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MECHANISMS OF MUTATGENESIS
AND DAMAGE AVOIDANCE
IN HUMAN FIBROBLASTS

presented by

Ziqiang Li

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Molecular Genetics

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MECHANISMS OF MUTAGENESIS AND DAMAGE AVOIDANCE IN HUMAN
FIBROBLASTS
BY

Ziqiang Li

A DISSERTATION

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

DOCTOR OF PHILOSOPHY
Microbiology and Molecular Genetics

2002

ABSTRACT

MECHANISMS OF MUTAGENESIS AND DAMAGE AVOIDANCE IN HUMAN
FIBROBLASTS

BY

Ziqiang Li

The main focus of this research concerns the mechanisms by which human cells
tolerate spontaneous or induced DNA damage that blocks the replication fork.
Damage tolerance consists of two sub-pathways, i.e., translesion synthesis and
damage avoidance. Translesion synthesis involves a series of newly discovered
specialized DNA polymerases that, in contrast to the major DNA replication
polymerases, can use damaged DNA as a template. It can be error-prone or
error-free depending on the nature of the lesion and the availability of a particular
DNA polymerase or set of DNA polymerases. The alternative pathway for cells to
deal with fork-blocking lesions is to use an undamaged homologous copy of the
sequence, i.e., the newly replicated daughter strand of the sister duplex or an
allelic copy, as a template temporarily to circumvent the lesion. This error-free
pathway is referred to as damage avoidance. To determine whether hMmsZ
protein is involved in the damage avoidance pathway, an antisense strategy was
used to eliminate hMms2 protein in the target cells. The results demonstrated
that hMms2 is essential for damage avoidance in human cells dealing With UV-
or BPDE-induced replication fork-blocking lesions (providing direct evidence for

use of the allelic copy and indirect evidence for use of the newly synthesized

daughter strand of sister duplex). Loss of hMmsZ protein eliminated the ability of
human cells to use the undamaged copy as the template dealing with fork-
blocking lesions and significantly increased the frequency of UV— or BPPDE-
induced mutants. Analysis of the damage avoidance products revealed that they
are predominantly caused by copying a homologous sequence using gene
conversion not associated with crossing-over. Furthermore, the results indicated
that damage avoidance and translesion synthesis are used almost equally for
tolerating BPDE-induced replication fork-blocking lesions. In addition, the role of
hREV3 in error-prone translesion synthesis, i.e., carcinogen-induced
mutagenesis, was also determined. Compared to their parental cell strain, the
strains expressing high levels of hREV3 antisense exhibited a significantly
reduced UV- and BPDE-induced mutation frequency (up to 6 fold). These data
indicate that hRev3 protein, the catalytic subunit of DNA polymerase zeta, plays

a causal role in generating mutations during translesion synthesis.

This work is dedicated to:
My wife, Min Zhuang

My son, Jack Hang Li

iv

ACKNOWLEDGEMENTS

First of all, I sincerely want to thank my major advisor, Dr. Veronica M. Maher, for
her endless support, advice, guidance, discussion, encouragement, and
friendship throughout the course of my PhD. study. Her hard-working habits and
dedication to science continually remind me what it takes to be a real scientist.
Her warm-hearted and considerate personality is always what I look up to. I am
also grateful to the co-director of the Carcinogenesis Laboratory, Dr. J. Justin
McCormick, for his invaluable suggestions and effort for my projects and my

career.

I am indebted to my other thesis committee members, Dr. Vilma Yuzbasiyan-
Gurkan, Dr. Patrick D. Storto, Dr. Patrick J. Venta for their invaluable suggestions
for my projects. I want to thank Dr. Katheryn Meek for her tremendous help with
my experiments and invaluable advice for my projects and my career. I would
also like to acknowledge Dr. Wei Xiao and Dr. Christopher W. Lawrence for their
collaboration on the hMMSZ project and the hREV3 project, respectively. Their

suggestions and contributions are an integral part of my thesis.

I also want to thank previous and current members of the Carcinogenesis
Laboratory. l have benefited from their suggestions, friendship, and unselfish
help. They include Dr. Hong Zhang, Dr. Kim-Hien (Jackie) Dao, Michele A.

Battle, Yun Wang, Dr. Susanne Kleff, Lijuan Wang, Katherine Bergdolt, Bethany

Heinlen, Dr. Hongyan Liang, Dr. Xi-De Wang, Dr. Sandra O'Reilly, and Suzanne
Kohler. I also obtained valuable suggestions from Kristin L. McNally, Piro Lito,
Zhenjun Lou, and Jie Zhang. Special thanks go to Ling Qin, Terrence P.
McManus, Clarissa J. Dallas, and Andrew McCoy for their significant

contributions to my projects.

In addition, I want to thank my parents who taught me to cherish the opportunity
of being educated, who supported me through all these years, who love me, and
who always remind me where I am from. I want to thank my brother, my sister,
my brother-in-Iaw, and my sister-in-law for their love and tremendous support.
Special thanks go to my parents-in-law for their love, sacrifice, and tremendous

support of my family.

In conclusion, I want to thank my lovely wife, Min, for her constant love, support,
patience, understanding, and tolerance for not having a good vacation during the
past four years. I want to thank my son, Jack, because his smile is always the

source of my happiness and my biggest motivation.

vi

TABLE OF CONTENTS

LIST OF FIGURES ................................................................................. x
LIST OF ABBREVIATIONS ..................................................................... xi
INTRODUCTION ................................................................................... 1
References ...................................................................................... 4
CHAPTER I. LITERATURE REVIEW ......................................................... 5
A. Introduction of Damage Tolerance ......................................................... 5
B. Damage Tolerance Mechanisms in Prokaryotes ....................................... 6
1. Overview of the SOS System ............................................................ 6

2. Translesion DNA Synthesis: The UmuC/DinB family .............................. 7
2.1. UmuC and UmuD (Pol V) ........................................................... 7

2.2. DinB (Pol IV) ........................................................................... 9

3. SOS-Daughter Strand Gap Repair ................................................... 11

C. Damage Tolerance Mechanisms in Eukaryotes ....................................... 13
1. Overview of the RAD6-RAD18 Pathway ............................................ 14
1.1. RAD6 .................................................................................. 15

1.2. RAD18 ................................................................................. 17

2. Translesion Synthesis: The UmuC/DinB Superfamily ........................... 19
2.1. Yeast and human polymerase zeta and Rev1 ............................... 20
2.1.1. Polymerase zeta ........................................................... 20

2.1.2. Rev1 .......................................................................... 25

2.2. Yeast and human polymerase eta .............................................. 29

vii

2.3. Polymerase iota (human/mouse) ............................................... 34
2.4. Polymerase kappa (human/mouse) ............................................ 36

2.5. Other DNA polymerases potentially involved in

translesion synthesis ............................................................... 37

3. Damage Avoidance ....................................................................... 38
3.1. Overview of damage avoidance ................................................. 38

3.2. Double strand break repair ........................................................ 40

3.3. The ch13-Mm32 pathway ....................................................... 44
3.3.1. MMS2 and UBC13 in yeast ............................................... 44

3.3.2. MMSZ and UBC13 in human cells ...................................... 48
References ......................................................................................... 51

CHAPTER II. IDENTIFICATION OF A PROTEIN
ESSENTIAL FOR A MAJOR
PATHWAY USED BY HUMAN CELLS

TO AVOID UV-INDUCED DNA DAMAGE ............................... 70
Abstract ............................................................................................. 71
Introduction ......................................................................................... 72
Materials and methods .......................................................................... 74
Results and discussion .......................................................................... 78
Acknowledgements .............................................................................. 92
References ......................................................................................... 93

CHAPTER III. TRANSLESION SYNTHESIS AND
DAMAGE AVOIDANCE PLAY VIRTUALLY
EQUAL ROLES IN TOLERATING BPDE-

ADDUCTS IN HUMAN FIBROBLASTS ................................. 96
Abstract ............................................................................................. 97
Introduction ........................................................................................ 98

viii

Materials and methods ........................................................................ 101

Results and discussion ....................................................................... 104
Acknowledgements ............................................................................. 1 1 1
References ....................................................................................... 1 12

CHAPTER IV. hREV3 IS ESSENTIAL FOR ERROR-PRONE
TRANSLESION SYNTHESIS PAST UV OR
BENZO[A]PYRENE DIOL-EPOXIDE-INDUCED

DNA LESIONS IN HUMAN FIBROBLASTS ........................... 117
Abstract ............................................................................................ 118
Introduction ....................................................................................... 1 19
Materials and methods ........................................................................ 122
Results and discussion ........................................................................ 125
Acknowledgements ............................................................................. 1 38
References ....................................................................................... 1 38

ix

LIST OF FIGURES

CHAPTER II

Figure 1: Schematic diagram of the two possible paths for avoiding
DNA damage by making use of an undamaged homolog

to continue replication .............................................................. 81
Figure 2: Northern and Western blots showing the correlation

between expression of antisense hMM82 RNA

and loss of hMmsZ protein ........................................................ 85

Figure 3: The biological effects of inhibition of hMmsZ expression .................. 87

CHAPTER III

Figure 1: The biological effects of loss of
hMmsZ protein for tolerating BPDE-

fork-blocking induced lesions ................................................. 107

CHAPTER IV

Figure 1: Northern blot analysis of the level of
expression of hRE V3 antisense ................................................ 127

Figure 2: UV-induced cytotoxiclty and mutagenicity
in the parental cell strain 9N and cell

strains 6|, BB, and 12C .......................................................... 131

Figure 3: BPDE-induced cytotoxiclty and mutagenicity
in the parental cell strain 9N
and cell strains 6| and 12C ..................................................... 135

Figure 4: BPDE-induced mutagenicity versus percent
survival in the parental cell strain 9N
and cell strains 6| and 12C ...................................................... 136

AP:
AAF:
AAF-G:

BPDE:

BPDE-G:

8-oxoG:
HPRT:
HR:
Hyg :
MMS:
NHEJ:
06-MeG:
Pol:
PCNA:
TG:
TLS:

T-T:

T-T (6-4):

ch:

XPV:

LIST OF ABBREVIATIONS

apurinic/apyrimidinic
N-2-acetyl-2-aminofluorene
AAF-adducted guanine

(:)-7B, 8a-dihydroxy-9a, 10a-epoxy-7, 8, 9, 10-tetrahydro-
benzo[a]pyrene or benzo[a]pyrene diol-epoxide

BPDE-adducted guanine
8-oxoguanine

hypoxanthine phosphoribosyltransferase
homologous recombination
hygromycin resistant

methyl methanesulfonate
non-homologous end joining
06-methylguanine

polymerase

proliferating cell nuclear antigen
6-thioguanine

translesion DNA synthesis

T-T cis-syn cyclobutane dimer
T-T pyrimidine (6-4) pyrimidinone
ubiquitin-conjugating enzyme

xeroderma pigmentosum variant

xi

INTRODUCTION

Prokaryotic and eukaryotic cells are able to replicate their DNA even when it
contains DNA damage. This so-called “damage tolerance" ensures that DNA
replication can continue even when DNA damage has not been efficiently
removed by DNA repair mechanisms. Damage tolerance is considered to
encompass two pathways, i.e., translesion synthesis and damage avoidance.
Translesion synthesis involves the newly discovered specialized DNA
polymerases that are able to insert nucleotides across from the damaged DNA
template when the major replicase, e.g., polymerase delta, is blocked by DNA
lesions. This process can be either error-prone or error-free depending on the
nature of the lesion and the characteristics of the particular DNA polymerase
used. Damage avoidance is the alternative pathway by which cells deal with fork-
blocking lesions by using an undamaged homologous copy of the replicating
sequence as a temporary template to circumvent the lesion. This error-free
pathway has been hypothesized (Higgins et al., 1976), but the exact mechanism

and various gene products involved remain obscure.

One of my dissertation research aims was to determine whether the hMMS2
gene is involved in damage tolerance in human cells and if so, to identify the
pathway(s) in which it is involved. The yeast MMSZ gene was cloned by Xiao
and colleagues (Broomfield et al., 1998). The mmsZ deletion mutants in yeast

exhibited an elevated UV-induced mutation frequency and spontaneous

mutations, although the exact mechanism was not understood (Broomfield et al.,
1998). The human counterpart of MMSZ, hMMSZ, was cloned (Xiao et al.,
1998a), which made it possible to determine the role of hMmsZ in damage
tolerance in human cells. The second part of my dissertation research was to
determine the role of hREV3 in error-prone translesion synthesis, i.e.,
carcinogen-induced mutagenesis. In yeast, the deletion mutants of rev3 exhibit
virtually no carcinogen-induced mutations and loss of the majority of
spontaneous mutations (Lawrence and Hinkle, 1996). Rev3 protein, the catalytic
subunit of DNA polymerase zeta, is able to perform translesion synthesis for
thymidine—thymidine dimers in vitro (Nelson et al., 1996). The human homolog,
hREV3, was cloned (Gibbs et al., 1998; Xiao et al., 1998b, Lin et al., 1999),
which made it possible to determine the role of hREV3 in error-prone translesion

synthesis in human cells.

Chapter I reviews the background literature of translesion synthesis and damage
avoidance in prokaryotic and eukaryotic cells, with the focus on eukaryotes.
Chapter II consists of a manuscript by Li et al. published in the April 2002 issue
of Proceedings of the National Academy of Science U. S. A., 99:4459-4464. It
describes the results of experiments I carried out using an antisense strategy to
eliminate hMmsZ protein in a human fibroblast cell strain and to identify the role
of hMmsZ protein in damage avoidance. In that study, I showed that hMmsZ is
essential for damage avoidance in human cells dealing with UV-induced

replication fork-blocking lesions (providing direct evidence for use of the allelic

copy and indirect evidence for use of the newly synthesized daughter strand of
sister duplex). The loss of hMms2 protein eliminated the ability of human cells to
use an undamaged homologous copy as the template dealing with fork-blocking
lesions and increased UV-induced mutant frequency. Chapter III consists of a
manuscript by Li et al. submitted to Mutation Research. It describes the
determination of the relative contribution of translesion synthesis versus damage
avoidance for the tolerance of DNA damage induced by benzo[a]pyrene diol-
epoxide (BPDE). I showed that hMmsZ is essential for damage avoidance
dealing with BPDE-induced fork-blocking lesions, and more importantly that
translesion synthesis is used almost equally to damage avoidance for tolerating
BPDE-induced fork-blocking lesions. Chapter IV consists of a manuscript by Li et
al. being prepared for submission to Mutation Research. It describes the
research I carried out using an antisense strategy to investigate the role of

hRE V3 in error-prone translesion synthesis for UV- or BPDE-induced lesions.

References

Broomfield, S., Chow, BL. and Xiao, W. (1998) MMSZ, encoding a ubiquitin-
conjugating-enzyme-Iike protein, is a member of the yeast error-free
postreplication repair pathway. Proc Natl Acad Sci U S A, 95, 5678-5683.

Gibbs, P.E., 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 U S
A, 95, 6876-6880.

Higgins, N.P., Kato, K. and Strauss, B. (1976) A model for replication repair in
mammalian cells. J Mol Biol, 101, 417-425.

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

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

Nelson, J.R., Lawrence, CW. and Hinkle, DC. (1996) Thymine-thymine dimer
bypass by yeast DNA polymerase zeta. Science, 272, 1646-1649.

Xiao, W., Lin, S.L., Broomfield, S., Chow, BL. and Wei, Y.F. (1998a) 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, 3908-3914.

Xiao, W., Lechler, T., Chow, B.L., Fontanie, T., Agustus, M., Carter, KC and
Wei, Y.F. (1998b) Identification, chromosomal mapping and tissue-specific
expression of hREV3 encoding a putative human DNA polymerase zeta.
Carcinogenesis, 19, 945-949.

CHAPTER I

LITERATURE REVIEW

A. Introduction

Cells are continuously exposed to endogenous and exogenous DNA damaging
agents. Certain types of DNA damage, e.g., ultraviolet (UV)-induced cyclobutane
pyrimidine dimers (CPDs) and pyrimidine-pyrimidone (6—4) photoproducts,
benzo[a]pyrene diol-epoxide (BPDE)—induced guanine adducts, and N-2-acetyl-
2-aminofiuorene (AAF)-induced lesions, will block normal DNA replication
carried out by the major DNA polymerases (Friedberg et al., 1995). Cells have
developed mechanisms to deal with such INA damage. For example, cell cycle
checkpoints are the mechanism by which cells temporarily inhibit normal DNA
replication in order to allow time for DNA repair before replication starts.
Checkpoints also prevent cells from entering mitosis until the full complement of
DNA has been replicated. Excision repair mechanisms are responsible for
physically removing such DNA lesions and restoring damaged DNA to its original
state. In addition, cells possess damage tolerance mechanisms i.e., translesion
synthesis (TLS) and damage avoidance (DA). TLS is a special replication
process by which cells are able to temporarily use various specialized DNA
polymerases to incorporate nucleotides opposite the damaged template.
Damage avoidance is a special replication process by which cells temporarily

use an undamaged alternative template to circumvent DNA lesions. I will briefly

discuss these two mechanisms in prokaryotes in Section B, and then I will

discuss these two mechanisms in detail in eukaryotes in Section C.

B. Damage Tolerance Mechanisms in Prokaryotes

1 . Overview of the SOS System

The SOS response is a mechanism by which E. coli cells deal with DNA damage,
particularly fork-blocking lesions. Approximately 30 gene products are induced in
the SOS response after E. coli cells are exposed to DNA damaging agents, and
the majority of these gene products are used for damage tolerance and DNA
repair (Friedberg et al., 1995; Fernandez De Henestrosa et al., 2000). Among
these gene products, RecA and LexA proteins play important roles in regulating
gene expression of other SOS regulon components. The LexA protein is a
repressor of the SOS regulon. It binds to the SOS operon promoter region and
prevents transcription of SOS response genes. Autodigesion of LexA, a process
assisted by RecA protein, triggers transcription of SOS regulon genes. RecA is a
single strand DNA (ssDNA) binding protein and it forms a nucleoprotein filament
with ssDNA (Friedberg et al., 1995). The RecA/ssDNA filament can assist
autocleavage of LexA protein, which activates the SOS-regulon. Dinl, a protein
that is also induced as part of the SOS response, inhibits co-protease activity of
the RecA/ssDNA nucleoprotein filament by inhibiting the autodigestion of LexA
protein. In summary, RecA is the activator of the SOS-regulon, whereas Dinl and

LexA act as repressors of the SOS regulon.

Once the SOS response is activated, the important gene products that are
induced are DNA polymerase V, which is encoded by the umuDC gene, and
polymerase IV, which is encoded by the dinB gene. Both polymerases IV and V
are the key components in E. coli translesion synthesis and mutagenesis.
Polymerase II, which is involved in recombination-mediated replication re-start, is

also induced (see below).

2. Translesion DNA Synthesis: The UmuDCIDinB proteins

Translesion DNA synthesis (TLS) is the mechanistic basis of the SOS response.
TLS allows cells to tolerate DNA damage to a certain degree by replicating DNA
containing lesions. Specifically, translesion synthesis is a special type of DNA
replication by which specialized DNA polymerases are able to insert nucleotides
opposite the damaged template that would otherwise block normal DNA
replication carried out by major replicase. In E. coli, TLS increases cell survival at
the expense of elevated mutations. This error-prone TLS requires the umuDC
operon, which was originally identified by screening for mutants that exhibited an
extremely low frequency of UV- and other carcinogen-induced mutations,
compared to the wild type (Steinbom, 1978). Translesion synthesis also requires

the E. coli dinB gene (Wanger et al., 1999) (see below).

2.1. UmuC and UmuD’ (Pol V)
E. coli DNA pol V is composed of one molecule of UmuC protein and two

molecules of UmuD’ protein, the post-translationally processed form of UmuD

protein. The biochemical nature of this processing is similar to that of LexA
autocleavage which is assisted by the RecA/ssDNA nucleoprotein filament. The
interaction between the RecA/ssDNA filament and UmuD protein activates the
autodigestion of UmuD, generating a protein that is missing its N-terrninal 25
amino acids (Nohmi et al., 1988; Shinagawa et al. 1988). The Dinl protein inhibits

this process by inhibiting the co-protease activity of RecA/ssDNA.

Goodman and colleagues (Tang et al., 1998; Tang et al., 1999; Tang et al., 2000)
and Livneh and colleagues (Reuven et al., 1998; Reuven et al., 1999) have
independently demonstrated in vitro that pol V is required for error-prone TLS
past abasic sites, thymidine-thymidine cis-syn dimers (T-T dimers), and T-T
pyrimidine (6-4) pyrimidinone [T-T (6-4) ] photoproducts. Both groups found that
UmuD’z alone is unable to carry out translesion synthesis in vitro (Tang et al.,
1998; Tang et al., 1999; Reuven et al., 1998; Reuven et al., 1999). However,
Reuven et al. (1999) showed that UmuC, by itself, is able to insert nucleotides
across from an undamaged template with a limited efficiency in vitro. However, it
needs UmuD’z, RecA, and single-strand DNA binding protein to carry out
translesion synthesis on a damaged template. These results indicate that the pol
V protein complex and associated factors are the replication machinery inserting
nucleotides opposite the damaged template. It is uncertain whether DNA Pol III
plays an essential role in translesion synthesis, although it is considered to carry

out the normal DNA replication once the lesion is bypassed.

In addition to their roles in translesion synthesis, the umuDC gene products may
also play important roles in DNA damage checkpoints (Opperman et al., 1999).
Opperrnan et al. (1999) observed that uncleaved UmuD in complex with UmuC
acts, in vivo, to inhibit DNA replication after UV-irradiation. Sutton et al. (2001)
showed the genetic interaction between the umuDC gene products and the beta
processivity clamp of the replicative DNA polymerase III. In vitro evidence
demonstrated that UmuD interacts with the pol llI beta processivity clamp at its
N-terminus domain, the domain that UmuD' protein is missing (Sutton et al.,
2002). This interaction is essential for the role of UmuD in checkpoints. After E.
coli cells are exposed to DNA damaging agents, the UmuD protein is probably
initially involved in prohibiting cell-cycle progression before DNA replication and
then it is degraded into UmuD' protein and the UmuCD’z complex triggers
translesion DNA replication. Therefore, regulation of the autodigestion of UmuD

is critical for cells to switch from checkpoint to translesion synthesis.

2.2. DinB (Pol IV)

The E. coli dinB gene is a part of the SOS regulon (Kenyon and Walker, 1980). It
was later shown to encode a novel E.coli DNA polymerase IV (pol IV) involved in
spontaneous mutagenesis (Wagner et al., 1999; Strauss et al., 2000) and
induced mutagenesis (Napolitano et al., 2000). The importance of the dinB gene
in mutagenesis was originally discovered in studies with lambda phage
untargeted UV-mutagenesis (Brotcorne-Lannoye and Maenhaut—Michel, 1986),

indicating that it is important for UV-induced error-prone translesion synthesis. In

addition, over-expression of the DinB protein enhanced spontaneous mutations
(Kim et al., 1997). DinB was shown to be distributive, devoid of proofreading
activity, and able to elongate a DNA substrate with a bulged-out nucleotide in the
template in vitro, which resulted in -1 frameshift mutations (Wagner et al., 1999).
DinB was named DNA polymerase lV (pol IV) (Wagner et al., 1999). This implies
that pol IV is important for bypassing certain type of DNA lesions, although the
lesion specificity for pol IV both in vivo and in vitro is not well understood.
However, Kim et al. (2001) reported that DNA pol IV is involved in -1 frameshift
mutagenesis induced by 4-nitroquinoline N-oxide (4-NQQ) and in E. coli the
expression level of pol IV protein is 6 to12 times higher than those of other SOS-

inducible DNA polymerases in E. coli such as DNA pol II and DNA pol V.

The interaction between pol IV and the processivity factor of replicative pol lll
strongly increases its processivity (Wanger et al., 2000) and this interaction is
presumably essential for pol lV’s role in translesion synthesis and mutagenesis.
Lenne-Samuel et al. (2002) recently showed that five amino acids at the C-
terrninal of pol IV are essential for the interaction between pol IV and the beta-
clamp of pol Ill. Disruption of this interaction obliterates the function of pol IV in

both spontaneous and induced mutagenesis in vivo (Lenne-Samuel et al., 2002).

The crystal structure of the Sulfolobus solfataricus pol IV homolog in complex

with DNA and an incoming nucleotide has been determined (Ling et al., 2001;

Zhou et al., 2001). The results revealed that, despite having a conserved active

10

site and a palm-like configuration similar to all known DNA polymerases, pol IV
has very limited and nonspecific contact with the replicating bases (Zhou et al.,
2001). This could explain why pol IV has a low processivity and has a low fidelity

in choosing the incoming nucleotides (Zhou et al., 2001).

3. SOS-regulated daughter-strand gap repair (Recombination)

In E. coli, recombination-mediated replication restart is a major DNA tolerance
mechanism. It is an error-free pathway. Early studies demonstrated that
recombination is involved in the tolerance of UV-induced DNA damage in uvr E.
coli mutants. In 1971, Howard-Flanders and colleagues using density transfer
experiments showed that recombinants form between irradiated DNA strands
and strands synthesized after irradiation (Rupp et al., 1971). Later, it was shown
that T-T dimer exchange occurs between strands with a DNA lesion and strands

without damage (Ganesan, 1974).

In E. coli, when the major DNA replicase pol III is blocked by UV-induced DNA
damage, the DNA replication machinery can re-establish downstream of the
block and resume DNA synthesis. This results in a single-strand DNA gap
between the two newly synthesized DNA fragments. The gap is then filled by a
DNA recombination mechanism (Rupp and Howard-Flanders, 1968; Rupp et al.,
1971) termed daughter-strand gap repair. It is the gaps in the daughter-strand,
rather the lesion itself, that are repaired by this mechanism. Therefore, this is a

mechanism for tolerating DNA damage rather than physically removing the

11

lesion. The recombination-mediated single-strand gap repair provides a
mechanism that avoids the fork-blocking lesions, in contrast to translesion
synthesis in which specialized DNA polymerases incorporate nucleotides across

from the damaged template.

The existence of this recombination-mediated single-strand gap repair is further
supported by the following observations. E. coli cells carrying mutations in umuD
or umuC, which encodes pol V, are still capable of resuming DNA synthesis
shortly after UV-irradiation (Witkin et al., 1987; Rangarajan et al., 1999). This
indicates that, in addition to the translesion synthesis pathway, an additional
pathway, which was later found to be dependent on damage-inducible DNA
polymerase pol II, is present to deal with fork-blocking lesions in E coli
(Rangarajan et al., 1999). Rangarajan et al. (2002) recently showed that both pol
II and the origin-independent primosome-assembling function of PriA are
essential for the efficient recovery of DNA synthesis after UV-irradiation,
indicating that pol II and PriA are important for the recombination-mediated
replication restart. They also showed that umuDC priA or umuDC poIB (poIB
encodes pol ll) double mutants fail to resume DNA synthesis after UV-irradiation
(Rangarajan et al., 2002). These data further indicate that translesion synthesis
involving pol V and the recombination-mediated error-free pathway involving
RecA, pol II, and PriA are alternative pathways for tolerating fork-blocking lesions

and to resume DNA replication.

12

Recently, increasing evidence has been obtained in prokaryotes that
homologous recombination plays an important role in an error-free process
dealing with replication fork-blocking lesions. DNA single-strand gaps formed
during DNA replication and possibly double strand breaks caused by replication
fork arrest are excellent substrates for homologous recombination (Haber, 1999;
Cox et al., 2000; Kowalczykowski, 2000; Kuzminov, 2001). Thus, DNA
replication, repair, and recombination are coordinated events that prevent

genomic instability caused by DNA damage-induced replication arrest.

C. Damage Tolerance Mechanisms In Eukaryotes

Damage tolerance mechanisms in eukaryotes are quite different from those in
prokaryotes with regard to the protein components involved. However,
prokaryotic and eukaryotic organisms share the same basic strategy to deal with
fork-blocking lesions induced by DNA damaging agents, i.e., translesion
synthesis and damage avoidance are the major mechanisms dealing with
replication fork-blocking lesions. In prokaryotes, as discussed above, damage
tolerance mainly relies on translesion synthesis by pol V and pol IV, and the
error-free recombination-mediated gap “repair" pathway. In eukaryotes,
translesion synthesis is also a major pathway to bypass DNA damage, but it is
more complex and has more components involved than the equivalent pathway
in prokaryotes. Damage avoidance, similar to the recombination-mediated error-

free pathway in E. coli, is the alternative pathway to deal with fork-blocking

13

lesions by circumventing DNA lesions (Higgins et al., 1976; Li et al., 2002). In
eukaryotic cells, damage tolerance mechanisms are dependent on the RADB-
RAD18 pathway (Montelone et al., 1981; di Caprio and Cox, 1981). In this
pathway, multiple DNA polymerases are involved in translesion synthesis (see
below). There is another sub-pathway in the RADB-RAD18 epistasis group called
the MMSZ-UBC13 pathway. This pathway contains proteins shown to be
involved in the damage avoidance pathway in human cells (Li et al., 2002) and is

an error-free pathway in S. cerevisiae (Broomfield et al., 1998; Xiao et al., 1999).

1. Overview of RAD6-RAD18 Pathway

In S. cerevisiae, both rad6 and rad18 mutants are sensitive to various DNA
damaging agents and deficient in damage tolerance (Montelone et al., 1981; di
Caprio and Cox, 1981). These mutants were initially identified by Cox and Parry
(1968) in a screen of UV-sensitive S. cerevisiae mutants. It was later confirmed
that the mutagenic effect of UV irradiation indeed depends on Rad6 and Rad18
proteins (Cassier-Chauvat and Fabre, 1991). The RAD6-RAD18 epistasis group
in yeast includes proteins involved in translesion synthesis, e.g., pol eta (Johnson
et al., 1999a; Johnson et al., 1999b), pol zeta (Nelson et al., 1996a; Lawrence
and Hinkle, 1996), and proteins involved in an error-free pathway, i.e., MMSZ
(Broomfield et al., 1998), UBC13 (Brusky et al., 2000), RADS (Johnson et al.,
1992), and POL30 (T ones-Ramos et al., 1996). In human cells, in addition to the
human homolog of pol eta (Masutani et al., 1999a; Masutani et al., 1999b) and

zeta (Gibbs et al. 1998, Gibbs et al., 2000, Murakumo et al. 2000), there are

14

additional kinds of DNA polymerases that yeast cells do not possess, e.g., pol
iota (Tissier et al., 2000a; Tissier et al., 2000b), pol kappa (Gerlach et al., 1999;
Gerlach et al., 2001), pol mu (Dominguez et al., 2000; Aoufouchi et al., 2000),
and pol lambda (Garcia-Diaz et al., 2000; Aoufouchi et al., 2000). Most, if not all,
of these polymerases, are involved in translesion synthesis for different DNA
replication fork-blocking lesions. The damage avoidance pathway in human cells
is not well understood, but a recent report indicates that the human homolog of

MMSZ, hMMSZ, is involved (Li et al., 2002).

1.1. RADG

ficchgmmvces cerevisiafi In budding yeast, Rad6 protein is a ubiquitin-
conjugating enzyme (Jentsch et al., 1987). Rad6 forms a stable complex with
Rad18 and is involved in diverse cellular functions. rad6 mutants are extremely
sensitive to various DNA damaging agents such as UV, X-ray, y-ray, bleomycin
and alkylating agents and are defective in carcinogen-induced mutagenesis
(Prakash et al., 1993). The RADG gene product is also important for sporulation
(Morrison et al., 1988), telomere silencing (Huang et al., 1997), and N-terrninal

rule based protein degradation (Dohmen et al., 1991).

The ubiquitin-conjugating enzyme (ch) activity of Rad6 resides on the active
site Cys88. All the cellular functions of Rad6 require its ch activity because
mutation of the active site Cys88 results in a rad6 mutant phenotype (Sung et al.,

1990). Significant biochemical studies have focused on discerning regions of the

15

protein required for specific biological functions. The C-terrninal acidic residues
are important for the ubiquitination of histone HZB (Sung et al., 1988; Robzyk et
al., 2000), which is required for sporulation (Morrison et al., 1988). The N-
terrninal region of Rad6 is involved in N-end rule protein degradation (Dohmen et
al., 1991) and sporulation (Morrison et al., 1988). Deletion of N-terrninal amino
acids of one to nine does not disrupt ch-Ubiquitin thiolester formation, but
prevents the interaction between Rad6 and a ligase protein (E3), Ubr1 (Watkins
et al., 1993), which is required for N-end rule protein degradation (Dohmen et al.,
1991; Sung et al., 1991). Compared to rad6 deletion mutants, which are
extremely sensitive to the cytotoxic effect of UV and deficient in UV-induced
mutagenesis, cells expressing Rad6 with an N-terrninal deletion of amino acid
one to nine are moderately sensitive to the cytotoxic effect of UV but proficient in

UV-induced mutagenesis (Watkins et al., 1993).

Human cells Schneider et al. (1990) discovered that the human ubiquitin carrier
protein E2 is homologous to yeast Rad6, indicating the existence of a human
homolog of RADB. Subsequently, two human homologs of RADG, hHR6A and
hHRBB, were cloned (Koken et al., 1991). hHR6A is located on chromosome
Xq24-25 and hHR6A is located on chromosome 5q23-31 (Koken et al., 1992).
Co-expression of hRAD18 and hHR6 in yeast cells revealed the stable hRad18-
hHR6A and hRad18-hHR63 protein complexes (Xin et al., 2000), indicating that
the human homologs of Rad6 interact with hRad18, which is similar to what is

observed in yeast.

16

Inactivation of HRGB in mice causes male sterility associated with chromatin
remodeling (Roest et al., 1996; Koken et al., 1996). Interestingly, hHR6B protein
is up-regulated in metastatic mammary tumor cell lines (Shekhar et al., 2002).
Constitutive over-expression of exogenous hHRGB cDNA induces cell-cell fusion,
centrosome amplification, multipolar mitotic spindles, aneuploidy, and
anchorage-independent growth (Shekhar et al., 2002). These findings indicate
that hRad6 plays an important role in the maintenance of genomic stability, and
the loss of regulation of hRad6 protein may lead to chromosomal instability and
transformation. Recently, hHR6A was shown to be phosphorylated in vitro by
cyclin-dependent kinase 2 (CDK2) at residue Ser120. This is the first study to
demonstrate regulation of ubiquitin conjugation by the phosphorylation of this
conserved residue (Sarcevic et al., 2002). It suggests that CDK-mediated
phosphorylation of hHR6A is an important regulatory event in the control of cell

cycle progression (Sarcevic et al., 2002).

1.2. RAD18

Saccharomyces cerevisiae In budding yeast, Rad18 forms a complex with Rad6,
a ubiquitin-conjugating enzyme, through its RING finger domain (Jentsch et al.,
1987). Rad18 has an ATPase activity and is able to bind single-strand DNA
(Bailly et al., 1994; Bailly et al., 1997a; Bailly et al., 1997b). The RING finger
domain of Rad18 is also important for the interaction between Rad18 and the
Mms2-ch13 complex (Ulrich and Jentsch, 2000), bringing the Rad6-Rad18

complex into contact with the Mms2-ch13 complex. Unlike Rad6, the role of

17

Rad18 seems to be limited to the damage tolerance pathway. rad18 mutants are
defective in the repair of single-stranded gaps induced by y-rays, but proficient in
repairing double-stranded breaks created during the same treatment (Geigl and
Echardt-Schupp, 1991). Rad18 is also involved in maintaining the integrity of
single-stranded and linear DNA created after a DNA damaging treatment (Mowat

et al., 1983).

A homolog of RAD18 in fission yeast Schizosaccharomyces pombe, rhp18, was
characterized by Verkade et al. (1999). As in Saccharomyces cerevisiae, rhp18
deletion mutants are hypersensitive to DNA damaging agents, and have a long
DNA damage checkpoint arrest (Verkade et al., 2001). This result indicates that

the function of RAD18 is conserved in yeast.

Humai cells The human homolog of RAD18, hRAD18, was cloned by Tateishi et
al. (2000). hRAD18 is located on chromosome 3p24-25. The human Rad18
protein has 484 amino acids with a calculated molecular weight of 54 kDa
(Teteishi et al. 2000). hRad18 protein binds to hHR6 proteins through a
conserved RING finger motif. Stable transfectants expressing hRad18 protein
mutated in this RING finger motif (cystein 28 to phenylalanine) become more
sensitive to the cytotoxic effect of UV, methyl methanesulfonate (MMS), and
mitomycin C, and are defective in replicating UV-induced DNA lesions (T ateishi

et al., 2000). These data indicate that the interaction between hRad18 and hHR6

l8

proteins is important for damage tolerance and mutagenesis, and also suggest

that hRad18 has a similar function to that of its yeast counterpart.

M_o_u_s_e_ The mouse homolog of RAD18, designated mRAD1BSc, was identified
and characterized by van der Laan et al. (2000). The mRAD188c gene is located
on mouse chromosome 6F. Its open reading frame encodes a protein of 509
amino acids with a molecular weight approximately 56 kDa. High homology was
found between yeast Rad18, mouse Rad18, and human Rad18, particularly with
the conservation of the RING finger domain. As yet there has been no report of

RAD18 knock out mice yet.

2. Translesion Synthesis: The UmuC/DinB Superfamily

Analogous to the Eco/i SOS system, eukaryotic cells utilize translesion synthesis
as a mechanism to deal with fork-blocking lesions. The Rad6 and Rad18 proteins
are essential for translesion synthesis in eukaryotic cells, although the exact
mechanism is not fully understood. Recent studies have confirmed that in
eukaryotic cells, translesion synthesis relies on newly discovered DNA
polymerases that belong to the UmuC/DinB superfamily. Both in yeast and in
human cells, polymerase zeta (pol Q), composed of Rev3 and Rev7 proteins, and
polymerase eta (pol n) are important for translesion synthesis past lesions
induced by various DNA damaging agents. In higher eukaryotes, e.g., mice and
humans, additional kinds of DNA polymerases exist that might be involved in

translesion synthesis past DNA lesions, e.g., polymerase iota (pol I), polymerase

19

kappa (pol x), polymerase mu (pol II), and polymerase lambda (pol I.) (see

below). However, pol II and pol 7L do not belong to UmuC/DinB family.

2.1. Yeast and human polymerase zeta and Rev1

2.1.1. Polymerase zeta

Saccharomyces cerevisiae One type of translesion synthesis carried out in yeast
is dependent on polymerase zeta (pol Q), which is generally considered an error-
prone polymerase dealing with a variety of DNA fork-blocking lesions. The rev3
mutants were initially isolated in S. cerevisiae, along with rev1 mutants, based on
their inability to revert arg4-17 and Iys1-1 alleles in response to UV irradiation
(Lemontt 1971). Studies in yeast strongly suggest that almost all of damage-
induced mutations and a large portion of spontaneous mutations are generated
by Rev3 protein (Lawrence and Hinkle, 1996), the catalytic subunit of pol C,
(Morrison et al., 1989). Recently, Pavlov et al. (2001) reported that replacing
each tyrosine residue with an alanine in the conserved motif of the catalytic
subunit of pol g in yeast causes the loss of UV-induced mutations. This result
confirms that the catalytic activity of pol t; is essential for mutagenesis. The pol Q
translesion pathway is dependent the gene products of RADG and RAD18
(Lawrence and Hinkle, 1996). Genetic evidence showing that REV3 belongs to
the RAD6-RAD18 pathway comes from the finding that rad6-1 is epistatic to
rev3-1 for the cytotoxic effect of UV (Lawrence and Christensen, 1976). rad6 and
rad18 mutants are extremely sensitive to the cell killing by a variety of DNA

damaging agents and are defective in UV-induced mutagenesis. In contrast, rev3

20

mutants display only slightly increased sensitivity to the cytotoxic effect of UV but

a greatly reduced frequency of UV-induced mutations (Lemontt 1971).

In addition to Rev3, pol Q contains another component, the Rev7 protein. The
rev7 mutants were isolated for their deficiency in reverting the UV-induced IysZ
allele (Lawrence et al., 1985a). The effect of the rev7 mutation was found to be
target allele-specific and mutagen-specific (Lawrence et al., 1985b), indicating
that, in contrast to Rev3, Rev7 is less important in some carcinogen-induced

mutagenesis.

The REV3 gene in yeast encodes a protein that has 1,504 amino acids and a
molecular weight of 173 kDa with conserved DNA polymerase motifs (Morrison et
al., 1989). The REV7 gene encodes a protein with a molecular weight of 28 kDa
(T orpey et al., 1994). Biochemical studies showed that the dimerization of Rev3
and Rev7 forms functional pol Q (Nelson et al., 1996a). Translesion synthesis
past T—T dimers is carried out more efficiently by DNA pol Q than by yeast DNA
pol or, indicating that pol Q is indeed capable of inserting nucleotides across from
DNA lesions. Pol Q was also shown to perform error-prone translesion synthesis
in vitro past a T-T (6-4) photoproduct and an acetylaminofluorene (AAF)-
adducted guanine (AAF-G) (Guo et al., 2001). In addition, pol Q extends
mismatches with high efficiency including nucleotides inserted opposite a lesion
(Johnson et al, 2000; Guo et al., 2001). Another example of yeast pol Q being a

mismatch extender comes from the in vitro efficient bypass of an abasic (AP) site

21

by the sequential combined action of pol 6 and pol Q (Haracska et al., 2001 a).
Pol 8 is able to insert an A nucleotide opposite an AP site, and pol Q
subsequently extends from the inserted nucleotide, but it is not efficient in
incorporating nucleotides across from the AP site (Haracska et al., 2001a).
These results indicate that pol Q has a dual-function: it can function as a special
DNA polymerase that incorporates nucleotides opposite some DNA lesions, and
it can sometimes function as an extension DNA polymerase. However, whether
this is true in vivo is unclear. Nevertheless, pol Q must be acting as either an
error-prone polymerase by itself or an extender of other error-prone
polymerases, or both, during translesion synthesis because deletion of REV3

virtually eliminates carcinogen-induced mutations.

Human cells The human homolog of the yeast REV3 gene, hREV3, was
independently cloned by three groups (Xiao et al., 19983; Gibbs et al., 1998; Lin
et al., 1999a) and the mouse homolog was also identified (Van Sloun et al.,
1999). The hREV3 gene is localized on chromosome 6q21 (Morelli et al., 1998;
Kawamura et al., 2001). Its cDNA consists of 10,716 base pairs and encodes for
a predicted protein of 3,130 amino acids with a predicted molecular weight of 350
kDa, about twice the size of the yeast counterpart that has 1,504 amino acids.
The deduced hRev3 protein also contains in the right order all six motifs that are
the characteristic of eukaryotic DNA polymerases. The yeast and human Rev3

proteins share 29% identity at the amino terminal region of ~350 residues, 39%

22

identity within the carboxyl-tenninal region of ~850 residues, and 29% identity

within an internal region of 55 residues (Gibbs et al., 1998).

In addition to the start codon ATG for the main open reading frame (ORF), the
hREV3 gene possesses an out-of-frame ATG codon at —58 at the 5’ end of
untranslated region (Gibbs et al., 1998). Furthermore, a common variant
transcript with an insertion of 128-bp between nucleotides +139 and +140 was
found both in human (Gibbs et al., 1998; Kawamura et al., 2001) and in mouse
REV3 cDNAs (Kawamura et al., 2001). Neither the insertion variant nor the out-
of-frame start codon will produce the predicted Rev3 protein, indicating that the
Rev3 protein level in higher eukaryotes is strictly regulated. hREV3 and its
insertion variant transcripts are ubiquitously detected in all normal human
tissues, with an additional variant species found in tissues with relatively high

levels of hRE V3 expression (Kawamura et al., 2001).

Murakumo et al. (2000) reported the discovery of a putative human homolog of
yeast RE V7, hRE V7, using a yeast two-hybrid screen. The deduced hRE V7 gene
product displays 23% identity and 53% similarity with yeast Rev7 protein, as well
as 23% identity and 54% similarity with the human mitotic checkpoint gene
product hMad2 (Murakumo et al., 2000). hREV7 is located on human
chromosome 1p36 in a region exhibiting frequent loss of heterozygosity in human
tumors (Murakumo et al., 2000). The precise domain of hRE V3 that interacts with

hREV7 is within the region of amino acid residues 1847-1892 and the precise

23

domain for hREV7 that interacts with hREV3 is within the region of amino acid
residues 21-155 (Murakumo et al., 2001). Interestingly, an interaction between
hRev7 and hMad2 was identified, indicating that hREV7 may act as an adapter
between damage tolerance and the spindle assembly checkpoint (Murakumo et

al., 2000). The functional of this putative hREV7 in vivo is yet to be determined.

The translesion polymerase Q was recently shown to be involved in
immunoglobulin gene somatic hyperrnutation (Zan et al., 2001). In mammals, in
order to produce high affinity antibodies against a specific antigen, the DNA
coding for the V-region of an antibody gene has to be vigorously mutated, such
that all kinds of different antibodies will be produced and ultimately one of these
antibodies will be selected. This process of mutating DNA in the V-region is
referred to as somatic hypennutation. Inhibition of the catalytic subunit of pol Q,
REV3, by specific phosphorothioate-modified oligonucleotides was found to
impair immunoglobulin hyperrnutation (Zan et al., 2001). In addition, transgenic
mice that express REV3 antisense showed a delay of generating high affinity
antibodies and a decreased somatic hyperrnutation in the V-region (Diaz et al.,
2001). These results suggest that translesion synthesis carried out by pol Q plays

an important role in the process of generating high affinity antibodies.
Unfortunately, to date, there has been no success in knocking out the REV3

gene in mice. Four different groups attempted to knock out the REV3 gene in

mice and discovered that REV3"‘ mice exhibited early embryonic lethality

24

(Esposito et al., 2000; Wittschieben et al., 2000; Bemark et al., 2000; Van Sloun
et al., 2002). Mouse fibroblasts derived from the REV3" embryos could not be
propagated. A significant increase in double-strand DNA breaks as well as
chromatid and chromosome aberrations was observed in cells from REVT‘
embryos (Van Sloun et al., 2002), indicating that REV3 might be involved in
dealing with double-strand breaks and maintaining genomic stability. These
studies with REV3 provide the evidence that an enzyme involved in translesion

synthesis and mutagenesis is critical for mammalian embryonic development.

Because no one has succeeded in knocking out REV3 gene in mice, it is very
difficult to study an in vivo model system to fully explore the biological function of
REV3 gene. There has been no report of Rev3 protein expression, purification,
and biochemical characterization in mammalian cells, either. It is very important

to break through these two barriers to extend the REV3 research.

2.1.2. REV1

Mmmyces cerevisiae rev1 mutants were initially isolated in S. cerevisiae
along with rev3 mutants one the basis of their inability to revert arg4-17 and Iys1-
1 alleles in response to UV irradiation (Lemontt 1971). The 3.1 Kb REV1
transcript encodes a 985 amino acid-protein with a molecular weight of 112 kDa.
rev1 deletion mutants exhibit a reduced frequency of UV-induced mutations
compared to wild type cells (Larimer et al., 1989). REV1 is a nonessential gene

and its gene product is an auxiliary factor of pol Q (Lawrence and Hinkle, 1996).

25

A 152-residue internal segment of Rev1 protein shows 25% identity with E. coli
UmuC protein (Larimer et al., 1989). Rev1 has a deoxycytidyl transferase activity
on a template containing an AP site (Nelson et al., 1996b). Insertion of cytosine
by Rev1 opposite an AP site produces a terminus that is efficiently extended by
pol Q (Nelson et al., 1996b). In addition to its role as a deoxycytidyl transferase,
Rev1 appears to have a second function. Nelson et al. (2000) reported that Rev1
function is needed for the bypass of a T-T (64) UV photoproduct, a process in
which dCMP incorporation occurs very rarely. Replication past this lesion is much
reduced in the G193R rev1-1 mutant, which retains substantial levels of
deoxycytidyl transferase activity. This mutant is, therefore, deficient principally in
the second function which might be the DNA polymerase activity. Consistent with
this, Haracska et al., (2002a) showed recently that Rev1 is a G template-specific
DNA polymerase, specifically inserts a C residue opposite template G, and that
Rev1 is approximately 25-, 40-, and 400-fold less efficient at inserting a C
residue opposite an AP site, an 06-methyllguanine (Os-MeG), and an 8-
oxoguanine (8-oxoG) lesion, respectively. Rev1 misincorporates G, A, and T
residues opposite template G with a frequency of approximately 10’3 to 10“

(Haracska et al., 2002a).

Human cells The human homolog of yeast REV1 gene, hREV1, was identified by
two different groups (Lin et al., 1999b; Gibbs et al., 2000). hREV1 is localized
between 2q11.1 and 2q11.2 (Lin et al., 1999b). Its cDNA consists of 4,225bp and

codes for 1,251 amino residues with a calculated molecular weight of 140 kDa.

26

Significant homology exists between hRev1 and Rev1 proteins. In two regions of
their N-terminus (about 100 amino acids in length), these proteins exhibit 41%
and 20% identity. A centrally located motif that is highly conserved in Rev1
proteins is also found in E. coli pol IV (Gibbs et al., 2000). The level of hREV1
mRNA is very low (Gibbs et al., 2000). Like the hREV3 transcript, hRE V1 mRNA
has a 5’ out-of-frame ATG at position -35 upstream of the main translation start

codon, which could explain why the hRev1 protein level is low in human cells.

hREV1 interacts with hREV7 in a yeast two-hybrid system using hREV7 as the
bait (Murakumo et al., 2001). hREV1 interacts with the region of amino acid
residues 21-155 of hREV7 and the minimum interaction domain of hREV1 is
within amino acid residues 1130-1251. It appears that hRev7 interacts with
hRev3 and hRev1 proteins in the same region (Murakumo et al., 2001). The
interaction between hRev1 and hRev7 was confirmed by in vitro and in vivo
binding assays (Murakumo et al., 2000). Although hREV7 interacts with both
hREV1 and hREV3, a stable complex formation of these three proteins has not
been detected in vitro, and no direct interaction between hREV1 and hREV3 has
been found (Murakumo et al., 2000). These findings suggest that hRev7 plays an
important role in bringing hRev1 and hRev3 proteins together for translesion

synthesis.

Like yeast Rev1, hRev1 protein was shown to have a deoxycytidyl transferase

activity and to efficiently insert a dCMP opposite a DNA template with an AP site

27

(Lin et al., 1999b). Deletion and point mutation analysis of the hRev1 protein
revealed that the domain required for deoxycytidyl transferase and DNA binding
activities is located in a conserved domain of translesion DNA polymerase
(Masuda et al., 2001). This result indicates that structure of the catalytic site of
deoxycytidyl transferase closely resembles a translesion DNA polymerase.
However, this result is contradictory to what is described in yeast in which it
seems that dCTP transferase domain and polymerase domain are separate
(Nelson et al., 2000). Recently, Zhang et al. (2002) showed in vitro that purified
hRev1 lacks a 3' to 5' proofreading exonuclease activity and exhibits a DNA
polymerase activity on a repeating template G sequence, similar to that of its
yeast counterpart (Haracska et al., 2002a). hRev1 is able to insert a dCMP
efficiently opposite a template containing 8-oxoG, BPDE-adducted guanine
(BPDE-G), or 1,N6-ethenoadenine adducts, and very inefficiently opposite an
AAF-G, but is unresponsive to a template containing a T-T dimer or T-T (6-4)
photoproduct (Zhang et al., 2002). This is not consistent with what is described in
yeast (Nelson et al., 2000). Nevertheless, both the yeast Rev1 and the human

Rev1 possess a polymerase activity on a repeating G-template.

Pioneering biological function studies of hREV1 in vivo were carried out using the
expression of hREV1 antisense and the results showed that human cells
expressing a high level of antisense hREV1 have a greatly reduced UV-induced

mutation frequency (Gibbs et al., 2000). The biological data strongly support the

28

idea that hRev1 is an auxiliary factor of human pol Q that is involved in an error-

prone translesion synthesis.

2.2. Yeast and human polymerase eta

Saccharomyces cerevisiae The alternative translesion synthesis pathway in
yeast is the RAD30 or pol n pathway. Earlier studies showed that mutations in
RAD30 increased the cytotoxic effect of UV and enhanced UV-induced mutability
(McDonald et al., 1997; Roush et al., 1998) but showed minor sensitivity to other
DNA damaging agents (Roush et al., 1998). Rad30 is a DinB-like protein. Like
dinB in E. coli, RAD30 is UV-inducible in yeast. Genetic analysis showed that
RAD30 belongs to the RADG epistasis group because rad6 or rad18 deletion
mutants are epistatic to rad30 deletion mutants, and rad30 mutants are additive
to rev1, rev3, and rev7 mutants for UV-sensitivity (McDonald et al., 1997). These
results indicate that for UV-irradiated DNA, Rad30 protein is involved in an error-
free pathway parallel to the error-prone translesion synthesis pathway of pol Q
dealing with fork-blocking lesions. Later, Johnson et al. (1999b) demonstrated
that Rad30 is able to carry out error-free translesion synthesis past T-T dimmers,

and therefore, Rad30 protein was named polymerase eta (pol n).

The polymerase activity of pol TI is required for the resistance to cell killing and
prevention of mutagenesis in response to UV-irradiation in yeast (Johnson et al.,
1999a), indicating the error-free nature of pol n in damage tolerance.

Interestingly, it was suggested that pol 8 is more error-prone than pol Tl during

29

the translesion synthesis past N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-
induced Iesions (Os-MeG) in vivo (Haracska et al., 2000a) because a rad30
pol32 double mutant shows an abolished MNNG-induced mutagenesis whereas
a rad30 single mutant shows no defect [Pol32 is a non-essential subunit of the
pol 8 complex (Gerik et al., 1998)]. Pol 11 is ten times more efficient inserting
bases across from 06-MeG in vitro than is pol 8. Pol 8 has strong stall sites both
immediately preceding the lesion, and directly across from it, whereas these stall
sites are much weaker for pol Tl (Haracska et al., 2000a). Pol n inserts a C
across from 06-MeG twice as frequently as pol 8, although both polymerases can
be error-prone for this kind of lesion (Haracska et al., 2000a). These results
indicate that, when pol 8 fails to replicate DNA lesions, pol TI might be recruited to
bypass these lesions with a reasonable accuracy. Washington et al. (2001a)
showed that pol n utilizes an induced-fit mechanism to selectively incorporate the
correct nucleotide, which explains the error-free nature of pol 11 during translesion
synthesis for a lot of DNA lesions. During the initial nucleotide-binding step, pol n
is not selective between the correct and incorrect nucleotide. The subsequent
“induced-fit” conformational change step allows pol TI to select the correct base
(Washington et al., 2001a). Pol TI interacts with the proliferating cell nuclear
antigen (PCNA), and this interaction is essential for the function of pol n in vivo.
Pol 11 is highly inefficient at inserting a nucleotide opposite an AP site, but

interaction with PCNA greatly stimulates its ability for nucleotide incorporation

opposite this lesion (Haracska et al., 2001b).

30

The accuracy of pol Tl replicating a T-T dimer is almost the same as replicating
an undamaged T-T template (Washington et al., 2000). Genetic studies also
indicate that pol n is error-free replicating cyclobutane dimers and (6-4)
photoproducts formed at CC and TC sites in vivo (Yu et al., 2001). Opposite the
3' T of a (6-4) T-T photoproduct, both yeast and human 11 preferentially insert a G
residue, but they are unable to extend from the inserted nucleotide (Johnson et
al., 2001). Pol 11 can also efficiently insert C and A opposite 8-oxoG, a C opposite
an AAF-G, and a G or less frequently an A opposite an AP site, but it is unable to
extend in both cases (Yuan et al., 2000). These results indicate that pol n can be
error-prone when replicating certain types of lesions. However, Haracska et al.
(2000b) showed that yeast and human pol Tl replicate 8-oxoG efficiently by
inserting a cytosine across from the lesion (error-free). This result is not

consistent with the finding of Yuan et al. (2000).

Domains essential for pol 11 function were identified. Deleting the last 54 amino
acids of pol 1'] has no effect on its DNA polymerase or T-T dimer bypass activity
in vitro, but abolishes its biological function in vivo (Kondratick et al., 2001).
There is a bipartite nuclear targeting sequence within C-terminal, indicating that
portion of Rad30 is for nuclear localization. Acidic residues Asp30, Glu39, and
Asp155 are critical for the catalytic activity and biological function of pol n
(Kondratick et al., 2001). These residues are able to bind two metal ions required
for the reaction of the incoming deoxynucleoside 5'-triphosphate with the 3'-

hydroxyl in the primer terminus. Trincao et al. (2001) reported the crystal

31

structure of pol n, which revealed a novel polydactyl right hand-shaped molecule
with a unique polymerase-associated domain. The fingers and thumb domains
are unusually small and stubby. The openness of the active site is the critical

feature that enables pol 'fl to replicate past DNA lesions, such as T-T dimers.

Human cells The human homolog of RAD30, hRAD30, was discovered by two
research groups (Masutani et al., 1999a; Johnson et al., 1999c). hRA030 gene is
located at chromosome 6p21.1-6p12, has 11 exons covering the entire coding
sequence, and lacks a TATA sequence in the upstream region of the
transcription initiation site (Yuasa et al., 2000). Like Rad30 in yeast, hRad30 is a
DNA polymerase 11 (hpol n) that is able to perform accurate TLS past T-T dimers
in vitro (Masutani et al., 1999b; Washington et al., 2001b). Interestingly, the
hRAD30 gene is either truncated or has a reduced expression in xeroderma
pigmentosum variant (XPV) (Masutani et al., 1999a; Johnson et al., 19990). This
explains why XPV cells are hyperrnutable in UV—induced mutagenesis (Maher et
al., 1976) and exhibit a greatly reduced ability to perform TLS in vitro (Ensch-
Simon et al., 1998; Svoboda et al., 1998; Cordonnier et al., 1999). It is generally
considered that, for reactions in vitro, hpol n is error-free for UV-induced lesions
(Masutani et al., 1999b) and error-prone for some other lesions (Zhang et al.,

2000a; Chiapperino et al., 2002).

Although hpol n is able to perform accurate TLS past certain types of lesions in

vitro (Masutani et al., 1999b; Washington et al., 2001b; Haracska et al., 2000b), it

32

replicates undamaged DNA with much lower fidelity than any other template-
dependent DNA polymerases. It lacks an intrinsic proofreading exonuclease
activity and makes one base substitution error for every 18 to 380 nucleotides
synthesized (Matsuda et al., 2000). In studies designed to elucidate the
mechanism of the error-free nature of hpol n, Masutani et al. (2000) discovered
that hpol n preferentially puts A or G opposite AP sites and C opposite AAF-G
and cisplatin-GG lesions. However, if hpol TI incorporates an incorrect nucleotide
opposite a lesion, it is not able to continue chain elongation. If hpol TI
incorporates a correct nucleotide opposite a lesion, elongation continues
(Masutani et al., 2000). This result indicates that hpol II has a special mechanism
to ensure the correct nucleotides can be incorporated during translesion
synthesis. However, increasing evidence suggests that hpol TI may be acting in
an error-prone fashion past some DNA lesions in vitro. Zhang et al. (2000a)
demonstrated that hpol TI preferentially inserts A or C opposite a template 8-
oxoG, inserts A opposite AP sites, and inserts A opposite BPDE-G lesions.
Chiapperino et al. (2002) also showed that hpol TI preferentially incorporates G or

A opposite BPDE-G adducts.

hpol 11 is mainly localized in the nucleus and is associated with replication foci
during 8 phase. After cells are exposed to UV light, hpol Tl accumulates at
replication foci stalled at DNA lesions (Kannouche et al., 2001). The C-terminal
70 amino acids are required for nuclear localization, and 120 amino acids are

needed for relocation into foci. hpol I] missing this domain fails to correct XP-V

33

defects (Kannouche et al., 2001). Glick et al. (2001) showed that Tyr52 and
Ala54 are the key residues for hpol 11 function in translesion synthesis.
Interestingly, they described an hpol 11 mutant S62G that has an increased
activity to bypass lesions. The physical interaction of hpol TI with PCNA is
important for the function of hpol n, and mutations in the PCNA binding motif of

hpol n abolish this interaction and decrease the activity of hpol n (Haracska et

al,2001c)

2.3. Polymerase iota (human/mouse)

Human and mouse cells contain a second RAD30 homolog, RAD3OB (McDonald
et al., 1999). hRAD3OB is localized on chromosome 18q21.1 in a region that is
often implicated in the etiology of many human cancers. The mouse homolog
Rad30b is located on chromosome 18E2. The second human RAD30 homolog,
hRADSOB, encodes a novel DNA polymerase, pol iota (pol 1) (Tissier et al.
2000a). Pol I is a highly error-prone and distributive DNA polymerase when
replicating undamaged DNA. At template G or C, the average error frequency
was approximately 1 x 102. Most errors occurred at template T, where the
misincorporation of G was significantly preferred (T issier et al. 2000a). Zhang et
al. (2000b) confirmed that pol l preferentially inserts G opposite an undamaged
template T and inserts T, A, or C opposite an undamaged template C. Pol 1 is
also a very error-prone DNA polymerase replicating damaged DNA. Tissier et al.
(2000b) showed that pol 1 replicates T-T dimers and T-T (6-4) photoproducts in

an error-prone fashion. Pol l. predominantly inserts T or G opposite the 3' T of T-T

34

dimers and readily misinserts any two bases opposite T-T (6-4) photoproducts
(T issier et al., 2000b). Zhang et al. (2001) demonstrated that pol I at a low
concentration is not able to incorporate nucleotides across from 8-oxoG lesions,
but incorporates C or A opposite these lesions at a higher concentration. Pol I
efficiently incorporates a G opposite an AP site, a C opposite an AAF-G, and an
A opposite a T-T (6-4) photoproduct (Zhang et al., 2001). However, contrary to
the finding of Tissier et al. (2000b), Zhang et al. (2001) reported that pol 1 was
not responsive to T-T dimmers. Taken together, the data indicate that pol I. might
play a significant role in the error-prone and error-free translesion synthesis past
UV-induced and other carcinogen-induced lesions. Haracska et al. (2001d)
reported the physical interaction of pol I with PCNA and showed that PCNA,
together with replication factor C (RFC) and replication protein A (RPA),
stimulates the DNA synthetic activity of pol 1. Whether pol I is involved in

carcinogen-induced mutagenesis in vivo is yet to be determined.

Although Johnson et al. (2000) reported that pol I is not able to extend mispairs
after it incorporates incorrect bases opposite DNA lesions, Vaisman et al. (2001)
showed that pol 1 extends all 12 possible mispairs and four correct pairs in
different sequence contexts. They showed that extension from both matched and
mismatched primer termini is generally efficient and accurate when A is the next
template base. In contrast, extension occurs less efficiently and accurately when

T is the target template base.

35

Pol l. was also found to contain an intrinsic 5’-deoxyribose phosphate lyase
activity in vitm, an important enzymatic activity for polymerase beta which is
involved in base excision repair (Bebenek et al., 2001). This result implies that

pol 1 may have multiple functions in vivo.

2.4. Polymerase kappa (human/mouse)

The human homolog of DINB has been identified. It encodes DNA polymerase
kappa (pol x). The human DINB1 gene is localized to chromosome 5q13 and is
ubiquitously expressed (Gerlach et al., 1999). Interestingly, pol K is over-
expressed in lung cancer patients (O-Wang et al., 2001). The mouse and human
pol x share significant identity with E. coli DinB, including distinct motifs
implicated in catalysis (Gerlach et al., 1999). Ogi et al. (1999) transiently
expressed the mouse DINB cDNA in cultured mouse cells and discovered that it
resulted in a nearly 10-fold increase in the incidence of spontaneous point
mutations, ~30% of which were frameshift mutations, indicating that pol K plays a

role in translesion synthesis and mutagenesis.

Gerlach et al. (2001) reported that human pol x lacks detectable 3' to 5'
proofreading exonuclease activity and is not stimulated by recombinant PCNA in
vitro. However, Haracska et al. (2002b) provided evidence for the physical
interaction between pol K and PCNA and showed that PCNA along with RFC and

RPA promotes the DNA synthesis activity of pol K. These results contradict to

36

each other. The difference could be due to the different conditions used in those

assays.

Human pol K is able to incorporate mismatched bases on an undamaged
template with a high frequency (Ohashi et al., 2000a; Zhang et al., 2000c). With
damaged templates, pol x is unable to bypass a cisplatin adduct, a cis-syn T-T
dimer, or a T-T (6-4) photoproduct (Gerlach et al., 2001; Ohashi et al., 2000b;
Zhang et al., 2000d; Suzuki et al., 2002). Pol x can bypass of an AAF-G with low
efficiency and incorporate C or T across from this lesion, suggesting that this
process is potentially mutagenic (Zhang et al., 2000d; Gerlach et al., 2001;
Suzuki et al., 2002). Moreover, pol x incorporates A opposite an AP site (Ohashi
et al., 2000b; Zhang et al., 2000d; Suzuki et al., 2002), and efficiently inserts A
opposite an 8-oxoG and C opposite a BPDE-G (Zhang et al., 2000d; Suzuki et
al., 2002). These in vitro studies indicate that pol x can perform both error-prone

and error-free translesion synthesis for different DNA lesions.

2.5. Other DNA polymerases potentially involved in translesion synthesis

Recently, two additional kinds of DNA polymerases were identified in human
cells: pol II and pol 9.. These polymerases do not belong to the
UmuC/DinB/Rad30/Rev1 superfamily, but belong to the X-family. Pol p is similar
to terminal deoxynucleotidyl transferase and has a high rate of error in vitro.
Because of its elevated level in tissues of the immune system, it was considered

to be involved in the generation of somatic hypermutations in the immunoglobulin

37

genes (Dominguez et al., 2000; Aoufouchi et al., 2000). However, Recent work
indicates that pol p. and pol I. are not involved in somatic hyperrnutation because
mice inactivated for either of these two polymerases are viable and fertile and
display a normal hyperrnutation pattern (Bertocci et al., 2002). Pol I. is a pol B-
Iike DNA polymerase and an accurate polymerase. It might be functioning in

error-free repair in meiosis (Garcia-Diaz et al., 2000).

3. Damage Avoidance

3.1. Overview of damage avoidance

Rupp and Howard-Flanders, 1968 first showed that in E.coli, recombinational
strand transfer of DNA from the damaged duplex to the homologous strand of the
undamaged duplex is one mechanism for tolerating UV damage. Recently,
increasing evidence has been obtained that the homologous recombination
pathway plays an important role in this error-free process for dealing with
replication fork-blocking lesions in prokaryotic cells. DNA gaps and possibly
double strand breaks caused by replication fork arrest are excellent substrates
for homologous recombination (Haber, 1999; Cox et al., 2000; Kowalczykowski,
2000; Kuzminov, 2001). In eukaryotes, in addition to the translesion synthesis
pathway that is carried out by the newly discovered specialized DNA
polymerases, cells have an additional error-free pathway that is also dependent
on Rad6 and Rad18 proteins (see below). This pathway is usually referred to as
damage avoidance by which cells temporary utilize an undamaged homologous

copy as the alternative template to circumvent DNA lesions that block normal

38

replication carried out by the DNA polymerase 8 complex. The undamaged copy
can be either the newly synthesized daughter strand of the sister duplex or the
homologous allele. Sometimes referred to as “copy choice” or “strand switch”,
this pathway was originally proposed as a mechanism by which DNA
polymerases (e.g., pol 8) temporarily use the newly synthesized daughter strand
of the undamaged complementary sequence as a template to avoid the lesion
(Higgins et al., 1976). Once the replication fork has moved beyond the lesion, the
replicating pol 8 switches back to continue replicating using the original template
strand. However, the exact mechanism of this process and the various gene
products involved remain obscure. Recently, Nikolaishvili-Feinberg and Cordeiro-
Stone (2000), using bisulfite treatment followed by methylation-specific
polymerase chain reaction in vitro, were able to distinguish the nascent DNA
strand synthesized from the damage avoidance pathway and the strand
replicated from the plasmid itself. The results of their studies indicated the use of

a template switch mechanism.

The phenotype of several newly identified mutants in the RAD6-RAD18 epistasis
group clearly suggests that these genes are involved in the damage avoidance
pathway, e.g., MMS2 (Broomfield et al., 1998), UBC13 (Brusky et al., 2000),
RAD5 (Johnson et al., 1992), and POL30 (T orres-Ramos et al., 1996) (see
below). In this section, I will discuss the homologous recombination repair in
yeast and in mammalian cells as a mechanism to deal with double-strand breaks.

I will also discuss the error-free damage tolerance pathway that depends on the

39

RADG-RAD18-RAD5—POL30 epistasis group dealing with replication fork-

blocking lesions, although the majority data are from S. cerevisiae.

3.2. Double-strand break repair

§accha_romycg§ cerevisiae Double-strand breaks (083s) are potent inducers of
mutations and of cell death and are predominantly repaired by homologous
recombination (HR) and non-homologous end joining (NHEJ) in yeast (Paques
and Haber, 1999). During HR, the damaged chromosome is repaired using the
genetic information from an undamaged DNA molecule with which it shares
extensive sequence homology. Thus, it is a relatively error-free repair process. In
contrast, NHEJ ligates two DNA DSBs without requiring the extensive sequence
homology between the DNA ends and thus it often alters the DNA sequence of

the involved molecules.

Homologous recombination in yeast can involve gene conversion and/or crossing
over. The preferred substrate for homologous recombination repair is the sister
chromatid (Kadyk and l-lartwell, 1992). Since sister chromatids are identical to
each other, DNA damage is repaired faithfully, with no genetic consequences.
Although the sister chromatid is preferred, homologs can serve as the repair
template at a frequency 2-3-fold less lower (Kadyk and Hartwell, 1992). Gene
conversion is defined as a non-reciprocal transfer of genetic information from one
molecule to its homolog. Usually this occurs between two alleles of a gene, but it

can embrace many contiguous genes, including the entire distal part of a

40

chromosome arm. Szostak et al. (1983) proposed a model to account for the
frequent association of gene conversion with crossing-over. Central to this model
is the formation of a recombination intermediate containing two Holliday junctions
and the resolution of these Holliday junctions results in either non-crossover or
crossover gene conversion products. It is now taken for granted that most, if not
all, crossovers arise from the same transfers of DNA strands that cause gene

conversion.

Genes important for the homologous recombination repair of 0883 were
identified primarily by identifying mutants sensitive to ionizing radiation. These
genes were classified as the RAD52 epistasis group. A mutation in rad52 was as
radiation sensitive as a double mutant consisting of rad52 and one of the other
rad mutations (Game and Mortimer, 1974), indicating that RAD52 is epistatic to
the other RAD genes in the same pathway. Currently 10 genes fall into this
group: RAD50, RA051, RAD52, RAD54, RA055, RAD56, RA057, MRE11, and
XRSZ (Paques and Haber, 1999). RAD52 is required for all homologous
recombination events. RAD51, RAD54, RAD55, RAD57, RAD50, MRE11, and
XRSZ are required for some homologous recombination events but dispensable

or less necessary for others (Paques and Haber, 1999).

Non-homologous end joining (NEHJ) is the alternative pathway to repair double-

strand breaks. It requires the second DNA ligase of yeast (Schar et al., 1997;

Wilson et al., 1997), termed LIG4 (T eo and Jackson, 1997). Re—ligation also

41

requires RAD50, XRSZ, and MRE11 (Milne and Weaver, 1996; Moore and
Haber, 1996; Tsukamoto et al., 1996) and the two subunits of the yeast
homologs of the mammalian Ku80 and Ku70 proteins, HDF1 and HDF2,
respectively (Boulton and Jackson, 1996a; Boulton and Jackson, 1996b; Milne
and Weaver, 1996). Ku protein, a heterodimer of Ku70 and Ku80, binds DNA In a
non-sequence-dependent fashion and in a manner that relies on DNA 0833
(Dynan and Yoo, 1998). Ku forms an open ring-type structure that can be
threaded onto a DNA end. One side of ring forms a cradle to protect the surface
of the DNA double helix, whereas the other side is to allow other NHEJ

components to access DSB.

Mammals In mammalian cells, the repair of DNA double-strand breaks (DSBs)
also occurs by both homologous recombination and non-homologous end joining.
It was considered that non-homologous mechanisms were used significantly
more frequently than homologous one. However, more recently, homologous
recombination has also been shown to be a major DSB repair pathway in

mammalian cells (Liang et al., 1998).

The homologous recombination pathway is conserved in mammalian cells
although the details of HR are considerably more complex. Homologs of the S.
cerevisiae gene products in RADSZ epistasis group are identified. The
mammalian homologs of RAD51, RAD52, RAD54, RAD54B, RAD50, MRE11,

RAD51B, RAD51C, RAD51D, XRCCZ, and XRCC3 have been shown to be

42

implicated in HR (Johnson and Jasin, 2001). In addition, the breast cancer
susceptibility genes, BRCA1 and BRCA2 (Scully and Livingston, 2000), which do
not appear to have yeast homologs, are also involved (Moynahan et al., 1999;
Moynahan et al., 2001). A deficiency in homologous recombination is correlated
with high rates of spontaneous chromosomal abnormalities (Johnson et al.,

1999d; Pierce et al., 1999).

To introduce a double strand break at a specific locus, the l-Scel endonuclease
experimental assay in mammalian cells commonly used to investigate the
molecular events of homologous recombination and the protein components
involved makes use of l-Scel, a rare cutting endonuclease. l-Scel recognizes an
18—bp DNA substrate, which allows one to generate a site-specific double-strand
break on an artificial DNA substrate without cutting other chromosomal DNA.
Using this system, Jasin and colleagues showed that homologous recombination
accounts for 30-40% of the double-strand break repair products, whereas NHEJ
accounts for the remainder (Liang et al., 1998; Johnson and Jasin, 2000),
indicating that homologous recombination is a prominent pathway for double-
strand break repair. As in yeast, homologs, as well as sequence repeats located
at ectopic positions on heterologs, are able to serve as repair templates for HR in
double-strand break repair. However, in contrast to yeast, the frequency of using
the repair templates from homologs and heterologs is 2-3 orders of magnitude
lower than that of using the sister chromatid (Richardson et al., 1998; Johnson

and Jasin, 2000). In addition, unlike in yeast, gene conversion events in

43

mammalian cells are not associated with crossing-over. This lack of crossing-
over is observed whether repair involves sister chromatids or two different

chromosomes (Richardson et al., 1998; Johnson and Jasin, 2000).

Non-homologous end joining (NHEJ), the rejoining of DNA ends with the use of
little or no sequence homology, involves the processing of ends such that
nucleotides are often deleted or inserted at the break site prior to ligation (Jeggo,
1998). Several processes exist in which repair of a double-strand break is
restricted to either NHEJ or HR. For example, double-strand breaks introduced
by the RAG proteins to generate antigen receptor diversity during V(D)J
rearrangement are repaired by the NHEJ pathway (Jeggo, 1998), whereas those
introduced during meiosis by the mammalian homolog of Spo11 are repaired by
the HR pathway (Keeney, 2001). The proteins involved in NHEJ include Ku70,
Ku80, DNA-PKcs and the XRCC4/DNA ligase lV complex. Ku70, Ku80, and
DNA-PKcs form the DNA-dependent protein kinase complex (Jackson, 2002).
The XRCC4IDNA ligase IV complex has been implicated more broadly in the

general repair of chromosomal DSBs.

3.3. ch13-Mmsz pathway

3.3.1. MMS2 and UBC13 in yeast

ln yeast, tolerating UV-induced DNA damage needs an additional pathway that is
separate from error-free or error-prone translesion synthesis. This error-free

pathway relies on the UBC13 and MMSZ gene products. Xiao and colleagues

44

(Broomfield et al., 1998; Brusky et al., 2000) cloned MMSZ gene and UBC13
gene in S. cerevisiae. MMSZ, codes for a ubiquitin-conjugating-enzyme-like
protein. Although Mms2 shares strong homology with most ubiquitin conjugating
(ch) enzymes (E2), it does not possess the defining E2 active-site residues and
thus a ch activity (Broomfield et al., 1998). Mmsz forms a specific heteromeric
complex with ch13, a real E2. This interaction is required for the ch13-
dependent assembly of polyubiquitin chain formation via Lys-63 (Hofmann and
Pickart, 1999) instead of the conventional Lys-48 chain assembly mediated by
most other chs (Dubiel and Gordon, 1999). Evidence that MMSZ functions in
the RA06 pathway comes from the fact that rad6 and rad18 mutations are
epistatic to mmsZ for UV— and MMS—sensitivity. Loss of Mmsz significantly
increases the rate of spontaneous mutations in S. cerevisiae but only moderately
increases the cytotoxic and mutagenic effects of UV254nm radiation (Broomfield et
al., 1998). These data indicate that MmsZ plays a role in an error-free damage

avoidance pathway.

Brusky et al. (2000) showed that UBC13 belongs to the RADG-RAD18 pathway,
and is not involved in the same pathway as REV3, which is required for
mutagenesis. The ubc13 mutant displays up to a 30-fold increase in the
spontaneous mutation rate and is proficient in UV-induced mutagenesis. These
data indicate that UBC13 is a member of the error-free damage tolerance
pathway. Further genetic evidence showed that UBC13 and MMSZ belong to the

same error-free damage tolerance pathway (Brusky et al., 2000). However, there

45

are no data yet in yeast demonstrating that the MMSZ and UBC13 gene products

are involved in a damage avoidance pathway dealing with DNA lesions.

The crystal structure of the MmsZ/ch13 heterodimer indicates that the active
site of ch13 is located at the intersection of two channels, potential binding sites
for two ubiquitins. Mutations that destabilize the heterodimer interface confer an
increased UV sensitivity, providing direct evidence that the intact heterodimer is
necessary for damage tolerance (VanDemark et al., 2001), although the
mutagenesis effect of this destabilization was not investigated. In this MMSZ-
UBC13 pathway, there are two sub-pathways, i.e., RAD5 and POL30 (Xiao et al.,
2000)

B_A_D_5 RAD5 encodes a protein of 1,169 amino acids with the molecular weight of
134 kDa that has a single-strand DNA ATPase activity (Johnson et al., 1992;
Johnson et al., 1994). Rad5 does not possess helicase activity, although it
contains all seven conserved domains associated with DNA helicases (Johnson
et al., 1994). Deletion of rad5 mutants in yeast results in a ten-fold increase in
spontaneous mutagenesis as indicated by the instability of poly(GT) repeat
sequences (Johnson et al., 1992). rad5 mutants are moderately sensitive to UV-
irradiation and proficient in UV-induced mutagenesis, but are synergistic with
rev3 mutants for killing by UV. RAD5 was assigned to the error-free branch of the
RADG pathway (Johnson et al., 1992). rad5 mutants show an increased

frequency of non-homologous end joining of DNA double-strand breaks (Ahne et

46

al., 1997). rad5 and rad18 mutants are hyper-recombinant, and the level of
recombination of the rad5 rad18 double mutant does not exceed levels seen in

either single mutant (Liefshitz et al., 1998).

Ulrich and Jentsch (2000) showed that in yeast, Rad5 recruits the Mms2/ch13
complex to DNA through its RING finger domain, and that Rad5 interaction with
Rad18 brings the MmsZ/ch13 complex into contact with the Rad6/Rad18
complex. This result indicates that Rad5 acts as the adaptor protein to bring

Mms2/ch3 into the contact of Rad6/Rad18.

m POL30 encodes, proliferating cell nuclear antigen (PCNA), an auxiliary
subunit of DNA polymerase delta. The yeast POL30 gene, cloned by Bauer and
Burgers (1990), codes for an essential protein of 29 kDa and has 35% homology
with human PCNA. Ayyagari et al. (1995) reported an interesting pol30 mutant,
pol30-46, which exhibits an increased sensitivity to the cytotoxic effect of UV and
MMS but does not exhibit a defect in replication. In addition, the pol30-46
mutation has no effect on growth or cell cycle progression, and the mutant PCNA
protein interacts normally with DNA polymerase 8 and a. Genetic studies indicate
that the pol30-46 mutation is specifically defective in the RADG-dependent
pathway, and that this mutation impairs the error-free mode of post-replication
repair. Furthermore, poI30-46 does not affect UV-induced mutagenesis and is not
involved in the same pathway as REV3 (T orres-Ramos et al., 1996). These

observations strongly indicate that POL30 plays a role in an error-free pathway

47

dealing with DNA damage, similar to the role of RAD5. Furthermore, epistasis
analyses reveal a synergistic effect of UV sensitivity between rad5 and pol30-46,
and this effect is similar to that of mmsZ mutants (Xiao et al., 2000). Taken
together, these data indicate that MM82 and UBC13 promote both error-free

sub-pathways of RAD5 and POL30.

3.3.2. MMSZ and UBCf3 in human cells

The human homolog of MMSZ, hMMSZ, was cloned by Xiao et al. (1998). It
encodes a 145 amino acids, 16.4 kDa protein with 50.4% identity to MmsZ
throughout the entire length. In particular, the N-terminal half of the two proteins
have 65% identity, including two highly conserved stretches with 13/13 and 13/16
amino acid sequence identity (Xiao et al., 1998). As in yeast, the human MmsZ
and ch13 proteins form a stable complex in vitro (McKenna et al., 2001).
hMms2 can associate with a ch13-ubiquitin thiol ester to form an activated
heterodimer, which is capable of covalently binding of an untethered ubiquitin
molecule, resulting in di-ubiquitin formation through Lys-63 (McKenna et al.,
2001). The crystal structure of the hMms2-hch13 complex and a free hMms2
molecule reveals that the hMmsZ monomer undergoes a conformational change
upon interaction with hch13 (Moraes et al., 2001). The crystal structure also
indicates that hMms2 prefers hch13 as a partner over a variety of other
structurally similar ubiquitin-conjugating enzymes (Moraes et al., 2001). These
relationships are very similar to what is known about the yeast homolog.

However, no human homolog of RAD5 has been identified yet.

48

Evidence of use of a homologous copy of a gene for damage avoidance in
human cells comes from the earlier studies by Maher and McCormick and
colleagues (T sujimura et al., 1990; Bhattacharyya et al., 1990a and b; Zhang et
al., 2000e). Bhattacharyya et al., (1990a) utilized the thymidine kinase (tk)-
deficient human cell lines containing a single integrated copy of a plasmid
carrying two copies of the herpes simplex virus thymidine kinase (Htk) gene,
each containing an 8-base-pair Xhol linker inserted at a unique site. Expression
of a functional Htk enzyme requires a productive recombinational event between
the two nonfunctional genes. Following UV or BPDE treatment, wild type tk (tk+)
cells were formed in a dose dependent manner, and analysis of tk+ cells
indicated that majority of these cells were generated through gene conversion
events unaccompanied by crossing-over. These results indicate that human cells
are able to use a homologous allelic copy as an alternative template to
circumvent DNA lesions. Cells containing a functional copy of the tk gene were
resistant to HAT medium. A similar system in human cells that select for the
formation of a wild type hyg gene generated from two non-functional hyg copies
by homologous recombination provided similar results (Tsujimura et al., 1990).
Using that assay system, they showed that un-excised DNA lesions, rather than

excision repair processes, stimulate intrachromosomal recombination.

Using a human fibroblasts cell strain containing a hyg intrachromosomal

recombination substrate stably integrated into the genome and using antisense

RNA technology to eliminate hMmsZ protein in human cells, Li et al. (2002)

49

recently showed that cells lacking hMmsZ are devoid of UV-induced
intrachromosomal gene conversion. The loss significantly increased the
frequency of UV-induced HPRT mutants. These results indicate that when the
ability to use an undamaged homologous copy as an alternative template is
eliminated, human cells make increased use of translesion synthesis to tolerate

DNA lesions.

50

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CHAPTER II

Identification of a protein essential for a major pathway used by human

cells to avoid UV-induced DNA damage
Ziqiang Li*, Wei XiaoT, J. Justin McConnick* and Veronica M. Maher* *

*Carcinogenesis Laboratory, Department of Microbiology and Molecular Genetics
and Department of Biochemistry and Molecular Biology. Michigan State
University, East Lansing, MI 48824, USA; and TDepartment of Microbiology and

Immunology, University of Saskatchewan, SK, S7N 5E5, Canada

1Corresponding author: Veronica M. Maher, Carcinogenesis Laboratory, Food
Safety and Toxicology Building, Michigan State University, East Lansing, 48824-
1302

Tel.: (517)353-7785 Fax: (517) 353-9004; E-Mail: maher@msu.edu
Abbreviations: HPRT, hypoxanthine phosphoribosyltransferase gene; hyg,

hygromycin phosphotransferase gene; hyg“, hygromycin resistant; TG, 6-

thioguanine; TGR, 6-thioguanine resistant.

7O

Abstract

When DNA replication stalls at a fork-blocking lesion, cells use damage
tolerance pathways to continue replication. One pathway, “translesion
synthesis”, involves specialized DNA polymerases that can use damaged
DNA as a template. Translesion synthesis can result in mutations (i.e., can
be error-prone), but it can also be error-free. An alternative pathway,
sometimes called “damage avoidance”, has been hypothesized, by which
cells make temporary use of an undamaged copy of the blocked sequence
as a template, i.e., the newly-synthesized daughter strand of the sister
duplex or the allelic copy. This pathway is error-free. Direct evidence of the
use of this alternative pathway in intact mammalian cells has not been
available. To determine whether hMmsz, a ubiquitin-conjugating-enzyme-
like protein [Xiao, W., Lin, S. L., Broomfield, 8., Chow, B. L. 8. Wei, Y. F.
(1998) Nucleic Acids Res. 26, 3908-3914], plays a critical role in such
damage avoidance, a human fibroblast cell strain in which both error-prone
translesion synthesis and error-free damage avoidance can be detected
and quantified simultaneously, and several derivative strains in which
expression of hMms2 protein had been eliminated or greatly decreased,
were compared for their ability to avoid translesion synthesis past uvm...-
induced DNA photoproducts. Loss of hMms2 protein eliminated the ability
of the latter strains to use an allelic copy of a target gene for damage
avoidance, i.e., to produce a wild type gene from two non-functional allelic

copies of that gene. Molecular analysis of the wild type gene showed that

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this process involves gene conversion unassociated with crossing-over.
That loss of hMms2 also eliminated use of the daughter strand of the sister
duplex as a template for damage avoidance could be inferred from the fact
that the frequency of mutations induced by UV in the single copy HPRT
gene of the derivative strains was significantly higher than that observed in
the parental strain. These data indicate that hMMSZ is essential for human
cells to carry out damage avoidance using either type of homolog and that
damage avoidance and translesion synthesis are alternative pathways for

tolerating fork-blocking photoproducts.

Introduction

DNA is constantly exposed to damaging agents. If the damage is not removed
e.g., by excision repair, prior to onset of S-phase, certain kinds of lesions can
block replication by the major INA polymerase complex (1). The damage
tolerance mechanisms developed by prokaryotic and eukaryotic cells to
overcome such replication blocks fall into two categories: translesion synthesis
and damage avoidance. Evidence suggests that translesion synthesis is a
process in which specialized, distributive DNA polymerases take over for the
major DNA polymerase complex to carry out DNA replication using the damaged
DNA as a template (2-5). After distributive incorporation of nucleotides past the
damage, the major DNA replication complex resumes its replication activity.
Translesion synthesis can be either “error-prone” or “error-free" depending upon
such factors as the type of damage, its sequence context in the DNA, and the
availability of specialized distributive translesion synthesis polymerases, e.g.,

hPol zeta, hPol eta, Pol iota (6-9) or their auxiliary proteins (10). The alternative

72

pathway to tolerate DNA damage, sometimes referred to as damage avoidance,
temporarily uses a homologous undamaged copy of DNA instead of the
damaged DNA as a template to continue replication (11-13). By its very nature,
damage avoidance is error-free. The mechanisms involved in mammalian cells’
avoidance of fork-blocking lesions are not well characterized, but unlike what
occurs in bacteria (12), mammalian cells do not transfer parental DNA strands
containing UV photoproducts into daughter strands (14). It has been proposed
that in mammalian cells, the 3’ end of a blocked leading strand separates from its
original template and makes use of an intact homologous copy of the DNA as a
template for continuing replication past the block. If the newly-synthesized
daughter strand of the sister duplex is to provide the temporary template for the
blocked leading strand, synthesis of the lagging strand must have continued for a
short distance beyond where the leading strand was blocked, and this lagging
strand must become dissociated from its partner. After replication has proceeded
past the block, the paired structure must uncouple so that normal DNA replication
using the original template can resume. Although the newly-synthesized
daughter strand of the sister duplex may provide the most convenient
homologous copy for the blocked fork, the homologous allelic gene could also
serve as an alternative template for circumventing such a replication block.
Evidence that intact mammalian cells make use of such damage avoidance
mechanisms to circumvent fork-blocking lesions and identification of the various

gene products has not been available.

Xiao and his colleagues (15) cloned a gene from S. cerevisiae that
complements the sensitivity of yeast mutant mm32-1, originally identified (16) for
its sensitivity to methyl methanesulfonate. This gene, MMSZ, codes for a

ubiquitin-conjugating-enzyme-like protein (15) that forms a stable complex with

73

ch13 protein (17-19), enabling in vitro di-ubiquitin formation via Lys-63 instead
of the conventional Lys-48 chain assembly (20). Loss of Mms2 significantly
increases the rate of spontaneous mutations in S. cerevisiae and moderately
increases the cytotoxic and mutagenic effects of UV254nm radiation (15), indicating

that it plays a role in an error-free pathway.

Xiao et al. (21) isolated the human homolog of the S. cerevisiae MMSZ
gene, designated hMMSZ. The present study was designed to determine
whether the hMMSZ gene plays a critical role in the ability of human cells to
tolerate DNA damage, and if so, to identify the pathway in which it functions and
determine the nature of the molecular events involved. To do so, an infinite life
span, chromosomally-stable, human fibroblast cell strain (22) was engineered to
contain a chromosomally-integrated substrate that allows detection of the
production of a selectable wild type gene from two non-functional homologous
genes (23). Three strains in which expression of hMmsZ protein was eliminated
or greatly reduced using antisense were identified and compared with the
parental strain for their ability to carry out damage avoidance following the
exposure to UV254,,m irradiation. The results indicate that human cells can use
both kinds of homologous templates to avoid fork-blocking lesions and that

hMms2 protein is essential for the damage avoidance process.
Materials and Methods

Media Used. Cells to be used for experiments involving assaying cell survival, or
selection for resistance to 6-thioguanine (TG, Sigma), or hygromycin
(Calbiochem), were cultured in Eagle’s minimal medium supplemented with L-
aspartic acid (0.2 mM), L-serine (0.2 mM), sodium pyruvate (1 mM),

hydrocortisone (1 pg/ml), and supplemented calf serum (10% vol/vol) (culture

74

medium). This medium containing the appropriate selection drug, i.e., TG (40
pM) or hygromycin (100 Ulml) was used for selection of TG resistant or
hygromycin resistant cells, respectively. The parental cell strain, which contains a
transfected tTAk gene that was transfected on a plasmid carrying the hisD gene
as its selectable marker (see text), was cultured in McM medium (24) lacking
histidine, but containing histidinol (1mM), supplemented calf serum (10% vol/vol)
(Hyclone), penicillin (100 Ulml), streptomycin (100 pglml), and hydrocortisone (1
uglml). Cell strains that contain a transfected antisense hMMSZ gene that was
tranfected on a plasmid carrying the gene coding for puromycin resistance (see
text) as its selectable marker, were cultured in this same McM medium, but also

containing puromycin (1 pg/ml).

DNA Transfection. Cells were transfected using LipofectAMINE (Life
Technologies, GibcoBRL). After 48 h, transfectants were selected in medium
containing the appropriate drug. The selection medium was renewed every five
days. When drug-resistant clones had formed, they were isolated, expanded in

the appropriate selection medium, and stored frozen until needed.

Preparation of Purified hMms2 and Mouse Polyclonal hMmsZ Antibodies.
The human hMMSZ open reading frame was PCR amplified and cloned into
pGEX6 (Pharmacia). The GST-hMms2 fusion protein was over-produced in
Epicurian coli BL21-CodonPlus-RIL cells (Stratagene), and standard protocols
were followed to purify the fusion protein using a 5ml GSTrap column
(Pharmacia). After PreScission protease (Pharmacia) cleavage, hMms2 was
eluted from a GSTrap column (Pharmacia) and further purified using a Superdex-
75 size column (Pharrnacia). Polyclonal mouse serum was raised against the

purified hMms2 protein in five BALBIc mice. Three weeks after the second boost,

75

the mice were bled, and the anti-serum was pooled and characterized by ELISA

and Western analysis using the purified protein.

Northern and Western Analysis. For Northern analysis, RNA from each cell
strain was extracted using RNAzoITMB (TEL-TEST, INC.) according to
manufacturer's recommendations. For each sample, 15 ug of total RNA was
fractionated in 1.2% formaldehyde agrose gel and transferred to Hybond-N+
(Amersham) nylon membranes. The RNA was crosslinked to the membrane
using a UV stratalinker 2400 (Stratagene). To determine the level of antisense
RNA expression, hybridization was performed according to the standard
procedures using a probe specific to hMMSZ. The membrane was stripped and
re-probed with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) to
compare the relative amount of total RNA loaded for each sample. For Western
analysis, nuclear protein was extracted from the cell strains as described (25).
For each cell strain, 50 pg of nuclear protein was subjected to Western analysis
using hMmsZ mouse polyclonal antibody. Purified hMmsZ protein was included
as the positive control, and Ku-80 protein was detected using rabbit polyclonal
Ku-80 antibody to compare the relative amount of total nuclear extracts loaded

for each sample.

Assay for Cytotoxicity. Cells in exponential growth were detached from the
dishes using trypsin, plated at cloning density, and allowed 12 h to attach before
being irradiated by UV254nm as described (26). The culture medium was renewed
one day after irradiation and again after seven days. After 14 days, when the
surviving cells had formed clones, they were stained. The survival was
determined by comparing the cloning efficiency of the irradiated cells with that of
the sham-irradiated control cells, and the value was expressed as percent of that

of those control cells.

76

Assay for the Frequency of UV-lnduced Wild Type Hygromycin Resistant
Cells. This assay was carried out as described previously (27). Briefly, sufficient
sets of cells plated at 0.5><106 cells per dish were used to have at least 2x106
surviving target cells per dose and allowed 12 h to attach. The number of target
cells attached to the dishes at the time of irradiation was determined
electronically. Forty-eight hours after irradiation, the culture medium was
changed to medium containing hygromycin (100 U/ml). This selection medium
was replaced every five days, and the clones were stained 3 wk after irradiation.
The frequency was calculated as follows: total hygromycin resistant clones
divided by total number of surviving cells, determined as described in the above
paragraph, taking into account the number of attached target cells. The induced

frequencies were calculated by subtracting the background frequencies

Characterization of hyg Genes. Hygromycin resistant clones were analyzed
using PCR and Hindlll digestion as described previously (23). Briefly, cells from
each clone were used for PCR analysis using a set of primers that amplify a
1,167-bp fragment from each copy of the hyg gene. The PCR products were
digested with Hindlll and subjected to gel electrophoresis. PCR products from
wild type copies of the hyg gene are resistant to Hindlll digestion. When gene
conversion events occur, one or other of the mutant hyg genes is restored to wild
type while the second mutant hyg gene remains unchanged. Hindlll digestion of
PCR products generates 1,167-bp, 278-bp and 889-bp fragments or 1,167-bp,
695-bp and 472-bp fragments depending on which hyg gene has been restored
to the wild type. However, if a single reciprocal exchange were to occur between
two mutant hyg genes, this would result in a single wild type copy of the hyg

gene.

77

Assay for the Frequency of UV-induced Mutations in the HPRT Gene. The
methods used to determine the frequency of UV-induced TG resistant (TGR) cells
has been described (28). Briefly, sufficient sets of cells, plated at 0.5x106 to
2x106 per 150-mm diameter dish were used to have at least 1x106 surviving
target cells per dose. The culture medium was changed the day after irradiation.
Cells used to detect induced mutations were allowed an 8-day expression period
before at least 1x106 cells were plated at 25,000 cells per 100-mm diameter dish
to assay for TG resistance. Selection medium containing 40 uM TG was replaced
after seven days. At the time of selection, for each dose, a separate set of cells,
plated at cloning density and fed with medium lacking TG, was used to determine
the cloning efficiency of the cells. The frequency of mutants was calculated as
follows: total TG resistant clones divided by the total number of clonable cells
selected. The induced frequencies were calculated by subtracting the

background frequencies in the sham-irradiated control population.
Results and Discussion

Assay System for Detecting Damage Tolerance in the Same Target Cell
Strains. Damage tolerance pathways allow human cells with fork-blocking
lesions to continue DNA replication and avoid cell death. To detect use of
translesion synthesis, we measure the frequency of error-prone translesion
synthesis, using the HPRT gene as the target for mutations and quantifying the
frequency of induced 6-thioguanine-resistant (T GR) cells (28). [Error-free
translesion synthesis cannot be detected directly, but can be inferred by
comparing the frequency of mutations induced in cells that lack such a process
with that of cells that have it]. To detect use of damage avoidance, we employ a
substrate (stably integrated into the genome of the target cells) that allows

quantification of the frequency with which cells produce a selectable wild type

78

copy of a gene by combining information from two defective homologous genes.
The substrate (diagrammed in Fig. 1A) consists of two copies of the hygromycin
phosphotransferase (hyg) gene conferring resistance to hygromycin, each
inactivated by a 10-bp Hindlll linker inserted at a different site (27). The genes,
separated by ~6000 bp (23), are arranged in cis with one another, mimicking the
situation for allelic genes in human cells. If a fork-blocking DNA lesion
(represented by the triangle) interrupts replication of one of the genes, e.g., the
hng gene shown in Figure 1A, use of the corresponding section of the newly-
synthesized daughter strand (shown in blue in Figure 1B) of the sister duplex as
a template will allow the cell to avoid the damage and continue DNA replication.
However, the newly-synthesized DNA strand (shown in brown) of the hyg2 gene
resulting from such damage avoidance will still contain the 10-bp Hindlll linker
(Fig. 1C, top panel), even after the lesion has been removed or after another
round of replication has occurred (Fig. 10). Therefore, this process will not yield
a cell that is resistant to hygromycin. However, as diagrammed in Fig. 1E, if cells
use the homologous copy of the gene, hyg1, as a template to avoid the blocking
lesion, the newly-synthesized strand (shown in brown) can obtain the information
from the corresponding section of the hyg1 gene. The strand will no longer
contain the 10-bp Hindlll linker (Fig. 1F, top panel). The next round of replication
of that strand will yield a selectable wild type copy of the hyg gene (Fig. 16). An
added value of this substrate is that it allows one to infer the nature of the
mechanisms involved by analyzing the structure of the hyg gene(s) in the

hygromycin resistant (hng) cells.

79

 

Figure 1:The two possible paths for avoiding DNA damage by making use of an
undamaged homolog to continue replication, Le, a daughter strand of the sister
duplex or a homologous copy. (This image in this dissertation is presented in
color). (A) The intrachromosomal substrate contains two copies of the hyg gene
(hyg1, red; and hng, green), orientated in the same direction. Each has a Hindlll
linker insertion (I) at the different position. The open triangle in the hyg2 gene
represents a UV-induced lesion. (B) The blocked strand of the hyg2 gene is
shown copying information from the newly-synthesized daughter strand (blue) of
the sister duplex, rather than the damaged template. (C) The outcome of this
process. The newly-replicated leading strand (brown) of the hng gene still
contains the Hindlll linker sequence (top panel). The replicated product not
containing the UV lesion also retains the original Hindlll linker DNA sequence
(bottom panel). The section containing the hyg1 gene (red) is not shown for
these products. (D) The outcome after one round of replication. The hng gene
has been replicated, but still contains the linker. (E) The hng gene is shown
copying information from the homologous copy, hyg1, rather than the damaged
template. (F) The outcome of this process. The DNA sequence of the hyg1 gene
remains intact. The newly replicated leading strand of the hng gene (brown) has
obtained information from the corresponding section of the hygf gene, so it no
longer contains the Hindlll linker sequence (top panel). The replicated product
not containing the lesion retains its original DNA sequence (bottom panel). (G)
The outcome after one round of replication. A wild type (w.t.) copy of the hyg

gene has been generated.

80

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81

Preparation and Identification of Human Cells Expressing Antisense
hMMSZ and Devoid of hMms2 Protein. We successfully used an antisense
hMMSZ RNA strategy to deplete human cells of hMms2 protein. To do so, we
transfected an MSU-1.2 human fibroblast-derived cell strain, designated E72
(23), containing the substrate as diagrammed in Figure 1A, with a plasmid
designated pTet-tTak containing the tetracycline promotor (tetP) (29, 30), the
tetracycline transactivator (tTA) gene under the control of tetP, and the hisD gene
coding for histidinol resistance. A clone of histidinol—resistant cells that expressed
tTA at a high level and exhibited the same UV—induced frequency of TGR cells
and hng cells as cell strain E7.2 was chosen as the parental strain for the
present study. This parental cell strain expressing tTA was transfected with a
plasmid carrying antisense hMMSZ gene under control of the tetP promoter. The
hMMSZ antisense expression construct contains a 430-bp Spel-Hindlll fragment
carrying the first 400-bp hMMSZ open reading frame and 20-bp 5’ untranslated
region of the hMMSZ cDNA. This fragment was cloned into the Spel-Hindlll sites
of pTet-Puro plasmid (6, 10), in antisense orientatation. The construct also
contains a gene coding for puromycin resistance. Sixty puromycin—resistant
transfectants were isolated and screened for expression of antisense hMMSZ
RNA by RT-PCR using a set of antisense-specific primers. Sixteen clones were
found to express hMMSZ antisense RNA. These clones were expanded into
large populations, and the cell strains were further analyzed by Northern blotting
to quantify the level of expression of antisense RNA (data not shown). Nuclear

extracts from the parental cell strain and the 16 antisense RNA-expressing cell

82

strains were analyzed by Western blotting for the level of hMmsZ protein. Purified
hMmsZ protein was included as the positive control. Three cell strains,
designated 2A-M2, 3C-M2, and 13D-M2, which express high levels of antisense
hMMSZ RNA (Fig. 2A), showed an undetectable level or a very low level of
expression of hMms2 protein compared to the parental cell strain (Fig. 28). We
verified that these three cell strains lacking hMms2 protein still retained an intact
copy of the substrate shown in Figure 1A and then used them to investigate the

role of hMmsZ in damage tolerance.

Effect of Depletion of hMms2 Protein on Cell Survival. These three cell
strains were compared with their parental strain for the effect of loss of hMms2
on UV-induced cytotoxicity. As shown in Figure 3A, there was no significant
difference between cells expressing or not expressing hMms2 protein in their
sensitivity to the cytotoxic effect of UV. This indicated that cells expressing or not
expressing hMmsZ protein were equally capable of tolerating the UV
photoproducts. The implications of this finding are discussed below. The majority
of the data points shown in Figure 3A were derived from experiments in which
cell strains were also assayed for the frequency of UV-induced hng cells and

TGR cells (see below).

Effect of Depletion of hMmsZ Protein on the Frequency of UV-Induced HygR
Cells. As expected (27), there was a dose-dependent increase in the frequency
of UV-induced hng cells in the parental cell strain (Fig. 38). However, in the
three cell strains lacking hMmsZ protein, that response to UV was virtually

eliminated. The frequency of UV-induced hng cells was not significantly greater

83

 

Figure 2: Northern and Western blots showing the correlation between
expression of antisense hMMSZ RNA and loss of hMmsZ protein. (A) Northern
analysis showing that clones 2A-M2, 3C-M2, and 13D-M2 express high levels of
hMMSZ antisense RNA. GAPDH transcript was used as the loading control for
all the samples. hMMSZ AS indicates the transcript of hMMSZ antisense. (B)
Western analysis showing the level of expression of hMmsZ protein in nuclear
extracts from these hMMSZ antisense-expresssing transfectants and their
parental strain. Ku-80 was used as the loading control. Purified hMmsZ was

included as the positive control.

84

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85

Figure 3: The biological effects of inhibition of hMMSZ expression. Open symbol,
the parental cell strain; closed symbols, transfectant cell strains showing an
undetectable or very low level of hMmsZ protein: circle, strain 3C-M2; triangle,
strain 2A-M2; and square, strain 13D-M2. (A) The UV-induced cytotoxicity in
these cell strains. Some points have been offset slightly in order to be visible. (8)
The frequency of hng cells induced by UV in the parental cell strain and the
three cell strains lacking hMmsZ protein. Some points have been offset slightly in
order to be visible. (C) The frequency of TGR cells induced by UV in the parental
cell strain and the three cell strains lacking hMmsZ protein. For the majority of the
data points generated, at least 2x106 cells were assayed. The observed
frequency of TGR clones has been corrected for the cloning efficiency of the cells
at the time of selection as described (28). For the majority of the data points, the
cloning efficiency of the cells at the time of selection ranged from 45-65%, so the
correction factor was less than 3-fold. The induced frequencies were calculated
by subtracting the background frequencies, observed with the sham-irradiated
populations. The latter were less than or equal to 10 per 106 clonable cells. The
solid line represents the least-squares line for the data obtained from the parental
cell strain, and the dashed line represents the least-squares line for all the data
points generated from the three cell strains deficient in hMmsZ protein, taken

together.

86

 

 

 

 

 

 

 

 

 

 

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than the background frequency, i.e., < 1x106. This decrease is not the result of
mere expression of antisense, rather than the loss of hMmsZ protein, because a
cell strain exhibiting a very high level of antisense RNA, but a normal level of
hMmsZ protein, gave an UV-induced frequency of hng cells equal to that found
in the parental strain (data not shown). The data in Figure 38 indicate that
hMms2 protein plays an essential role in the damage avoidance pathway dealing
with photoproducts, i.e., during DNA replication, human cells can make
temporary use of the allelic copy of a gene as a template to avoid fork-blocking
lesions, but cells lacking hMmsZ protein are virtually incapable of using this
damage avoidance pathway. PCR and restriction enzyme digestion was used to
analyze 30 of the hng clones induced by UV, or arising spontaneously, for the
structure of their hyg genes. The results showed that each wild type hyg gene
had been generated by a gene conversion event unaccompanied by crossing-

over.

Effect of Depletion of hMms2 Protein on the Frequency of UV-lnduced TGR
Cells. As expected, UV radiation caused a dose-dependent increase in the
frequency of TGR cells in the parental cell strain (Fig. 30), indicating that these
cells had carried out translesion synthesis. The three cell strains lacking hMmsZ
protein also gave a dose-dependent induction of TGR cells for doses up to 8
Jlmz, but the frequency of TGR cells was twice as high as that induced in the
parental cells, indicating that the strains lacking hMms2 carried out translesion
synthesis more frequently. This increased use of translesion synthesis as a
means of tolerating photoproducts explains why the cells lacking hMms2 were

not more sensitive to than the parental cells to the cytotoxic effect of UV.

88

Increased sensitivity to cell killing in the cells lacking hMmsZ should only be seen
if they are unable, or less able than the parent cells to continue to replicate DNA
containing fork-blocking lesions. If the human cells lacking hMms2 were to
increase their use of various specialized translesion synthesis polymerases, they
could continue to replicate their DNA even though all use of an undamaged
homologous copy of the DNA as a template had been eliminated. S. cerevisiae
cells lacking MmsZ are slightly more sensitive to the cytotoxic effect of UV than
are wild type cells. However, human cells have additional translesion synthesis
polymerases, e.g., they have DNA pol iota (9) and should be more capable than

yeast of replicating past fork-blocking lesions.

At higher doses of UV, the frequency of mutants in the three cell strains
lacking hMms2 leveled off. The dashed line is the least-squares line for all the
data points obtained from these three cell strains. One possible explanation for
why cells lacking hMms2 protein did not exhibit a dose—dependent increase in
mutant frequency at higher UV doses is that at those doses, the number of
mutations continued to increase, but some of these occurred within the same
cell. We tested that possibility by sequencing the coding region of the HPRT
gene of 8 independent mutants from these cell strains irradiated with the highest
doses of UV. There was only one mutation in each gene. Another possible
explanation is that, at those higher closes, the level of a DNA polymerase(s)
involved in error-free translesion synthesis past photoproducts is increased in
cells lacking hMms2 protein. Western analysis of the level of expression of
polymerase eta after UV-irradiation did not demonstrate such induction (data not

shown).

89

 

Indirect Evidence That Human Cells Use the Newly-Synthesized Daughter
Strand of the Sister Duplex for Damage Avoidance. The data in Figure 3A
and 30 showed that the parental cells expressing hMmsZ and the three strains
lacking hMms2 are equally able to tolerate UV-induced damage, but the latter are
twice as sensitive to the induction of mutations in the HPRT gene. HPRT is
located on the X-chromosome and the target cells are from a male donor, there
is only one copy of this gene in the cell. Therefore, cells have only two options for
tolerating fork-blocking lesions in the HPRT gene: 1) use the newly-synthesized
daughter strand of the sister duplex of the HPRT gene as the template to avoid
the damage (error-free) or 2) carry out potentially mutagenic translesion
synthesis. Our data indicate that the parental cells must have used option 1 and

that this option was not available in cells lacking hMms2.

We recognize that UV induces several kinds of fork-blocking
photoproducts and that some types of lesions are more likely than others to
result in mutations. However, for the sake of simplicity, let us assume there is
only one type of fork-blocking lesion and that cells make equal use of the two
major pathways for tolerating such lesions, I.e., translesion synthesis and
damage avoidance. If the latter pathway is completely eliminated, e.g., by
knocking out of expression of hMms2 protein, the number of fork-blocking lesions
that have to be dealt with by translesion synthesis polymerases should double.
Even though some translesion synthesis might not result in mutations, the
frequency of mutations introduced during translesion synthesis should double
because the total number of lesions to be passed has doubled. This is the extent

of increase we observed when we eliminated hMms2 protein.

90

Possible Role of hMms2 plays in Damage Avoidance The role that the
hMms2 play3 in the observed loss of ability of the human cells to carry out gene
conversion events to avoid DNA fork-blocking lesions remains elusive. Hofmann
and Pickart (17) showed that in yeast, Mms2 forms a complex with ch13, and
the complex is required for the assembly of polyubiquitin chains linked through
lysine 63 in ch13-depepndent manner. They proposed that this complex acts as
a signal transducer to recruit target proteins to the site of DNA damage for repair.
Ulrich and Jentsch (18) later on showed that in yeast, Rad5 recruits the
Mms2/ch13 complex to DNA through its RING finger domain, and that Rad5
interaction with Rad18 brings the Mms2/ch13 complex into contact with the
Rad6/Rad18 complex. At the same time, Xiao et al. (31) proposed that in yeast
the Mms2/ch13 complex is required to promote both RAD5 and POL30 error-
free repair pathways. Recently, McKenna et al. (32) showed that in human cells,
hMmsZ forms a complex with hch13, and this interaction is required for
hch13-mediated polyubiquitination through lysine 63. The crystal structure of
this complex was also determined recently (33). Our data support the hypothesis
that the hMms2/hch13 complex is required to recruit or activate proteins
needed by human cells to be able to make use of an undamaged homologous
copy of DNA as an alternative template to avoid fork-blocking lesions and

continue DNA replication.

In summary, our data provide direct evidence that human cells can carry
out damage avoidance using an allelic copy of a gene as a temporary template

for DNA replication and that this process involves gene conversion,

91

unaccompanied by crossing-over. Our mutagenesis data provide indirect
evidence to support the model, proposed by Strauss and colleagues (11), that
the newly-synthesized daughter strand of the sister duplex can also be used as a
temporary template for DNA replication to avoid DNA damage. Our data indicate
that hMMSZ gene is essential in both cases. Our assay system provides a basis
for identifying other proteins involved in the damage avoidance pathway dealing

with fork-blocking lesions.

Acknowledgements

We thank L. Pastushok for hMmsZ protein purification and generation of hMmsZ
mouse polyclonal antibody, Dr. H. Zhang for preparing and characterizing the
parental cell strain, L. Qin for assisting with RNA extraction and Northern
analysis, and J. Dao, M. Battle, and Y. Wang for helpful suggestions on Western
blotting. We thank Dr. K. Meek for providing rabbit polyclonal Ku-80 antibody and
Dr. S. Kleff and M. Hanna for critical reading of the manuscript. This research
was supported by DHHS NIH grants ES-09822 and CA 91490 (V.M.M.) and

grant MT-15076 from the Medical Research Council of Canada (W.X.)

92

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33. Moraes, T. F., Edwards, R. A., McKenna, S., Pastushok, L., Xiao, W.,

Glover, J. N. & Ellison, M. J. (2001) Nat. Struct. Biol. 8, 669-673.

95

 

CHAPTER III

Translesion synthesis and damage avoidance contribute almost equally

to the tolerance of BPDE-adducts in human cells

Ziqiang Li 3, Wei Xiao b, J. Justin McCormick 3, Veronica M. Maher 3' *

" Carcinogenesis Laboratory, Department of Microbiology and Molecular
Genetics and Department of Biochemistry and Molecular Biology, Michigan State
University, East Lansing, MI 48824, USA

b Department of Microbiology and Immunology, University of Saskatchewan, SK,

S7N 5E5, Canada
*Corresponding author: Veronica M. Maher, Carcinogenesis Laboratory, Food
Safety and Toxicology Building, Michigan State University, East Lansing, 48824-

1302

Tel.: (517) 353-7785 Fax: (517) 353-9004; E-Mail: maher@msu.edu

96

Abstract

We showed recently (Li et al., Proc. Natl. Acad. Sci. U.S.A. 99, 2002:4459)
that hMms2 plays a critical role in a pathway by which human cells avoid
translesion synthesis past fork-blocking lesions induced by UV by making
temporary use of an undamaged copy of the blocked sequence, i.e., either
the newly synthesized daughter strand of the sister duplex or an allelic
copy, as a template to continue DNA replication. However, because UV
produces several different kinds of photoproducts, and cells may differ
significantly in their relative use of a particular pathway for each type of
photoproduct, it is not possible to use UV as the DNA-damaging agent to
determine the relative frequency with which human cells make use of
damage avoidance (DA), rather than translesion synthesis (TLS). The
present study was designed to estimate the ratio with which human
fibroblasts use TLS versus DA to continue DNA replication past a major
(~94%) type of fork-blocking adduct formed by (fl-7l3,8a-dihydroxy-9a,10a-
epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE). Three cell strains lacking
hMms2 were compared with their hMms2-proficient parental strain for the
ability to make temporary use of an undamaged homologous copy of DNA
as a template to avoid such damage, rather than using translesion
synthesis. The results show that loss of hMms2 did not increase the
cytotoxic effect of BPDE, but completely eliminated the cells’ ability to use
an undamaged homologous copy of a reporter gene as a template for DNA

replication past BPDE-adduct: and increased the frequency of BPDE-

97

 

induced mutants in the single copy HPRT gene ~2.5-fold. These results
indicate that human cells make approximately equal use of DA and TLS in

order to continue replicating DNA containing BPDE adducts.

Keywords: hMMSZ; damage avoidance; translesion synthesis; BPDE;

homologous recombination; mutagenesis

1. Introduction

Cells are continuously exposed to endogenous and exogenous DNA damaging
agents. Certain kinds of lesions block replication carried out by the major DNA
polymerase complex, but cells have developed mechanisms for tolerating such
damage. For example, they can make temporary use of specialized distributive
polymerases that are capable of inserting nucleotides opposite a damaged
template (translesion synthesis) [16]. Alternatively, cells can make temporary
use of an undamaged homolog of the blocked sequence, e.g., the newly
synthesized daughter strand of the sister duplex [7], which was suggested by
Higgins et al. [8], or the allelic gene [7], as a temporary template to circumvent
the lesion (damage avoidance). Damage avoidance is error—free. In contrast,
translesion synthesis can be either “error-prone” or “error-free” depending upon
the type of damage, its sequence context in the DNA, and the availability of these
various specialized distributive DNA polymerases in the cell. Evidence indicates
that after a nucleotide(s) has been incorporated opposite a fork-blocking lesion

and extended for a limited number of nucleotides, the major DNA replication

98

 

—-- I .1—-- a.“

complex can resume replication. The relative use of these two processes in

human cells is the subject of the present study.

Using a parental human fibroblast cell strain containing a chromosomally-
integrated substrate that allows quantification of the frequency by which cells
produce a wild type copy of a target hyg gene by combining genetic information
from two defective homologous hyg genes [9-11] and its three derivative cell
strains, we recently showed that hMmsZ, a ubiquitin-conjugating-enzyme-like
protein [12], is essential if cells are to make temporary use of an undamaged
homologous copy of a gene to circumvent UV photoproducts and continue DNA
replication. Elimination of hMmsZ protein from the derivative cell strains did not
increase their sensitivity to the cytotoxic effect of UV, but completely eliminated
the induction of Hyg-resistant cells, i.e., eliminated use of a homologous copy of
a blocked gene to carry out damage avoidance. What is more, the frequency of
UV-induced mutations in the target HPRT gene of the cells lacking hMmsZ
increased significantly above that induced in the parental cells, indicating
increased use of error-prone translesion synthesis [7]. Iecause the target gene
for detecting mutations is located on the X-chromosome, there is no allelic copy
of the HPRT gene in these male cells. Therefore, the increase in HPRT
mutations when hMms2 was eliminated strongly suggests that when it is present,
hMms2 facilitates temporary use of the newly synthesized daughter strand of the
sister duplex as an undamaged template for completing replication (damage

avoidance).

99

 

Because UV produces several different kinds of photoproducts, and cells the
potential for translesion synthesis, rather than temporary use of an undamaged
template, may well depend on the type of photoproduct encountered by the
replication fork [13], it is not possible to use UV as the DNA damaging agent to
determine cells’ relative use of these two pathways. Therefore, in the present
study, designed to estimate this ratio, BPDE was used as the DNA-damaging
agent. Approximately 94% of the adducts formed in DNA by BPDE involve
guanine [14, 15], and consist of a covalent bond between the C10 of BPDE and
the exocyclic 2-amino position of guanine [16]. This adduct strongly blocks the
replication fork [17, 18] and is highly mutagenic in human cells [18-21]. (Of the
mutations induced by BPDE in the HPRT gene of finite life span human
fibroblasts, 110 of the 121 analyzed (2 90%) involved G:C base pairs; 95% of
these were base substitutions, with the majority (70%) being GO to T:A

transversions [19-21]).

We compared the parental human cell strain and its three derivative strains
lacking hMmsZ for their response to BPDE. Loss of hMms2 did not increase the
sensitivity of the cell strains to the cytotoxic effect of BPDE. In the parental cell
strain, BPDE induced a dose-dependent increase in the frequency of
hygromycin—resistant (Hng) cells, indicating that the parental cells were able to
combine information from two non-functional homologous copies of the hyg gene
(damage avoidance). In the cell strains lacking hMmsZ, this process was

completely eliminated. BPDE-induced a dose—dependent increase in the

100

 

frequency of mutations in the single copy HPRT gene of the parent cell strain, but
in the cell strains lacking hMms2, the frequency of such mutations was 2.5-fold
higher. These results indicate that loss of hMms2 not only eliminates use of a
homologous allelic copy of a gene, but also use of the newly synthesized
daughter strand of the sister duplex. They also indicate that human cells make
approximately equal use of translesion synthesis and damage avoidance in order

to tolerate BPDE-induced fork-blocking lesions.

2. Materials and methods
2.1. Cell strains and media used

The parental cell strain and its three derivative strains 2A-M2, 3C-M2, and 13D-
M2, deficient in hMms2 protein, were described previously [7]. Unless otherwise
indicated, cells were cultured in Eagle’s minimal medium supplemented with L-
aspartic acid (0.2 mM), L-serine (0.2 mM), sodium pyruvate (1 mM),
hydrocortisone (1 pg/ml), penicillin (100 Ulml), streptomycin (100 pglml), and
supplemented bovine calf serum (Hyclone, Logan, UT) (10% vol/vol) (culture
medium). This medium containing the appropriate selection drug, i.e., TG (40
pM) or hygromycin (100 Ulml) was used for selection of TG resistant or
hygromycin resistant cells, respectively. When these cell strains were removed
from storage in liquid nitrogen to be used in biological experiments, they were
cultured for several days in selective culture medium containing histidinol or

histidinol plus puromycin [7] to be sure that they retained the transfected genes.

101

 

2. 2. BPDE treatment

Exposure of the cells to BPDE was carried out as described [21]. Briefly, cells
in exponential growth were plated at 10" cells/cm2 12 h before treatment to allow
cells to attach to the culture dishes. The medium was removed from the dishes,
cells were rinsed twice with phosphate-buffered saline (PBS), and Eagle’s
minimal essential medium lacking serum was added. BPDE was dissolved in
anhydrous dimethylsulfoxide (DMSO) immediately before treatment and
delivered to each dish by micropipette to yield the desired concentrations. An
appropriate amount of DMSO was added to the dishes to ensure that all the cells
were exposed to the same concentration of DMSO (<0.5%). After 1 h incubation
at 37°C, in a humidified 5% CO; incubator, the medium containing BPDE was

removed, and the cells were assayed for the biological effects of BPDE.

2. 3. Assay for cytotoxicity

Cells were washed with PBS, and detached from the dishes using trypsin,
suspended in culture medium, and plated into 100-mm diameter dishes at
various cloning densities. The culture medium was renewed 7 days later. When
the surviving cells had formed colonies, ~14 days after the plating, the cells were
stained and the number of clones on each dish was scored. Cell survival was
calculated as follows: for each dose, the cloning efficiency of the BPDE-treated
cells was divided by that of the untreated control cells, and the value was

expressed as percent of that of control cells.

102

 

2. 4. Assay for the frequency of BPDE-induced h ygromycin resistant cells

Cells were plated at 104 cells/cm2 and sufficient sets of dishes were used to
have at least 2x106 surviving target cells per dose. Just prior to exposure to
BPDE, the cells in at least two dishes were detached and counted electronically
to determine the percentage of cell attachment. After 1 h of BPDE treatment, the
medium was removed, the cells were rinsed twice with PBS, and the medium
was replaced with appropriate selective culture medium. Approximately 48 h after
BPDE treatment, the medium was changed to culture medium containing
hygromycin (100 Ulml), which was replaced every five days. Cells were stained 3
weeks after treatment. The frequency of recombination for each dose was
calculated as follows: total hygromycin resistant clones divided by total number of
surviving cells corrected by the percentage of attachment. The induced

frequencies were calculated by subtracting the background frequencies.

2. 5. Assay for the frequency of BPDE-induced mutations in the HPRT gene

Cells were plated at 104 cells/cm2 into sufficient dishes to have at least 1x106
surviving target cells per dose, according to the expected survival. After 1 h
exposure to BPDE, the cells were rinsed twice with PBS. The medium was
replaced with appropriate selective culture medium, and cells were allowed an 8-
day expression period to be depleted of wild type Hprt protein before at least
1x106 cells per dose were assayed for the frequency of TG resistance as
described [22]. Culture medium containing 40 [M TG was renewed after 7 days.

For each dose, a separate set of dishes with cells plated at cloning density in

103

 

medium lacking TG, was used to determine the cloning efficiency of the cells at
the time of TG selection. The culture medium in these dishes was replaced 7
days after the plating. The cells for determining cloning efficiency and TG
resistance were stained 7 days later. The number of clones on each dish was
scored. For each cell strain, the frequency of mutants was calculated as follows:
the number of colonies of TG resistant cells divided by the number of clonable
cells selected (determined as above). The induced frequencies for each cell
strain were calculated by subtracting the background frequencies determined

using the mock-treated cells.

3. Results and discussion
3.1. Determining the relative use of translesion synthesis vs. damage avoidance
The underlying assumption an approach is that if an agent forms only one
major type of fork-blocking adduct in DNA, and this adduct is mutagenic, and if
one can eliminate use of the damage avoidance, error-free pathway for dealing
with such damage, the relative use of translesion synthesis versus damage
avoidance can be determined by comparing the frequency of mutations induced
by that agent in cells in which use of damage avoidance has been blocked to that
found in the wild type parental cells that retain the use of this latter pathway. One
cannot estimate cells’ relative use of damaged DNA directly as a template versus
use of an undamaged homologous copy as a template to continue replication of
DNA containing fork-blocking lesions if the DNA damage consists of several

different kinds of fork-blocking lesions, e.g., UV-photoproducts, each with a

104

different potential for using one or the other of these two processes. Use of
BPDE as the DNA-damaging agent eliminates this difficulty. In contrast to UV,
BPDE induces only one very predominant adduct. Approximately 94% of BPDE
adducts involve guanine [14, 15], and consist of a covalent bond between the
C10 of the hydrocarbon molecule and the exocyclic N2 of guanine [16].
Therefore, BPDE was used for the present study designed to estimate how
frequently human cells make use of damage avoidance rather than translesion
synthesis. To detect use of an undamaged copy of the blocked sequence as a
template (damage avoidance), we employed as our target a substrate stably
integrated into the genome of the target cells that allows quantification of the
frequency with which cells produce a selectable wild type hyg gene, coding for
resistance to hygromycin (Hng), by combining information from two defective
hyg genes [7, 9, 11]. To detect error-prone translesion synthesis, we employed

as our target the HPRT gene, present as a single copy in our male target cells.

3. 2. Effect of depletion of hMmsZ protein to the cytotoxic effect of BPDE

As shown in Figure 1A, there was no significant difference between the
parental cell strain and the three derivative cell strains devoid of hMms2 in
sensitivity to the cytotoxic effect of BPDE. This indicates that the parental cells
containing hMms2 and the derivative cell strains lacking hMms2 are equally
capable of replicating the damaged DNA. This result is consistent with the
previous finding that these cell strains deficient of hMmsZ are not more sensitive

to the cytotoxic effect of UV than their parental strain [8].

105

 

 

Figure 1. The biological effects of loss of hMms2 protein on BPDE-induced
recombination and mutations. Open symbol, the parental cell strain; closed circle,
strain 3C-M2; closed triangle, strain 2A-M2; and closed square, strain 13D-M2.
Each data point is derived from the average of three independent experiments.
For some data points, the standard deviation is too small to be visible. Some
points are offset slightly in order to be visible. (A) The BPDE-induced cytotoxicity
in these cell strains. The solid line represents the least-squares line for all the
data points. (8) The BPDE-induced frequency of hng cells in the parental cell
strain and the cell strains lacking hMmsZ protein. For the data points generated,
at least 3x106 surviving cells were assayed in each experiment. The solid line
represents the least-squares line for the data points obtained from the parental
cell strain. (C) The BPDE-induced frequency of TGR cells the wild type cell strain
and the three cell strains lacking hMms2 protein. For the data points generated,
at least 1x106 cells were assayed in each experiment. The observed frequency of
TGR clones was corrected by the cloning efficiency of the cells at the time of
selection and the induced mutation frequency was obtained as described [22].
For the majority of the data points, the cloning efficiency of the cells at the time of
selection ranged from 40-52%, so the correction factor was less than 3-fold. The
solid lines represent the least-squares lines for the data obtained from the

parental cell strain and cell strains lacking hMmsZ, respectively.

106

 

 

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107

3. 3. Effect of depletion of hMmsZ protein on the frequency of BPDE-induced
h ng recombinant cells

As shown in Figure 1B, the parental cell strain exhibited a dose-dependent
increase in the frequency of BPDE-induced hng cells (recombinants), indicating
that this strain used the undamaged homologous copy of the hyg gene as a
template to circumvent BPDE-induced adducts in one or the other of the two
nonfunctional hyg genes. In contrast, this response to BPDE was eliminated from
cell strains 2A-M2, 3C-M2, and 13D-M2 which are virtually devoid of hMms2
protein. These data indicate that hMmsZ protein plays an essential role in
allowing human cells to make use of a homologous copy of a blocked sequence
as a temporary template to tolerate BPDE-induced fork-blocking lesions (damage
avoidance). This is consistent with our previous finding in human cells containing

UV-induced DNA damage [7].

3.4. Effect of depletion of hMms2 protein on the frequency of BPDE-induced TGR
cells

BPDE treatment also caused a dose-dependent increase in the frequency of
HPRT mutants (T GR cells) in the parental cell strain (Fig. 10), indicating that
these cells carry out translesion synthesis. The three cell strains lacking hMms2
protein also gave a dose-dependent induction of TGR cells, but the frequency of
TGR cells at each dose was ~2.5 times higher than that induced in the parental
cells, which are capable of carrying out damage avoidance. These data can be

interpreted as indicating that loss of hMms2 protein resulted in lack of the ability

108

to use an undamaged copy of the blocked HPRT sequence as a template and,
therefore, forced the cells to use the translesion synthesis pathway. Because
there is only one copy of the HPRT gene in these target cell strains, the
increased frequency of mutations in the HPRT gene provides indirect evidence
that when hMms2 is present, it not only allows human cells to make use of a
homologous copy, e.g., an allele, but also allows them to make use of the newly
synthesized daughter strand of the sister duplex as an undamaged template to
avoid DNA damage. The data in Figure 1A, 1B, and 1C strongly suggest that
when normal human cells replicate DNA containing BPDE adducts, they make
approximately equal use of damage avoidance and translesion synthesis, and
that loss of hMms2, with the resulting loss of the ability to copy an undamaged
homolog, channels DNA lesions into the translesion synthesis pathway, resulting

in a 2.5-fold increase in frequency of mutants.

3. 5. Significance for future studies

The present data obtained using BPDE, together with our earlier data using UV
as the DNA damaging agent, indicate that translesion synthesis and
recombination are the major means for tolerating fork-blocking lesions in human
cells. Our data with the hyg gene indicate that damage avoidance is achieved via
a recombination-mediated specialized DNA replication process that
predominantly involves gene conversion, unaccompanied by crossing-over [9-
11]. Both sets of data obtained using cells lacking hMms2, i.e., those exposed to

UV [7] or to BPDE, indicate that hMmsZ protein is absolutely essential for this

109

 

process, although the exact mechanisms and various components remain to be
explored. Use of homologous recombination by mammalian cells to repair
double-strand breaks has been extensively studied, and the mammalian
homologs of the yeast RAD52 epistasis group, RAD51, RAD52, RA054,
RAD548, RAD50, MRE11, N881, RAD51B, RAD51C, RAD51D, XRCCZ and
XRCC3, have been implicated in this latter process [23]. The breast cancer
susceptibility genes BRCA1 and BRCA2, which do not appear to have yeast
homologs, are also involved [24, 25]. It will be important to determine whether
these gene products are involved in damage avoidance as a means of tolerating

replication fork-blocking lesions.

How the hMms2/hch13 complex is connected to the recombination process is
not yet clear. It has been shown in yeast that ch13 is required for the assembly
of polyubiquitin chains linked through lysine 63 in a Mms2-dependent manner
[26], and similar findings were also obtained in human cells [27]. A most
intriguing finding was that by Ulrich and Jentsch [28] who demonstrated that in
yeast, Rad5 interacts with both ch13 and Rad18 through its RING finger
domain. These interactions bring the MmsZ/ch13 complex into contact with the
Rad6/Rad18 complex. They proposed that the Mms2/ch13 complex acts as a
signal transducer to recruit target proteins to the site of DNA damage for repair.
However, a human homolog of RAD5 has not been identified. It will be important
to find out whether a functional counterpart of yeast Rad5 exists in human cells.

In addition, the connection between the novel polyubiquitin pathway through

110

 

 

lysine 63 catalyzed by the MmsZ/ch13 complex and the damage tolerance
pathways demonstrated in the present studies needs to be addressed. Our data
suggest that the hMms2/hch13 complex is required to recruit and/or activate
proteins needed for human cells to avoid fork-blocking lesions by using an
undamaged homologous copy of DNA as an alternative template to continue
DNA replication. The data implicate the novel polyubiquitin pathway through
lysine 63, mediated by the hMms2/hch13 complex, as being essential for this

recombination/gene conversion process.

Acknowledgements

We are grateful to Dr. Hong Zhang for generating the parental cell strain and to
Clarissa Dallas for technical assistance. This work was supported by Department
of Health and Human Services/National Institutes of Health grants ES-09822 and
CA 91490 (to V.M.M.) and by grant MOP-53240 from the Canadian Institute of
Health Research (to W.X.)

111

 

 

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recombination in mammalian cells, Biochem Soc Trans 29 (2001) 196-
201.

M.E. Moynahan, A.J. Pierce, M. Jasin, BRCA2 is required for homology-
directed repair of chromosomal breaks, Mol Cell 7 (2001) 263-272.

M.E. Moynahan, J.W. Chiu, B.H. Koller, M. Jasin, Brca1 controls

homology-directed DNA repair, Mol Cell 4 (1999) 51 1-518.

115

[26]

[27]

[231

RM. Hofmann, C.M. Pickart, Noncanonical MMSZ-encoded ubiquitin-
conjugating enzyme functions in assembly of novel polyubiquitin chains for
DNA repair, Cell 96 (1999) 645-653.

S. McKenna, L. Spyracopoulos, T. Moraes, L. Pastushok, C. Ptak, W.
Xiao, M.J. Ellison, Noncovalent interaction between ubiquitin and the
human DNA repair protein Mmsz is required for ch13-mediated
polyubiquitination, J Biol Chem 276 (2001) 40120—40126.

H.D. Ulrich, S. Jentsch, Two RING finger proteins mediate cooperation
between ubiquitin- conjugating enzymes in DNA repair, EMBO J 19 (2000)
3388-3397.

116

 

CHAPTER IV

hREV3 is essential for error-prone translesion synthesis past
UV or benzo[a]pyrene diol-epoxide-induced

DNA lesions in human fibroblasts

Ziqiang Li a, Hong Zhang 3' 1, Terrence P. McManus 3, Yun Wang a, J. Justin
McCormick ‘, Christopher W. Lawrence b, Veronica M. Maher " *

‘ Carcinogenesis Laboratory, Department of Microbiology and Molecular
Genetics and Department of Biochemistry and Molecular Biology, Michigan State
University, East Lansing, MI 48824, USA

b Department of Biochemistry and Biophysics, School of Medicine and Dentistry,
University of Rochester, Rochester, NY 14642, USA

1 Present address: Department of Genetics, Stanford University School of

Medicine, Stanford, CA 94305-5120, USA

*Corresponding author: Veronica M. Maher, Carcinogenesis Laboratory, Food
Safety and Toxicology Building, Michigan State University, East Lansing, 48824-
1302. Tel.: (517) 353-7785; Fax: (517) 353-9004; E-Mail: maher@msu.edu

117

 

 

Abstract

In S. cerevisiae, the REV3 gene encodes the catalytic subunit of
polymerase zeta, which is involved in translesion synthesis and is required
for mutations induced by UV photoproducts and other DNA fork-blocking
lesions, as well as for the majority of spontaneous mutations. To determine
whether hREV3, the human homolog of yeast REV3, is similarly involved in
error-prone translesion synthesis past UV photoproducts and other kinds
of lesions that block DNA replication, we transfected an hREV3 antisense
construct under the control of the TetP promoter into an infinite life span
human fibroblast cell strain that expresses a high level of tTAk, the
activator of that promoter. Three cell stains expressing high levels of
hREV3 antisense RNA were identified and compared with the parental cell
strain for their sensitivity to the cytotoxic and mutagenic effects of UV- or
(;I-_)-78,80I-dihydroxy-9a,10a-epoxy-7,8,9,20-tetrahydrobenzo[a]pyrene
(BPDE). The latter strain gave a dose dependent increase in cell killing and
induction of mutations in the single copy HPRT gene. In contrast, the cell
strains expressing hREV3 antisense did not exhibit significantly increased
sensitivity to the cytotoxic effect of these agents, but the frequency of
mutants induced by UV and by BPDE in these cell strains was reduced to
~16% and ~25% that of the parental cell strain, respectively. These data
indicate that the function of hRev3 is not essential for cell survival, but is
absolutely required for the induction of mutations by UV or BPDE. Whether

this in vivo activity involves insertion of an incorrect nucleotide opposite

118

the fork-blocking lesion or extension beyond that position is not yet

known.

Keywords: hREV3; translesion synthesis; mutagenesis; antisense RNA;
UV; BPDE

1 . Introduction

DNA in prokaryotic and eukaryotic cells is continuously exposed to
endogenous and exogenous DNA damaging agents. It not removed, certain
types of DNA damage can impede normal DNA replication carried out by the
replication complex [1]. Excision repair mechanisms physically remove DNA
damage and restore the DNA to its original state. Nevertheless, cells possess
damage tolerance mechanisms which enable them to continue synthesizing DNA
containing fork-blocking lesions. In prokaryotes and eukaryotes, such damage
tolerance mechanisms include translesion synthesis and damage avoidance.
Damage avoidance is the term used to refer to various mechanisms by which
cells make temporary use of an undamaged homologous DNA of the blocked
sequence as a template to continue DNA replication. In mammalian cells, the
undamaged copy can be either the newly synthesized daughter strand of the
sister duplex [2], as suggested by Higgins et al. [3], or the homologous allelic
gene. The alternative pathway for tolerating DNA fork-blocking lesions, i.e.,
translesion synthesis, requires specialized distributive DNA polymerases that, in

contrast to the major DNA polymerases that are blocked by such damage, are

119

 

 

capable of incorporating nucleotides opposite the damaged template [4]. After
distributive incorporation of nucleotides, followed by extension of a limited
number of nucleotides, the major DNA replication complex is considered to
resume replication activity by using the original strand as the template.
Translesion synthesis can be either “error-prone” or “error-free” depending upon
such factors as the type of lesion, its sequence context in the DNA, the
availability of specialized distributive translesion synthesis polymerases in the
cell, and the characteristic of the polymerases used. At this time, polymerase
zeta, eta, iota, and kappa are considered to be the major DNA polymerases
carrying out translesion synthesis past different kinds of DNA lesions in

eukaryotes [5-10].

In S. cerevisiae yeast, RE V3 encodes the catalytic subunit of polymerase zeta
(pol g), and REV7 encodes the non-catalytic subunit. Compared to the wild type,
yeast rev3 mutants displayed only slightly increased sensitivity to the cytotoxic
effect of UV [11], but loss of function of Rev3 eliminated almost all of the
damage-induced mutations and a large portion of spontaneous mutations [12].
These studies indicate that Rev3 protein in yeast is not essential for preventing
cell death induced by UV, but is essential for error-prone translesion synthesis
past a variety of fork-blocking lesions. Lawrence and colleagues [5] showed that
when tested in vitro using primer extension assays, yeast Rev3 and Rev7
proteins form functional pol Q which is capable of inserting nucleotides opposite a

thymine-thymine (T -T) dimer. Yeast pol C in vitro also performs error-prone

120

translesion synthesis past a T-T (6-4) photoproduct or an acetylaminofluorene-
adducted guanine [13]. In addition, yeast pol q extends mismatches in vitro with

a high efficiency [13, 14].

The human homolog of the yeast REV3 gene, hREV3, was independently
cloned by three groups [6, 7, 15]. Studies using the yeast two-hybrid analysis
[16] indicates that the protein coded by hREV3 interacts with that coded by a
putative hREV7. One of these reports of the identification of hREV3 included
results from a pilot study of role of hRev3 in the induction of mutation in intact
human cells by UV irradiation. The data showed that in a cell strain expressing
hREV3 antisense the frequency of mutants induced by UV was significantly
reduced [7]. These results suggest that the function of hREV3 is similar to that of
its yeast counterpart. To examine the role of hREV3 in vivo more thoroughly and
expand the study to include additional types of fork-blocking lesions, in addition
to photoproducts of uvm...“ irradiation, we transfected hREV3 antisense
construct under the control of TetP promoter into an infinite life span human
fibroblast cell strain that expresses a high level of tTAk which is required for the
activation of that promoter. Three transfectants expressing high levels of hREV3
antisense RNA were identified. These cell strains were compared with their
parental strain for sensitivity to the cytotoxic and mutagenic effect of UV or
BPDE. Our data show that, compared to their parental strain, the cell strains
expressing high levels of hREV3 antisense did not exhibit significantly increased

sensitivity to the cytotoxic effect of W or BPDE, but exhibited ~84% and ~75%

121

 

 

reduction of the frequency of HPRT mutants induced by UV and by BPDE,
respectively. Our data indicate that the function of hRev3 is not essential for cell

survival, but is required for UV- and for BPDE-induced mutations.

2. Material and methods
2.1. Cell culture

Cells used for cytotoxicity and 6-thioguanine (T G, Sigma) assays were cultured
in Eagle’s minimal medium supplemented with L-aspartic acid (0.2 mM), L-serine
(0.2 mM), sodium pyruvate (1 mM), hydrocortisone (1 pg/ml), supplemented calf
serum (Hyclone, Logan, UT) (10% vol/vol), penicillin (100 Ulml), and
streptomycin (100 pg/ml) (culture medium). This medium containing TG (40 pM)
was used for selection of HPRT mutants. When these cell strains were removed
from storage in liquid nitrogen to be used in biological experiments, they were
cultured for several days in McM medium lacking histidine containing histidinol or
histidinol plus puromycin as described previously [2] to be sure that they retained

the transfected genes coding for these selectable markers.

2. 2. Transfection

The hREV3 antisense construct was transfected into the parental cell strain
using LipofectAMINE (Life Technologies, GibcoBRL). The same construct but not
containing the antisense DNA, was also transfected into the parental strain to
generate vector control transfectants. After 48 h, transfectants were selected in

McM medium lacking histidine but containing histidinol plus 1 ug/ml puromycin.

122

 

 

This medium was renewed every five days. When the clones had formed, they

were isolated, expanded in the selection medium, and stored frozen until needed.

2. 3. Northern analysis

RNA was extracted from cells using RNAzolTMB (TEL-TEST, INC.) according to
the manufacture’s recommendations. RNA was quantified and equal amount (15
pg) of total RNA was electrophoresed in 1.2% formaldehyde agrose gel. The gel
was washed and transferred to Hybond-N+ (Amersham) nylon membranes.
Hybridization was carried out according to the standard procedures using a
sequence-specific probe labeled by PCR to determine the level of antisense
hREV3 expression. The membrane was stripped and re-probed with
gcheraldehyde-3-phosphate dehydrogenase (GAPDH) to compare the relative

amount of total RNA loaded for each sample.

2. 4. Western analysis for hRev3 protein

Nuclear proteins from cell strains were extracted as described [17]. Standard
western analysis was carried out by running 4% acrylamide gel using 100 pg
nuclear protein for each sample, followed by transfer in 0.1 M CAPS (pH=11)

(Sigma) overnight and probing with five different antibodies against hRev3.

2. 5. UV treatment and BPDE treatment
Exposure of the cells to UV and to BPDE was carried out as described [18]

Briefly, cells in exponential growth were detached from the dishes using trypsin,

123

 

 

diluted in culture medium, plated at densities ranging from 0.5x106 to 1.5><106 per
150mm diameter dish or at cloning density for UV irradiation or plated at 104
cells/cm2 for BPDE treatment. They were allowed ~12 h to attach before being
exposed to the agent. The medium was removed from the dishes, and cells were
rinsed twice with phosphate-buffered saline (PBS) before the treatment. For UV,
cells were irradiated in a film of PBS. Immediately following irradiation the
medium was added to each dish. For BPDE, Eagle’s minimal essential medium
lacking serum was added to each dish after PBS was removed. BPDE was
dissolved in anhydrous dimethylsulfoxide (DMSO) immediately before treatment
and delivered to each dish by micropipette to yield the desired concentrations. An
appropriate amount of DMSO was added to the dishes to ensure that all the cells
were exposed to the same concentration of DMSO (<0.5%). After 1 h incubation
at 37°C, in a humidified 5% CO; incubator, the medium containing BPDE was

removed, and replaced with fresh cultural medium.

2. 6. U V- and BPDE-induced cytotoxicity assay

The UV254m-induced and BPDE-induced cytotoxicity was assayed as
described [18]. Briefly, for UV-induced cytotoxicity, the cells were plated cloning
densities and irradiated as above. The medium was renewed one day and seven
days after UV-irradiation. The cells were stained ~14 days after UV treatment.
The survival was determined by comparing the cloning efficiency of the irradiated
cells to that of the sham-irradiated control cells, and the value was expressed as

percent of that of those control cells. For determining BPDE-induced cytotoxicity,

124

 

 

the cells were plated and treated as above. After 1 h BPDE treatment, the cells
were washed with PBS, trypsinized, diluted in the culture medium, and plated at
desired cloning densities. The culture medium was renewed 7 days after plating,
and cells were stained ~7 days later. The survival was calculated from the
relative colony-forming ability of the BPDE-treated cells compared to the mock-

treated control.

2. 7. U V-induced and BPDE-induced HPRT mutation assay

The method used to determine the frequency of UV or BPDE-induced HPRT
mutants has been described [19]. Briefly, for UV experiments, sufficient cells
were plated at 0.5x106 to 2x106 per 150-mm diameter dish ensure to have at
least 1x106 surviving founder cells per dose. For BPDE experiments, cells were
plated into a series of 150mm diameter dishes at 104 cells/cmz. After treatment,
the cells were allowed an 8-day expression period to deplete the wild type Hprt
protein before being assayed for the frequency of TG resistant cells. A separate
set of cells plated at cloning density with medium lacking TG was used to
determine the cloning efficiency of the cells at the time of selection,. The
frequency of mutants was calculated as follows: total TG resistant clones divided
by the total number of clonable cells selected. The induced frequencies were

calculated by subtracting the background frequencies in the control population.

3. Results and discussion

125

 

3.1. Generation and characterization of cell strains expressing high levels of
hRE V3 antisense RNA

To generate cell strains expressing high levels of antisense hREV3 RNA, we
first generated an immortal, fibroblast-derived cell strain, designated 9N, by
transfecting MSU-1.2-E7.2 cell strain [20] with a tetracycline transactivator (tTAk)
construct under the control of TetP promoter [21, 22]. Western analysis indicated
that the 9N cells express a high level of tTAk protein (data not shown) which is
required for the activation of TetP promoter. An expression vector designated
pTet-Puro contains a TetP promoter and a gene for puromycin resistance was
designed. The antisense hRev3 DNA, a 1,506-bp Sall fragment of the hREV3
cDNA, containing 48 bp of 5'-untranslated sequence, 1,415 bp of coding
sequence, and polylinker sequences at each end, was cloned into pTet-Puro in
the antisense orientation under the control of the TetP promoter to generate the
plasmid pKS-2. pKS-2 was used to transfect the 9N parental cells to generate
transfectants expressing hREV3 antisense. A pTet-Puro vector lacking the
antisense DNA was used to transfect the 9N cell strain to obtain vector control
transfectants. At least 100 hREV3 transfectants and 10 vector control
transfectants were isolated and expanded for future study. RNA from these
strains and the parental cell strain, 9N, was isolated and subjected to Northern
analysis. Three transfectants were identified to express high levels of hREV3
antisense. As shown in Figure 1, as expected, the parental cell strain and vector
control transfectants (V1 and V2) do not express hREV3 antisense RNA,

whereas cell strains 6|, 88, and 12C expressed high levels of antisense.

126

9N V1 V2 61 BB 12C

hRE V3 ,
anfisense

  

GAPDH —’ u ‘

Figure 1

Figure 1. Northern blot analysis for detecting the expression of hREV3
antisense. The parental cell strain 9N and two vector control transfectants
V1 and V2 are not expressing antisense. Three transfectant cell strains 6l,
88, and 120 express high levels of hREV3 antisense. GAPDH was used as

a loading control.

127

 

 

Expression of endogenous hREV3 mRNA in the parental cell strain or the
transfectant cell strains was not detected, indicating that the mRNA level is very

low in human fibroblasts.

3. 2. Western analysis of hRev3 protein

We attempted to determine that cell strains 6|, 8B, and 12C have a significantly
lower amount of hRev3 protein than does the parental cell strain 9N. To do this,
we raised independent anti-rabbit polyclonal antibodies against three peptide
sequences in hRev3 and addition purchased two independent anti-goat
polyclonal antibodies against hRev3 from Santa Cruz Biotechnology, Inc. (Santa
Cruz, CA). Compared to the results from the pre-immune sera, no additional
band was detected between 250 kDa and 450 kDa with these polyclonal
antibodies against hRev3 which has a putative molecular weight about 350 kDa
(data not shown). These data indicate the level of hRev3 protein is too low to be
detected by these antibodies. Similar results were obtained using cell lysates
instead of nuclear extracts. Three possibilities could explain these results: 1)
none of these five different antibodies is sensitive enough to detect hRev3
protein; 2) the level of hRev3 protein level is too low to be detected; 3) the
combination of these two. According to our Northern results, the endogenous
mRNA of hREV3 was virtually undetectable. In addition, hREV3 mRNA has a
translation false start site [7] which may reduce the efficiency of translation.
Therefore, it is very possible that the level of hRev3 protein is very low in these

human fibroblast strains and is beyond the limit of detection using these available

128

 

 

antibodies. In any event, we are not able to compare the hRev3 protein level
directly between the parental cell strain and cell strains expressing hREV3

anfisense.

3. 3. The effect of hRE V3 antisense expression on U V-induced cytotoxicity

Cell strains 6|, 88, and 120, expressing high levels of hREV3 antisense, were
compared with their parental cell strain 9N for UV-induced cytotoxicity assays.
Strains 6|, 8B, and 12C were not more sensitive to the cytotoxic effect of UV than
the parental cell strain, (Fig. 2A), which is consistent with the results of the
preliminary study involving a separate set of cell strains [7]. In addition, two
vector control transfectants exhibited sensitivity to the cytotoxic effect of UV that
was similar to the parental cell strain 9N (data not shown). These results suggest
that hRev3 protein is not essential for preventing UV-induced cell killing, which is

consistent with what is found in S. cerevisiae yeast.

3. 4. The effect of hRE V3 antisense expression on U V-induced mutagenesis

As expected, the parental cell strain 9N showed a dose-dependent increase of
UV-induced HPRT mutant frequency. 6|, 8B, and 120 cell strains also showed a
dose-dependent increase of UV-induced HPRT mutant frequency, but they
exhibited >84% reduction of the frequency at each dose (Fig. 28), which is
consistent with the results found in the pilot study [7]. The two vector control
transfectants exhibited a frequency of UV-induced HPRT mutants similar to that

of the parental strain (data not shown), suggesting that these biological effects

129

 

Figure 2. The effect of expression of antisense hREV3 on the survival and
induced frequency of mutations observed with cell strains exposed to UV.
Parental cell strain 9N (open circle), cell strains 6| (closed circle), 88 (closed
triangle), and 120 (closed square). Some points have been offset slightly in order
to be visible. (A) The UV-induced cytotoxicity in the parental cell strain and cell
strains expressing antisense hREV3. The solid line represent the least square
line for all the data points. (8) The UV-induced frequency of HPRT mutants in the
parental cell strain and cell strains expressing antisense hREV3. The observed
mutant frequency was corrected by the cloning efficiency of the cells at the time
of selection, and the induced mutation frequency was obtained by subtracting the
background frequencies, which for the most part were less than 10 per million
clonable cells. For the majority of the data points, the cloning efficiency of the
cells at the time of selection ranged from 40-54%, so the correction factor was
less than 3-fold. In panel B, the solid lines represent the least-squares lines for
the data obtained from the parental cell strain and cell strains expressing hREV3

antisense, respectively.

130

Mal and
ad to UV
3B (closed
lly in only
in and set
Isl square
ants in the
observed
It the time
"actingtlre
)er millicr
Icy of the
actorwas
sllnesfor

Ig hREl’l

Percent Survival

Induced Mutants per10‘ Clonable Cells

0
100

80
70
60

5O
40

30

20'

10
240

200

160

120

80

40

6 8 10

12

14

 

 

 

 

 

 

 

4

6 8
UV (Jlmz)

Figure 2

131

 

observed in 6|, 88, and 120 were caused by the expression of antisense hRE V3
RNA and presumably caused by the loss of expression of hRev3 protein.
Moreover, studies involving growth curve to compare rate of replication of the
parental cell strain and vector control transfectants with that of cell strains, 6|, 88,
and 120, did not show any evidence of a growth disadvantage (data not shown).
This indicated that the biological effects were not caused by cells being sick or
growing too slowly. Flow cytometry analysis indicated that 6|, 8B, and 120 cell
strains are completely diploid (data not shown). Taken together, these data
suggest that 6|, 88, and 12C have a significantly reduced level of hRev3 protein,
and indicate that hRev3 protein is essential for the error-prone translesion

synthesis past UV-induced lesions in human cells.

3. 5. The effect of hRE V3 antisense expression on BPDE-induced cytotoxicity
Cell strains 6| and 120 were compared with the parental cell strain 9N for
BPDE-induced cytotoxicity. Because in BPDE-induced cytotoxicity experiments,
the survival is related to the amount of BPDE bound in the cell, and it was difficult
to be certain that the amount of BPDE weighed out each time was equal because
so little was used (20-30pg), the dose we gave was not consistent each time due
to weighing error. In addition, BPDE is chemically unstable, and the plated cell
density affects the cytotoxicity. Therefore, it is very difficult to compare the cell
survival between the experiments. However, within one experiment, we did not
observe a significant difference in BPDE-induced cytotoxicity between 9N, 6|,

and 120 cell strains. For example, in one experiment, the survival at 0.06pM of

132

 

 

BPDE for 9N, 6|, and 12C was 68%, 71%, and 68%, respectively. The survival at
0.1pM BPDE for 9N, 6I, and 120 was 47%, 50%, and 50%, respectively (Fig.

3A).

3. 6. The effect of hRE V3 antisense expression on BPDE-induced mutagenicity
As expected, the parental cell strain 9N showed a dose-dependent increase of
BPDE-induced HPRT mutant frequency. Cell strains 6| and 12C also showed a
dose-dependent increase of BPDE-induced HPRT mutant frequency, but at each
dose they exhibited >75% reduction of the frequency. Because the cell survival is
not always consistent between experiments, and the mutant frequency is
correlated to the cell survival, we plotted mutation frequency against the
percentage of survival. Our data showed that at the same survival, the BPDE-
induced mutation frequency in cell strains 6| and 120 was reduced to ~17% and
~23%, respectively, compared to the parental cell strain 9N (Fig. 4). These data
support the hypothesis that hRev3 protein is also essential for the error-prone

translesion synthesis past BPDE-induced fork-blocking lesions in human cells.

133

Figure 3. The biological effects of antisense hREV3 expression in the parental
cell strain 9N (open circle), and cell strains expressing hREV3 antisense 6|
(closed circle) and 12C (closed square). Some points are offset slightly in order
to be visible. (A) The BPDE-induced cytotoxicity in the parental cell strain and
cell strains expressing antisense hREV3. The solid line represents the least
square line for all the data points. (8) The BPDE-induced frequency of HPRT
mutants in the parental cell strain and cell strains expressing antisense hREV3.
The observed mutant frequency was corrected by the cloning efficiency of the
cells at the time of selection, and the induced mutation frequency was obtained
by subtracting the background frequencies, which mostly are less than 10 per
million clonable cells. For the majority of the data points, the cloning efficiency of
the cells at the time of selection ranged from 39-55%, so the correction factor
was less than 3-fold. The solid lines represent the least-squares lines for the data
obtained from the parental cell strain and cell strains expressing hREV3

antisense, respectively.

134

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:isenseg; 0.00 0.03 0.06 0.09 0.12 0.15
100 ' I F I I l I I
yin order g 90 ' a)
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strain and g 70 r 8
the least E 60 '
o
of HPRI’ 9 50 r 9 o
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ahRB’i. 2 '
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BPDE (IIM)
Figure 3

135

Induced Mutants Per 10' Clonable Cells

 

400

320

240

160

 

 

 

 

100 80 60 40

Percent Survival Cloning ability

Figure 4. BPDE-induced mutagenesis versus percent cell
survival in the parental cell strain 9N (open circle), and cell

strains 6| (closed circle) and 120 (closed square)

136

 

 

3. 7. Significance

The UV- and BPDE-induced mutagenesis data (Fig. 28 and 38) suggest that
the cell strains expressing hREV3 antisense have a significantly reduced level of
hRev3 protein compared to the parental cell strain, and that hRev3 protein is
essential for UV- and BPDE-induced error-prone translesion synthesis. Human
polymerase eta (pol 11), which is able to carry out the error-free translesion
synthesis for UV-induced T-T dimers [23] and is mutated in xeroderma
pigmentosum variant patients [8, 24], is functional in these cell strains expressing
hREV3 antisense. pol T] was shown to insert G across from T-T (6-4)s in vitro
[26] and may have contributed mutations. Polymerase iota (pol I), which is a
remarkable error-prone DNA polymerase in vitro for T-T dimers and T-T (6, 4s)
photoproducts [9], is presumably functional in these cells as well. Therefore, the
UV-induced mutation frequency observed in these cell strains expressing
antisense (~16% of the parental cell strain) could be due to these DNA
polymerases or due to the remaining hRev3 protein. Pol n was also shown in
vitro to be able to insert A across from BPDE-adducted guanine [26, 27]. It might
play a role in generating the BPDE-induced mutants in the cell strains expressing

hRE V3 antisense.

137

Acknowledgements

We are grateful to Clarissa Dallas for technical assistance. We also thank other
members of our research groups for helpful discussions. This work was
supported by Department of Health and Human Services/National Institute of
Health Grants ES-09822 and CA 91490 (to V.M.M.) and Grant GM21858 (to
OWL).

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