTRCQVHHLPVFLYHQHRD....RLWPPNOGG..ICPLCLKNATTCVVVLSDKTARLER...GLPTNSADM ....... PGLLRRLGSLOCDSLECPVLYVLEGKRR 2109
NSKNSHKTRIDYFYLSKHCILCG.. .EVVOESAQ...LCNRCLQNKSRRRATIVWKTSKLEREMOHLATICRHCG ...... GGDWVVOSGVKCN LRCSVFYERRKVQK 1871
PTEMAAOQRQOOQLQGVVPPDARGAVPPLFSFDTVSAPLSPRSRQMRLGQLASLMEGLRHHHSSSGVLSRLSGGAASAAATTAATREEISDDEDPSDEAAOPKEVADLTR 2830

200

,-' j

 

 

Figure 3. Sequence comparisons of conserved regions of Rev1p from various
species. Hs, Homo sapiens (accession No. AF206019); Mm, Mus musculus (P.
Gibbs and C. Lawrence, unpubl.); Dm, Drosophila melanogaster (CG12189,
accession No. AE003469); Sc, Saccharomyces cerevisiae (accession No.
M22222); Sp, Schizosaccharomyces pombe (accession No. AL035548); Ce,
Caenorhabditis elegans (accession No. 246812); At, Arabidopsis thaliana
(accession No. A0002342). Residues conserved in at least 5 of the sequences
are highlighted. Conserved sequence motifs (Gibbs et al., 2000) are overlined
and numbered. The conserved glycine mutated in rev1-1 is marked with an

asterisk.

201

 

 

m versus
sculus P
3612139.
“>8th N9.
548); 0a.

thaliana
equenoes
overlined

’ with an

*

HsREV1 4 8 iFSGVAIYVNGY'r DPSAEELRKLMM ..... LHGGQYHVYYSRSKT. .THI IATNLPNAKIK . BLKGBKVIRPBWIVES IKAGRLL

“MHREVW 48 IFSGVAIYVNGYTDPSAEELRNLMM ..... LHGGQYHVYYSRSKT..THIIATNLPNAKIK.ELKGEKVIRPEWIVESIKAGRLL
DnflREV1 39 LFQGISIFVNGRTDPSADELKRIMM ..... VHGGTFH.HYERSHT..TYIIASVLPDVKVR.NMNLSKFISAKWVVDCLEKKKIV
SCREVW 165 3IFKNCVIYINGYTKPGRLQLHBMIV ..... LHGGKF.LHYLSSKKTVTHIVASNL.PLKKRIEFANYKVVSPDWIVDSVKEARLL
SpREV1 63 LFHGLAIAINGYTKPSYTELRQMIV ..... SNGGTF.IQYVDGKTSISYLVCSFLTPSKAR.QWKHQKVVKPEWIVDCIKQKKIL
CBREV1 255 IMEGFSVFVNGYTDPPALVIRDLMI ..... SHGGEYHCYYQHGITSYT..IASSIATAKINRIRENEIFIKADWITESIAAGKPL
AIREV‘I 87 IFQGVSIFVDGFTIPSHQDIQQRITCCETYMDSGLYLCQQTSGLSHLTPQMASP ............................. VT

HsREV1 SYIPYQL-Y -(lllaa)-
MmREV1 SSAPYQL! - (109aa)-
(91aa)-
(63aa)-
(81aa)-
(36aa)-
AtREV1 SFQPDTGY -(110aa)-

DmREV1 DYKPYLLY -
ScREV1 ...PWQNY -
SpREV1 ...PWINY -
CeREV1 DYRDFLIY —

HsREV1 thKPVAVTs
MmREV1 LKGKPVAVTS
DmREV1 LRGLPIAVTH
ScREV1 FKRDPIWCH
SpREV1 LRLKPVAVAH
CeREV1 LKHKEVAITH
AtREV1 woxpvavca

Vl—

(57aa)-
(S7aa)-
(52aa)-
(4aa)-
(4aa)-
(9aa)-
(7aa)-

SDCNFISNFYSHSRLHHISMWKCELT -(49aa)- IMHVDM ......... DCFFVSVGIRNRP ...... D
SDCNFISDFYSRSRLHHISTWKCELT -(49aa)- VMHVDM ......... DCFFVSVGIRNRP ...... D
ADPNFLSEFYKNSRLHHIATLGAGFK -(37aa)- VMHIDM ......... DCFFVSVGLRTRP ...... E
DDPDFLTSYFAHSRLHHLSAWKANLK -(21aa)- IFHIDF ......... DCFFATVAYLCRSSSFSACD
QNQDPLENFFSSSRLHHLSTWKADFK -(26aa)- LLHVDF ......... DCFFASVSTR ..... FSH.E
RNPNFIRDYYARSRLHLISTLAQDMK -(36aa)- VFHVDL ......... DCFFVSVAVRNR ...... ID
EDPNFVENYFKNSRLHFIGTWRNRYR -(27aa)- IIHIDLLFESVKYFQDCFFVSVVIKNR ...... LE
'V — —'V — —VI

AEIASCSYEARQLGIKNGfiFFGHAKQLCPN.LQAv..PYDFHAYKEVAQTLYETLASYT..HN.IEAvsc
ABIASCSYEARQVGIKNGMFFGYAKQLCPN.LQAV..PYDFHACREVAQAMYBTLASYT..HS.IEAVSC
SEIASCSYEAREKGIRNGMFVGQALKLCPE.LKTI..PYDFEGYKEVAFTLYDTVAQYT..LN.IEAVSC
SDIASCNYVARSYGIKNGMWVSQAEKMLPNGIKLISLPYTFEQFQLKSEAFYSTLKRLN.IFNLILPISI
SEIASCNYEARKFGIKNGMYVGTAKNLCPS.LRVVD..YDFGAYESVSREFYTIL..VNTLHDYIKVISI
SEVASCSYAARDCGVKNGMLVRDALQKCP...QLTLLPYQFEDYVQVSRKIYEILASYTL...EVRAVSC
AEISSANIPARAYGVKAGMFVRHAKDLCE...QLVIVPYNFEAYEE .................... ALSC

VII

HsREV1 DEAL. . .VDITEILAETKLTP. . . .DEFANAVRMEIKDQT. KCAASVGIGSNILLARMATRKAKPDGQYHLKPEEVDD. . .E‘IRGQLVTN
MmREV1 DEAL. . .IDVTDILAETKLSP. . . .EEE‘AAALRIEIKDKT . KCAASVGIGSNILLARMATKKAKPDGQYHLQPDEVDD. . .E‘IRGQLVTN
DmREV1 DEME‘. . .VELTDLAHELNVDV. . . .MAFVSHLRQEVYSK‘!’ . GCPCSAGVAGNKLLARMATKEAKPNGQFLL. . DSSNDILAYMAPMSLDL

SCREVH DEAVCVRIIPDNIHNTNTLNA ..... RLCEEIRQEIFQGTNGCTVSIGCSDSLVLARLALKMAKPNG.YNITF.KSNLSEEFWSSFKLDD

SpREVH DEALL ...... DI..TSSVSSFQDCFBIAESIRSQVREKTN.CEVSVGIGPNVLLARLALRKAKPHNVYSLSI.E.NVFDVL.SPLSVQD

CBRENH DEMY...INMSSFCEKYEINDPTI...LAEHIRKVIREKTQ.CPASVGIGSTSLLARLATRHAKPDGVFWVNAHKKN...EFISEEKVKD

ARRENH DEAFLDVSDLSDV..ETEV ........ LASTIRNEILE.TTGCSASAGIGGTMLMARLATRVAKPAGQLYISAEKV...EEFLDQLPVGT
— VIII —' IX

HsREV1 LPGVGHSMESKLASL. .GIKTCGDLQ -(83aa)- KIMVRKPGAPVETAKFGGHGIcDNIARTVTLDQATDNAKIIGKAMLNMF
MmREV1 LPGVGRSMESKLASL. .GIKTCGDLQ -(83aa)— KIMVRKPGAPIETAKFGGHGICDNIARTVTLDQATDSAKIIGKATLNMF
DmREV1 LPGVGSSISHKLKQA. .GLNNCGDVQ —(83aa)- KIMVRAAEAPVETSKYMGHGVCDIINKSSLIKYATDDVNVITTVVLDLM
ScREV1 LPGVGHSTLSRLESTE‘DS PHSLNDLR - (91aa) - KLMRRCKDAPIEPPKYMGMGRCDS FSRSSRLGIPTNEFGIIATEMKSLY
SpREV1 LPGVGSSQAQKLFNLY . GVRTIGQLQ - (833a) - RILKRADGAPFSPPKYLGAGEVTSFTKSSTFTSATNSFDLIWKKVTSMY
CeREV1 LPGFGYEMMNRLTSFFGDITKCRELQ - (85aa) - RLMVRSANAPIQTSKFMGHGICDTFTKTCNLNVPTTRGESLTSEAMKLY
AtREV1 LPGVGSVLKEKLVK..QNIQTCGQL. - (4aa)- KIKKRKKDAE.EPTKYMGCGDCDNLSRSITVPAATDDIEVLQRISKKLF

202

804
802
656
715
612
782
557

 

BRCT domain of the BRCA1 gene, which may be concerned with protein-protein
interactions. This region contains G76, the residue homologous to 6193 in yeast
and the site of the G193R mutation found in the Rev1-1 mutant protein, which
substantially lacks the second function of yeast Rev1p, but retains much of the
dC transferase activity. The highly conserved DXD and DE sequences found in
motifs Ill and VI respectively appear to be concerned with polymerase function
(Johnson et al. 1999; Gerlach et al. 1999). As with the REV3 locus, the human
REV1 gene contains an upstream out-of frame ATG in a good context, again
suggesting that cellular levels of Rev1p may be low. Essentially identical REV3
and REV1 clones have also been isolated by other groups (Morelli et al. 1998;
Lin et al. 1999a,b).

The human Rev1p not only exhibit significant homology to the yeast
proteins, but also appear to perform similar functions to their fungal counterparts.
To investigate this issue, cells derived from MSU-1.2, an infinite life-span, non-
tumorigenic, near diploid, human fibroblast cell line, were transformed with
plasmids expressing high levels of either a REV1 or REV3 antisense RNA
fragment, and the cells assayed for UV—induced mutagenicity and cytotoxicity
(Gibbs et al. 1998, 2000). In cells expressing the antisense RNA, the frequency
of 6-thioguanine resistant mutants induced by UV was much reduced compared
to those seen in control cells in which antisense RNA was absent (Figure 4), a
phenotype similar to that of yeast rev mutants. Unlike yeast rev mutants,
however, the lower frequencies of UV-induced mutations were not accompanied

by an increased sensitivity to the cytotoxic effects of the UV radiation, perhaps

203

 

 

0 4 8 12 13 0 4 8 12 16
100 I I 100 I I I

 

 

  

   
 
 

Percent Survival

7AGM

 

 

 

 

A
A “A o 7AGM-17cm
. 10A-42-R3 I 7AGM-1ZB-R1
A
20 I l l A 1 I 1 20 1 l 1 l 1 l g
70 I I l I I I W 70 ' I ' l V l T

20"

we

 

 

 

 

Induced TG' Cells per 10‘ Clonable Cells
8
T

 

 

Figure 4. Percent survival and frequency of 6-thioguanine mutants in UV-
irradiated human cells expressing REV3 antisense RNA (A) or REV1 antisense

RNA (B), together with their antisense-free controls.

204

 

 

because the unrepaired damage was channeled into another repair or tolerance
pathway. In addition to the reduction in UV-induced mutagenesis, expression of
RE V3 antisense RNA also reduces spontaneous mutation frequencies in a msh6
mutant strain (X. Li and VM. Maher, unpublished data), suggesting that human
pol zeta also resembles its yeast counterpart with respect to involvement in
spontaneous mutagenesis. Reinforcing the similarity between the yeast and
human proteins, Zigang Wang and co-workers have additionally shown that
human Rev1p possesses a dC transferase activity (Lin et al. 1999b). Curiously,
however, although dCMP is predominantly inserted opposite an abasic site in
vivo in yeast (Gibbs and Lawrence 1995), incorporation of dAMP is the
predominant event in monkey cells (Takeshita and Eisenberg, 1994). Finally, a
putative human REV7 cDNA clone has been isolated (Murakumo et al. 2000);

whether the latter plays a role in mutagenesis remains to be determined.

DISCUSSION AND CONCLUSIONS

DNA polymerase zeta and Rev1 protein are employed in a translesion
replication pathway in yeast that has a wide range of substrates and is a major
source of DNA-damage induced mutations. Pol zeta is also responsible for the
majority of spontaneous mutations in yeast. Judging from the results with human
cells and from the widespread occurrence of homologs of the yeast REV1 and
REV3 genes in other organisms, pol zeta and Rev1p are probably found in most
eukaryotes. In addition to their presence in humans, REV1 and REV3 homologs
have been jointly found in the mouse (accession No. AL355632; P. Gibbs,

unpubl.), Arabidopsis thaliana (accession Nos. ACO11020, A0002342),

205

 

 

Drosophila melanogaster (J. Eeken, P. van Sloun, and R Romeijn, pers. comm.,
accession No. AE003469) and Schizosaccharomyces pombe (accession Nos.
AL355632, AL035548). In addition, Leishmania major has also been found to
possess a REV3 gene (accession No. AL122012). (see Figures 2 and 3).
Interestingly, Caenorhabditis elegans possesses a RE V1 gene (accession No.
246812) but not, apparently, a REV3 gene and since the entire genome of this
organism has been sequenced, it appears to lack an enzyme of the pol zeta type.
Translesion replication in the worm may therefore be accomplished by Rev1p in
combination with a different DNA polymerase such as pol delta, the enzyme to
which pol zeta is most closely related. If so, the eukaryotic ancestral pol delta
may perhaps have originally performed multiple functions in normal replication,
repair, and translesion replication with some, though not necessarily all, lineages
acquiring pol zeta by duplication of the gene encoding the catalytic subunit of pol
delta and functional specialization of the resulting Rev3p.

At the present, putative Rev7p homologs have been discovered only in
Drosophila (062948, accession No. AE003602) and humans (accession No.
AF157482), perhaps indicating poor conservation of sequence. Although future
work may uncover an enzymatic activity for this pol zeta subunit, its ability to
promote the activity of the Rev3p catalytic subunit may in fact represent its chief
function. Enhancing the activity of Rev3p may represent a mechanism for the
activation of pol zeta when unrepaired damage in the genome prevents the
completion of replication. The observation that the mutant phenotype of the yeast

checkpoint genes RADQ, RAD17, RADZ4, and MECS includes not only a failure

206

 

 

 

to observe checkpoints but also a lack of UV-induced mutability characteristic of
rev mutants (Paulovich et al 1998), suggests that a signalling pathway extends
from the detection of blocked replication forks to the mobilization of the enzymes
needed to remedy this situation. For pol zeta, such a mobilization might entail the
release of sequestered Rev7p and the formation of a more active enzyme.
Sequestering of Rev7p might involve association with the checkpoint gene
products themselves, or by gene products phosphorylated or othenrvise altered
by these proteins. A variety of genetic evidence, including the introduction of in-
frame stop codons by alternate splicing in human REV3 and the existence of out-
of-frame upstream ATG codons in several REV genes, suggests that cells
maintain only very low levels of pol zeta and Rev1 p, which may be necessary to
prevent competition with replicases and high frequencies of spontaneous
mutations.

Since pol zeta and Rev1p appear to be concerned with translesion
replication in both yeast and humans, they presumably perform the same
function in most eukaryotes. In the single-celled eukaryotes, such a process is
clearly useful; the advantage of completing replication is likely to outweigh the
disadvantage of possibly generating mutations. In mammalian cells, the balance
in favor of translesion replication is at first sight less clear, since such cells have
the additional option of apoptosis. Why, then, do mammals possess a translesion
replication pathway? Part of the reason may be that the adverse consequences
of lesion bypass are not necessarily high. The error—rate of translesion replication

is usually very much less than 100%, and may be quite low. Even if mutations

207

are produced, their occurrence in non-informational DNA, or the formation of
synonomous changes in coding sequences and the tolerance of many proteins
towards residue substitutions are all factors that diminish the adverse effects of
translesion replication. At the same time, the occurrence of unscheduled
apOptosis may also itself carry risks, particularly in early development. A REV3
knockout in the mouse is lethal (P. van Sloun, R. Romeijn, N. de Wind, and J.
Eeken, pers. comm), though the reasons for this are not yet known. By contrast,
cultured human cells that apparently contain very little Rev3p or Rev1p are fully
viable and grow at a normal rate (Gibbs et al. 1998, 2000). Significantly, such
cells appear to be relatively resistant to the induction of apoptosis.
Interrelationships of the regulation of pol zeta, apoptosis, tumor suppression, and
cell cycle control is perhaps suggested by the observation that p33’NG’, the
product of the human ING1 gene, strongly associates with Rev3p in a two-hybrid
assay (R. Murante, unpublished data). The candidate tumor suppressor p33’NG’
has been reported to negatively regulate cell proliferation, and apoptosis, to
facilitate transcription from the p21NVAF1 promoter, and to physically associate
with p53, indicating that it is part of the p53 signalling pathway (Garkavtsev et al.
1998). Lethality of the mouse REV3 knockout may also arise because the
mammalian enzyme provides another essential function, independent of its role
in translesion replication. The much greater size of the Rev3p in mammals
suggests that it may interact with a wider range of proteins or possess some
additional activity; it has been suggested, for example, that the mouse gene is

involved in a stress response (Kajiwara et al. 1996).

208

In addition to its role in translesion replication and the tolerance of
unrepaired DNA damage originating from exogenous mutagens, pol zeta is also
concerned with the production of spontaneous mutations; yeast rev3 mutants are
strong antimutator strains (Quah et al. 1980; Roche et al 1994; Datta and Jinks-
Robertson 1995; Holbeck and Strathern 1995), and spontaneous mutation
frequencies are reduced in msh6 mutant human cells producing high levels of
REV3 antisense RNA (Z. Li and V. M. Maher, unpub.). Although replication past
unrepaired spontaneously arising lesions may generate some of these mutations,
it is unlikely that sufficient damage occurs to explain more than a small fraction of
them. Instead, the majority may be produced by pol zeta when this enzyme is
called upon to restart replication at forks that are blocked for reasons other than
template damage. Roel Schaaper and co-workers (Fijalkowska et al. 1997) have
suggested that the enhanced spontaneous mutagenesis resulting from
derepression of the E. coli SOS regulon is caused by increased extension from
terminally mismatches that temporarily stall DNA polymerase III. Such extension
is presumably carried out by the UmuD'ZC complex, now known to constitute
DNA polymerase V (Tang et al. 1999). This observation raises the possibility that
E. coli pol V and its eukaryotic counterpart, pol zeta, may best be viewed as
enzymes engaged principally in normal replication. From this perspective, these
DNA polymerases are concerned with restarting replication at forks stalled for
any reason, whether it is by virtue of a terminal mismatch, hairpin structure, or
refractory DNA sequence, as well as by virtue of unrepaired DNA damage. Of

these, the terminal mismatch and template damage are likely to have the

209

 

 

greatest propensity to generate mutations, but the other causes of stalling may
also result in replication errors because pol zeta lacks proofreading.

Because it appears to be the major source of basepair substitution and
frameshift mutations, investigation of the pol zeta/Rev1p translesion replication
pathway is likely to have implications for human genotoxioology and for genetic
diseases such as cancer. Reduction in the activity of this pathway may reduce
spontaneous and induced mutation frequencies and therefore the risk of
developing cancer. It has been calculated that merely halving the spontaneous
mutation frequency might sufficiently delay the onset of cancer as to reduce its
incidence substantially (Loeb & Christians, 1996). Since the majority of
spontaneous mutations are likely to depend on pol zeta/Rev1p function, such a
reduction should be easily achievable. Although the pathway is essential for
viability during early development, it remains to be established whether it is also
essential in mature mammals, and whether ways of dissociating mutability and

viability can be found.

ACKNOWLEDGEMENTS

This work was supported by US Public Health Service grants GM21858 to
C.W.L. and CA56796 and E809822 to V.M.M., from the National Institutes of

Health.

REFERENCES

Bailly, V., Lamb, J., Sung, P., Prakash, S., and Prakash, L. 1994. Specific
complex formation between yeast RAD6 and RAD18 proteins: a potential
mechanism for targeting RAD6 ubiquitin-conjugating activity to DNA
damage sites. Genes and Development 8: 811-820.

210

Braithwaite, UK. and Ito, J. 1993. Compilation, alignment, and phylogenetic
relationships of DNA polymerases. Nucleic Acids Res. 21: 787-802.

Cassier—Chauvat, C., and Fabre, F. 1991. A similar defect in UV mutagenesis
conferred by the rad6 and rad18 mutations of Saccharomyces cerevisiae.
Mutat Res 254: 247-253.

Datta, A., and Jinks-Robertson, S. 1995. Association of increased spontaneous
mutation rates with high levels of transcription in yeast. Science 268:
1616-1619.

Fijalkowska, I.J., Dunn, RL, and Schaaper, RM. 1997. Genetic requirements
and mutational specificity of the Escherichia coli SOS mutator activity. J.
Bacteriol. 179: 7435-7445.

Garkavtsev, l., Grigorian, I.A., Ossovskaya, V.S., Chernov, M.V., Chumakov,
P.M., and Gudkov, A.V. 1998. The candidate tumor suppressor p33’NG’
cooperates with p53 in cell growth control. Nature 391: 295-298.

Gerlach, V.L., Aravind, L., Gotway, G., Schultz, R.A., Koonin, E.V., and
Friedberg, EC. 1999. Human and mouse homologs of Escherichia coli
UmuC/DinB superfamily. Proc. Natl. Acad. Sci. USA 96: 11922-11927.

Gibbs, REM, and Lawrence, CW. 1995. Novel mutagenic properties of abasic
sites in Saccharomyces cerevisiae. J Mol Biol 251: 229-236.

Gibbs, P.E.M., Borden, A., and Lawrence, CW. 1995. The T-T pyrimidine (6-4)
pyrimidinone UV photoproduct is much less mutagenic in yeast than in
Escherichia coli. Nucleic Acids Res 23: 1919-1922.

Gibbs, P.E.M., McGregor, W.G., Maher, V.M., Nisson, P., and Lawrence, CW.
1998. A human homolog of the Saccharomyces cerevisiae REV3 gene,

which encodes the catalytic subunit of DNA polymerase zeta . Proc. Natl.
Acad. Sci. USA 95: 6876-6880.

Gibbs, P.E.M., Wang, X.-D., Li, Z., McManus, T.P., McGregor, W.G., Lawrence,
C.W., and Maher, V.M. 1998. The function of the human homolog of
Saccharomyces cerevisiae RE V1 is required for mutagenesis induced by
UV light. Proc. Natl. Acad. Sci. USA 97: 4186-4191.

Glassner, B.J., Rasmussen, L.J., Najarian, M.T., Posnick, LM, and Samson, L.
D. 1998. Generation of a strong mutator phenotype in yeast by
imbalanced base excision repair. Proc. Natl. Acad. Sci. USA 95: 9997-
10002.

Holbeck, S.L., and Strathern, J.N. 1995. A role for REV3 in mutagenesis during
double-strand break repair in Saccharomyces cerevisiae. Genetics 147:
1017-1024.

211

 

 

 

Jentsch, S., McGrath, JP, and Varshavsky, A. 1991. The yeast DNA repair gene
RAD6 encodes a ubiquitin-conjugating enzyme. Nature 329: 131-134.

Johnson, R.E., Prakash, S., and Prakash, L. 1999. Efficient bypass of a thymine-
thymine dimer by yeast DNA polymerase, pol eta . Science 283: 1001-
1004.

Jones, J. S., Weber, 8., and Prakash, L. 1988. The Saccharomyces cerevisiae
RAD18 gene encodes a protein that contains potential zinc finger domains
for nucleic acid binding and a putative nucleotide binding sequence.
Nucleic Acids Res, 16, 7119-7131.

Kajiwara, K., Nagawawa, H., Shimizu-Nishikawa, K., Ookura, T., Kimura, M., and
Sugaya, E. 1996. Molecular characterization of seizure-related genes
isolated by differential screening. Biochem. Biophys Res. Commun. 219:
795-799.

Larimer, F.W., Perry, JR, and Hardigree, AA. 1989. The REV1 gene of
Sacchammyces cerevisiae: Isolation, sequence, and functional analysis.
J. Bacteriol., 171: 230-237.

Lawrence, CW. 1994. The RAD6 DNA repair pathway in Saccharomyces
cerevisiae: what does it do, and how does it do it? BioEssays 16: 253-258.

Lawrence, C.W., and Christensen, RB. 1978. Ultraviolet-induced reversion of
cyc1 alleles in radiation-sensitive strains of yeast. l. rev1 mutant strains. J
Mol Biol 122: 1-21.

Lawrence, C.W., and Christensen, RB. 1979. Ultraviolet-induced reversion of
cyc1 alleles in radiation-sensitive strains of yeast. lll. rev3 mutant
strains. Genetics 92: 397-408.

Lawrence, C.W., O’Brien, T., and Bond, J. 1984. UV-induced reversion of his4
frameshift mutations in rad6, rev1, and rev3 mutants of yeast. Molec Gen
Genet 195: 487-490.

Lawrence C.W., Das, G., and Christensen, R.B. 1985a. REV7, a new gene
concerned with UV mutagenesis in yeast. Molec Gen Genet 200: 80-85.

Lawrence C.W., Nisson, PE, and Christensen, R.B. 1985b. UV and Chemical
mutagenesis in rev7 mutants of yeast. Molec Gen Genet 200: 86-91.

Lawrence, C.W., Stewart, J.W., Sherman, F., and Christensen, RB. 1974.
Specificity and frequency of ultraviolet-induced reversion of an iso-1-
cytochrome c ochre mutant in radiation-sensitive strains of yeast. J. Mol.
Biol. 85: 137-162.

Lawrence, C.W., Borden, A., Banerjee, S.K., and LeClerc, JE. 1990. Mutation

212

frequency and spectrum resulting from a single abasic site in a single-
stranded vector. Nucleic Acids Res., 1 8, 2153-2157.

Lemontt JP 1971. Mutants of yeast defective in mutation induced by ultraviolet
light. Genetics 68: 21-33.

Lin, W., Wu, X., and Wang, Z. 1999a. A full-length cDNA of hREV3 is predicted
to encode DNA polymerase zeta for damage-induced mutagenesis in
humans. Mutat. Res. 433: 89-98.

Lin, W., Xin, H., Zhang, Y., Wu, X., Yuan, F., and Wang, Z. 1999b. The human
REV1 gene codes for a DNA template-dependent dCMP transferase.
Nucleic Acids Res. 27: 4468-4475.

Loeb, LA, and Christians, PC 1996. Multiple mutations in human cancers.
Mutat Res 350: 279-286.

McDonald, J.P., Levine, AS, and Woodgate, R. 1997. The Saccharomyces
cerevisiae RAD30 gene, a homologue of Escherichia coli dinB and umuC,
is DNA damage inducible and functions in a novel error-free
postreplication repair mechanism. Genetics 147: 1557-1568.

McKee, RH, and Lawrence, CW. 1979. Genetic analysis of gamma-ray
mutagenesis in yeast I. Reversion in radiation-sensitive strains. Genetics
93:361-373.

Morelli, C., Mungall, A.J., Negrini, M., Barbanti-Brodano, G., and Croce, GM.
1998. Alternative splicing, genomic structure, and fine chromosome
localization of REV3L. Cytogenet. Cell Genet. 83: 18-20.

Morrison, A., Christensen, R.B., Alley, J., Beck, A.K., Bernstine, E.G., Lemontt,
JF, and Lawrence, CW. 1989. REV3, a Saccharomyces cerevisiae gene
whose function is required for induced mutagenesis, is predicted to
encode a nonessential DNA polymerase. J Bacteriol. 171: 5659-5667.

Murakumo, Y., Roth, T., Ishii, H., Rasio, D., Numata, S.l., Croce, CM, and
Fishel, R. 2000. A human REV7 homolog that interacts with the
polymerase zeta catalytic subunit hREV3 and the spindle assembly
checkpoint protein hMAD2. J. Biol Chem. 275: 4391-4397.

Nelson, J.R., Lawrence,. C.W., and Hinkle, D.C. 1996a. Thymine-thymine dimer
bypass by yeast DNA polymerase zeta . Science 272: 1646-1649.

Nelson, J.R., Lawrence,. C.W., and Hinkle, D.C. 1996b. Deoxycytidyl transferase
activity of yeast RE V1 protein. Nature 382: 729-731.

213

 

Nelson, J.R., Gibbs, P.E.M., Nowicka, A.M., Hinkle, D.C., and Lawrence, CW.
2000. Evidence for a second function for Saccharomyces cerevisiae
Rev1p. Molec. Micro. (in press).

Paulovich, A.G., Armout, CD, and Hartwell, L.H. 1998. The Saccharomyces
cerevisiae RADQ, RAD17, RADZ4, and MEC3 genes are required for
tolerating irreparable, ultraviolet-induced damage. Genetics 150: 75-93.

Quah, S.-K., von Borstel, RC, and Hastings, PJ. 1980. The origin of
spontaneous mutation in Saccharomyces cerevisae. Genetics 96: 819-
839.

Roche, H.R., Gietz, RD, and Kunz, BA. 1994. Specificity of the yeast rev3
deletion antimutator and RE V3 dependency of the mutator resulting from a
defect (rad1 deletion) in nucleotide excision repair. Genetics 137: 637-
646.

Singhal, R.K., Hinkle, D.C., and Lawrence, CW. 1992. The REV3 gene of
Saccharomyces cerevisiae is transcriptionally regulated more like a repair
gene than one encoding a DNA polymerase. Molec. Gen Genet, 236, 17-
24.

Takeshita, M., and Eisenberg, W. 1994. Mechanism of mutation on DNA
templates containing synthetic abasic sites: study with a double strand
vector. Nucleic Acids Res. 22: 1897-1902.

Tang, M., Shen, X., Frank, E. G., O'Donnell, M., Woodgate, R., and Goodman,
M. F. 1999. UmuD'zC is an error-prone DNA polymerase, Escherichia coli,

DNA pol V. Proc. Natl. Acad. Sci. USA 96:8919-8924.

Torpey, L.E., Gibbs, P.E.M., Nelson, JR, and Lawrence, CW. 1994. Cloning
and Sequence of REV7, a gene whose function is required for DNA
damage-induced mutagenesis in Saccharomyces cerevisiae. Yeast 10:
1503-1509.

Woodgate, R. 1999. A plethora of lesion-replicating DNA polymerases. Genes &
Development 13: 2191-2195.

214