. . h.
v.4... u. .(Itm 12D..-
r.
.

.

-
. . .

r ”93.x...v fit
91.
film.
— A

 

v fun... )1.

IV « I‘M”. 0‘ J
f-u¢ .13 :
a. tum»: hafiraJWomvv
Revuufirwfi. is... ,.
. . .A (Law a"...
(A11.

.Iu¢ v fl
4‘...le1.

‘ull.
‘1;
a!

v

 

. ‘ . . y . I
. y it; . , u. . . . . . ’9” WHO? . .
1, ”$1.53“ . 7 .. , . . . , . , . J? .u. QUE“?
v . . I , . . . . . . . ‘ A . V ‘ . sh?‘wnv.q...av.k ‘2;
. ‘ . no“ drurdunfivho.“
- , , . _ .I.
$2....er .. . . . . ‘ , . .

t .
1-14%

I at-

n"... u.
a... Emmy,“ . «.04.. .. ....;
.1 .1 xi. ) tn. .1...
J. 9.1.43“! : ”.4 .QNfr 6P
. halal Levi): :05
, l..‘AJ4.!IL .t....lrv. #71:
t. on! .J... ‘ '5"!

Irdhyr

 

. u H.
, V 531
.mn ‘ .rw. .. . {:43 I»...
. ,...$.....ha . 2. 2.”... m

. . .. .. . . ,. A. . 2.2.1.4 Jr
(22”.. . ‘u! . . . .. . ‘ .. :1 , J. ‘ .. .
auxiKfiUfl. &!W\ .P A . . ,

(I o. .1511! glib! .
.u- . ‘ . H . . . .m«3_m,flr.m¢. .
.Ilwfltfihpflflfr . n . . . . . . . . m .2. . . run... . 494
Ah”... 5492.131} ,. , . , . .. . . . .
. (Jun... .. 3 ...P:I.V i . ‘ . . . ‘ .
._ . .

- «Law. «.9. b.5632 V
. .v. .. . f9 1 I
d. L - u. 3...¢K.W1.uf
hm? Luau.» Ar. _ . ‘. . yfimuWu/flihahfi.
. a .4. . {my}...

rim. ..r

 

 

PM?” 5”,.
r . .

x,
31, r." v!!!
50. . . . I
Ila Kr
HA
I '65.! _
n50! .

I“!

.lyc
u.tvb..... uh...
MmWinILuflod
I: - . .

smmuvmhunrdfiwfinvdhsn

o crrolod.

 

. .1 1. .2.
. n.
71.. H‘
ii}:
5.5.41

. u

.HflQ:

. n? hum/H .

I A
I-..
a»; A.
., ‘ rcfinuhuflhhuu.

31-3304... mun. ‘
.M.M.Nh l..11‘fl‘m¢wm¢n.u.fl..wv . h. tron! . .

A. , . r ,' bl.‘ n olnfrlrfh'
r’t: .» in l .c‘
‘rwpwu I‘lfiW‘ 0'. 1... . ‘ f ll‘

 

 

7": null. P31! .

 

. I ‘ ” R'EIHI“ ,III I

    

la I

 

”15813

This is to certify that the

dissertation entitled

The influence of IHF and F18 on the binding DnaA
protein to oriC, the E, coli
chromosomal origin

 

presented by

Carla Margulies

has been accepted towards fulfillment
of the requirements for

Ph .D . degree in Biochemistry

 

 

«it. WK ' r:

U Major professo

Date A?“ l 4. 5.51-

MSUi: an Affirmative Action/Equal Opportunity Institution 0-12771

 

 

Iliililfl'liiil i flit”? 1W Iil'ilsliflfifll’l‘l‘ll'iilelil

3 1293 01579 5929

 

LIBRARY 1

Michigan State a
Unlverslty i

 

 

PLACE ll RETURN BOX to remove this checkout from your record.
TO AVOID F INES rotum on or baton duo duo.

DATE DUE DATE DUE DATE DUE

 

 

 

 

 

 

 

   

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

i

 

MSU Io An Afflnnativo Action/Equal Opponunlly lmfltulon
m

 

ans-9.1 ~

THE INFLUENCE OF IHF AND FIS ON THE ORDERED BH‘IDING OF DnaA
PROTEIN TO oriC, THE E. coli CHROMOSOMAL ORIGIN

By
Carla Eva Margulies

A DISSERTATION
Submitted to
Michigan State University

in partial fulfillment of the requirements

of the degree of

DOCTOR OF PHILOSOPHY

Department of Biochemistry

1997

Abstract

THE INFLUENCE OF IHF AND FIS ON THE ORDERED BINDING OF DnaA
PROTEIN TO oriC, THE E. coli CHROMOSOMAL ORIGIN

By
Carla Eva Margulies

Initiation of Escherichia coli chromosomal replication from oriC is dependent
on the sequence specific DNA binding protein, DnaA protein. Although DnaA
protein is known to bind to four indirect repeats (called DnaA boxes R1, R2, R3,
and R4) within oriC, little is known of whether DnaA protein binds these
sequences in a random or ordered manner or whether other oriC binding proteins
affect the binding of DnaA protein. Gel mobility shift, footprinting and in vitro
replication assays, were used to study the binding of DnaA protein to oriC and
the effect of two sequence specific DNA binding proteins, integration host factor
(IHF) and factor for inversion stimulation (FIS), on the binding of DnaA protein.
From these studies, DnaA protein was found to bind to oriC in an ordered manner
in which DnaA boxes R1 and R4 are bound at low concentrations of DnaA
protein, and the interior boxes, R2 and R3, are bound at higher concentrations.

In addition replication of an oriC plasmid correlates with the binding of all four
sites. Neither IHF nor FIS affects the binding of DnaA protein to a linear oriC
fragment. However, IHF acts as a positive factor in in vitro replication, whereas

FIS acts as a negative factor.

To my parents and brother

Acknowledgments

I would like to take this opportunity to thank tne many who have made my
work possible. First, I would like to thank Jon Kaguni who has been my thesis
advisor. Next, I would like to thank Laurie Kaguni, my secondary advisor, for her
opinions and lab materials. Thanks to my committee members, John Wison, Lee
Kroos, Susan Conrad, Steve Triezenberg and alternate member Zack Burton for
their advice and guidance.

As both Kaguni labs work closely with each other, I have enjoyed the
company of the members of both labs. In particular, I enjoyed the sense of humor
of Dave Lewis, The Studier of Butterflies. Ice cream outings with Dave provided
an excellent forum for discussions on evolution, movies, books, and the opposite
sex. I had the good fortune to be co-founder of the MSU Biochemistry Brewing
Company with Dave and the Kaguni institution, Kevin “Mac” Carr. As his name
would suggest Kevin has been very helpful with my countless computer
problems. I thank Mark Sutton for the useful scientific discussions and teaching
me the importance of The Bears and Mike Ditka. From Mark I also learned of the
nutritional value of corn dogs. I enjoyed Yuxen Wang’s many stories of growing
up in China and his observations and sense of humor on American culture and
society. I have enjoyed discussions on the virtues of wine, travel and other
things of life with Matias Vicente Cruz, a fellow activist for the cause of federally
funded travel. Members of the lab long gone, Jarek Marszalek and Ted Hupp not
only indoctemated me to the workings of the Kaguni labs but also introduced me
to Bob Dylan. I would like to thank other member the Kaguni labs, past and
present including; Wenge Zhange, Nikki Mather, Scott Cherry, Carol Farr, Andrea

iv

Williams, Jianjun Wang, Angie Kolhoff, Matt Olson, Li Fan, Anthony Lagina and
the many undergraduates.

Outside of the lab many contributed to my work much more than they may
realize. Leaving here for New York, I will miss my Friday morning coffee/joumal
club with Drs. Cindy Petersen and Marty Reiger. They are friends who have
endured my bicycle rides and my rantings on many subjects. Janek Werel and
Geoff and Erin Williams are friends who sustained me with their company their
cooking and their appreciation of good red wines and whiskey, Thomas
Derderian, who I had the fortune to meet on one of my wanderings in the Lansing
area via bicycle, endured my rantings, provided endless support and
encouragement in addition to food and drink, and a room with a view. Lastly, I
thank those I met through Velocipede Peddler who have taught me about
cycling, accompanied me on many bicycling outings, and kept my wheels turning
smoothly.

Finally, I would like to thank Beth Gantt, Jerry Dietzer, Bibi Chromwell, and
Susan Gottesman who have influenced me long before I came to East Lansing.
Most importantly, I thank my parents for providing the opportunities and the
freedom for exploration. I thank my brother, Martin, often has provided me with

inspiration and reminders of real biology.

Table of Contents

Page

List of Tables .......................................................................................................... viii
List of Figures ....................................................................................................... ix
List of Abbreviations ............................................................................................. xii
Chapter I. Literature Review ............................................................................ 1
Introduction .................................................................................................... 2
Physiological characteristics of chromosomal replication ......................... 3
The origin of chromosomal replication, oriC .............................................. 6
Initiation of replication from oriC ................................................................ 12
The molecular model of initiation of chromosomal replication ............ 12

Other factors required for oriC-dependent replication ......................... 16
oriC-independent replication ................................................................. 17

DnaA protein .................................................................................................. 18
The dnaA gene ......................................................................................... 18

The promoter and regulation of the dnaA gene ................................... 21
Biochemical properties of DnaA protein .............................................. 22
Structural organization of DnaA protein .............................................. 24

Role in the cell other than initiation of chromosomal replication ...... 27
Replication of plasmids ..................................................................... 2 7
Regulation of Transcription .............................................................. 31
Regulation of initiation at oriC ..................................................................... 3 2
DnaA protein in the regulation of chromosomal replication ............... 3 2

vi

Other proteins which bind on’C .............................................................. 3 4

Inhibitor of chromosomal initiation (IciA) ........................................ 3 5
Histone like proteins ........................................................................... 3 5

Dam methylation ....................................................................................... 3 8
Transcription around oriC ....................................................................... 4O
Thesis overview .............................................................................................. 4 2

Chapter H. The ordered and sequential binding of DnaA protein to oriC

the chromosomal origin of Escherichia coli ............................................. 44
Abstract ........................................................................................................... 45
Introduction ................................................................................................... 46
Experimental Procedures .............................................................................. 51
Gel mobility shift assays .......................................................................... 51
In situ cleavage with 1,10 phenanthroline—copper .............................. 5 2
Complementiation replication assays .................................................... 5 3
Results .............................................................................................................. 54

Six discrete complexes are formed on binding of DnaA protein to
oriC ...................................................................................................... 54
Formation of Complex VI correlates with formation of an active
replication complex ............................................................................ 5 8
Relative binding affinities of DnaA protein to supercoiled and linear
DNA ...................................................................................................... 63
Ordered binding of DnaA protein to oriC ............................................. 66
DnaA protein binds to DnaA box R4 with greater affinity than to

 

R1 ......................................................................................................... 71

Discussion ........................................................................................................ 74

Chapter III. The influence of IHF on the binding of DnaA protein to

 

oriC ................................................................................................................... 80
Abstract ............................................................................................................ 81
Introduction .................................................................................................... 82
Experimental procedures ............................................................................... 8 8
Reconstituted replication assays ............................................................ 8 8
Probing complexes with antibodies ....................................................... 89

Determination of the dissociation rate constant of the IHF

Complex 1 ............................................................................................ 90
Results ............................................................................................................. 92
IHF substitutes for HU in purified oriC-dependent replication ......... 92

IHF forms a single major complex with oriC whereas HU forms

multiple complexes ............................................................................ 9 3
IHF binds oriC at a single site ................................................................ 97

Novel complexes with oriC form in the presence of IHF and DnaA
protein .................................................................................................. 97

IHF does not aid the binding of DnaA protein to the weaker inside

DnaA boxes within oriC ................................................................... 106
IHF does not stabilize DnaA-oriC complexes ..................................... 106
Discussion ....................................................................................................... 114

Although IHF and HU bind to oriC differently, they can replace

each other in oriC replication ......................................................... 114
IHF does not affect the affinity of DnaA protein for DnaA boxes
within oriC ........................................................................................ 115

Chapter IV. The influence of FIS on the binding of DnaA protein to oriC

and on replication from oriC ........................................................................ 117
Abstract ........................................................................................................... 1 18
Introduction .................................................................................................... 1 19
Experimental procedures .............................................................................. 125

Supercoiling assay ................................................................................... 125
Results ............................................................................................................. 1 26

FIS forms multiple complexes with a DNA fragment containing

oriC ..................................................................................................... 1 26
FIS binds several sites within oriC ....................................................... 126
FIS dissociated from oriC more slowly that DnaA protein ............... 133

Novel oriC complexes are formed in the presence of both FIS and

DnaA protein ....................................................................................... 134
F18 and DnaA protein binding are not mutually exclusive ................ 134
FIS does not affect DNA replication on binding of oriC .................... 140
FIS can not substitute for HU in in vitro replication assays ............... 147

Binding of FIS to oriC does not affect initiation from oriC in RNA

polymerase dependent initiation ..................................................... 147
Discussion ...................................................................................................... 1 55
FIS binds specifically to oriC ................................................................. 155

ix

Specific binding of FIS to oriC has no effect on initiation of

chromosomal replication in vitro ........................................................... 155
Roles of FIS in initiation at oriC ............................................................ 156
Chapter V. Summary and Perspectives ........................................................... 158
Bibliography ........................................................................................................ 163

List of Tables

Chapter II.
Page
Table 1. Sequences of DnaA boxes in oriC and the dnaA promoter

region .............................................................................................................. 5 5

xi

List of Figures

Chapter I. Page

Figure 1. Schematic presentation of the relationship between chromosomal
replication and cell division ........................................................................... 4

Figure 2. Consensus sequence of the minimal chromosomal origin ............. 8

Figure 3. A schematic representation of relevant sequences in and

flanking oriC ................................................................................................ ;.. 10
Figure 4. Model of initiation from oriC .............................................................. 14
Figure 5. A schematic of the dnaA operon and its promoter region ............. 19

Figure 6. E. coli DnaA amino acid deduced sequence compared with the

DnaA consensus sequence ........................................................................... 25
Figure 7. Structure-function of DnaA protein ................................................. 28
Chapter 11.

Figure 1. The on'C region .................................................................................... 47

Figure 2. DnaA protein bound to oriC forms six complexes that are
resolved by native polyacrylarnide gel electrophoresis .................................. 5 6
Figure 3. Formation of Complex VI correlates with DNA replication
activity .............................................................................................................. 59
Figure 4. Discrete complexes are formed by ATP- or ADP-bound form of
DnaA protein .................................................................................................. 61
Figure 5. Both supercoiled and linear oriC plasmids are comparable in

reducing binding of DnaA protein to DnaA box R4 ................................. 64

Figure 6. In situ footprinting of complexes with LID-phenanthroline-
copper ................................................................................................................ 67
Figure 7. DnaA protein binds to DnaA box R4 with greater affinity than

to R1 .................................................................................................................. 7 2
Chapter 111.
Figure 1. The oriC region ................................................................................... 85

Figure 2. IHF can replace HU in vitro oriC-dependent replication in the
presence or absence of RNA polymerase ................................................... 9 3
Figure 3. Both IHF and HU bind oriC .............................................................. 95
Figure 4. In situ footprinting of IHF complexes with LID-phenanthroline-
copper ............................................................................................................... 98
Figure 5. Novel complexes form in the presence of both IHF and DnaA
protein ............................................................................................................ 101
Figure 6. The novel complexes contain both DnaA protein and IHF ......... 104
Figure 7. The order of DnaA protein binding to oriC is not altered by
IHF ................................................................................................................... 107
Figure 8. Determination of the half-life of the IHF Complex 1 ..................... 111

Figure 9. The dissociation of DnaA protein from oriC was not altered by

IHF ................................................................................................................... 112
Chapter IV.
Figure l. The oriC region .................................................................................. 222

Figure 2. FIS bound to oriC forms multiple complexes that are resolved

by native polyacrylamide gel electrophoresis ............................................. 127
Figure 3. In situ footprinting of complexes with LID-phenanthroline-

copper .............................................................................................................. 1 29
Figure 4. The dissociation of DnaA-oriC and FIS-oriC complexes .............. 135
Figure 5. Novel complexes with oriC form in the presence of both DnaA

protein and FIS .............................................................................................. 138
Figure 6. In situ footprinting of the DnaA-FIS-oriC complexes with

phenanthroline-copper ................................................................................. 141
Figure 7. At high levels, FIS inhibits in vitro oriC-dependent

replication ..................................................................................... I .................. 143
Figure 8. FIS binds oriC in the presence of supercoiled vector DNA ......... 145
Figure 9. Inhibitory levels of FIS induce a highly negatively supercoiled

topoisomer of pBSoriC .................................................................................. 148
Figure 10. Only high levels of FIS inhibit oriC replication in the presence

of variouis amounts of HU ........................................................................... 150
Figure 11. FIS binding oriC does not affect initiation from oriC in RNA

polymerase dependent initiation ................................................................. 151

xiv

HEPES

kDa
PVA
rRNA
tRN A
SDS

Tn's

List of Abbreviations

ATP synthase

base pairs

deoxyadenosine methyltransferase

the 9-mer DNA consensus sequence recongized by DnaA protein
dithiothreitol

(ethylenedinitrilo)tetraacetic acid

factor for inversion stimulation
4-(2-hydroxyethyl)-1-piperazineethane-sulfonic acid
integration host factor

kilodalton

polyvinyl alcohol

ribosomal RNA

transfer RNA

sodium dodecyl sulfate

Tris(hydroxymethyl)aminomethane

XV

 

Chapter I

Literature Review

Introduction

Cell division is essential for the survival of Escherichia coli or any
organism. Cell division depends on the doubling of all the cell’s components; the
chromosome, the cytoplasm, the cell wall and the outer and cytoplasmic
membranes. Without duplication and proper segregation of chromosomal
material, daughter cells will not survive to pass on the instructions to allow the
two new daughter cells to carry out their functions. Therefore, chromosomal
replication must be tightly controlled and coordinated to growth of the cell. In
1963, Jacob proposed a model for the regulation of replication and the
segregation of the bacterial chromosome (Jacob et al., 1963). He envisioned that
replication initiated from a unique site on the chromosome and the bacterial cell
envelope served as a scaffold in much the same way that the eukaryotic spindle
functions. As new material was incorporated into the growing membrane, new
material would be inserted between the two new nucleoids.

The genetic material of E. coli is contained on a single circular
chromosome of 4,720 kilobase-pairs (Kohara et al., 1987). As Jacob speculated,
replication of this chromosome occurs from a unique site (now referred to as oriC)
located at approximately 85 minutes on the revised E. coli chromosome map
(Louarn et al., 1974; Marsh and Worcel, 1977). DNA synthesis then proceeds
bidirectionally and terminates near the trp gene located at 25 minutes on the E.
coli chromosome map (Bird et al., 1972). In early studies, Messelson and Stahl
determined that a newly replicated DNA molecule contained one newly
synthesized strand and one old strand (Messelson and Stahl, 1958). This type of

replication is referred to as semiconservative replication.

3

Physiological characteristics of chromosomal replication.

The frequency of initiation of replication and not the rate of elongation
regulates chromosomal replication. This conclusion is based on observations from
dividing the bacterial cell cycle into three periods and measuring the length of
these periods during various growth rates (Cooper and Helrnstetter, 1968;
Helrnstetter, 1968) (Figure 1). The minimum amount of time required to replicate
the entire chromosome, period C, is approximately 45 minutes (Trueba et al.,
1982). Because period C and the time between the completion of chromosomal
replication and cell division, period D, remains relativity constant regardless of
variation in generation times from 22 to 70 minutes (Skarstad et al., 1983), the
time required to achieved the capacity to initiate chromosomal replication, period
I, is concluded to be variable (Helrnstetter, 1994).

The fact that the doubling time of E. coli can proceed twice as fast as the
time required to replicate the chromosome seems to be contradictory. However,
initiation of subsequent rounds of chromosomal replication can occur before
previous replication cycles are completed (Figure 1). Thus, a fast growing cell
contains multiple copies of the chromosome (Skarstad et al., 1985). In rapidly
growing cultures with multiple chromosomal origins, all oriC within an individual
cell initiate at the same time (Skarstad et al., 1986). This is called synchronous
initiation. Mutations of factors involved in the regulation of initiation often result
in asynchronous timing of initiation (Boye and Lobner—Olesen, 1990; Lu et
al.,l994; Skarstad et al., 1988).

Central to models of the regulation of initiation is the idea that the ratio of

cell mass to the number of chromosomal origins within a cell triggers initiation

Figure 1. Schematic presentation of the relationship between chromosomal
replication and cell division (von Meyenburg and Hansen, 1987). tD represents
the generation time. The bacterial cell cycle can be divided into distinct periods,
C and D. C represents the time between initiation (ini) and termination (ter) of
chromosomal replication. D represents the time between termination of
chromosomal replication and cell division (div). The time of initiation varies as a
function of tD. a) When tD is greater than the sum of the periods C and D,
initiation occurs after the division event. b) When to is equal the sum of the
periods C and D, initiation occurs at the time of the division event. c) When tD is
less than the time for C and D, initiation occurs before the prior cycle is complete.
As a result a single cell will contain more than one oriC. All origins within a cell
initiate simultaneously.

a mac ,c-ss 0:20

2...... g:

 

V v
div div

l I
int ter ini

b t.-60 c-«o 0:20

‘1 ll
VV

 

 

 

 

VV
div div

: . . .
ini ter int ter

C 5835 C840 0820

 

6

(Donachie, 1968). This ratio is called the initiation mass and remains constant
irrespective of growth rate. Initiation of replication is dependent on de novo
protein synthesis, as blocking protein synthesis with antibiotics allows the
completion of replication but prevents additional initiation events (Lark and
Renger, 1969; Messer, 1972). These observations led to the speculation that the
accumulation of a specific protein may link initiation mass with initiation. DnaA
protein, a protein involved in the first steps of initiation, is an obvious candidate
for such a regulator of initiation.

Asynchronous initiation was mentioned as a common phenotype of
initiation mutants. Another characteristic which differentiates genes involved in
initiation of replication from those involved in chain elongation is slow and fast
stop phenotypes (Kohiyama et al., 1966). Temperature sensitive mutants
defective in chain elongation result in an immediate stop in DNA replication at
nonpermissive temperatures; they exhibit a fast stop phenotype. In contrast,
mutants of genes involved in initiation, such as dnaA and dnaC, result in the
completion of the round of replication but fail to initiate the next round of
replication; they exhibit a slow stop phenotype (Wechsler, 1975; Zyskind et al.,
1977).

The origin of chromosomal replication, oriC

The origin of chromosomal replication (oriC) located between the dnaA
and ngA genes was mapped by pulse labelling of synchronized cells (Louarn et
al., 1974; Marsh and Worcel, 1977). The minimal sequence of oriC required to

confer autonomous replication on non replicating DNA is a 245 base pair

7

sequence (Oka et al., 1980) (Figure 2). From mutagenesis (Oka et al., 1984) and
sequence comparison of oriC sequences from members of the
Enterobacteriaceae family (Zyskind et al., 1983), several key sequences are
identified as important for replication (Figure 2): 1) an A-T rich region located at
the left border of oriC, 2) four indirect repeats of the sequence 5’-
TTAT(C/A)CA(C/A)A-3’, called DnaA boxes, and 3) 8 Darn methylation sites (5’-
GATC-3’). The AT-rich region is composed of three 13-mers, identified as left,
middle and right (Figure 3). The four DnaA boxes are named, R1, R2, R3 and R4.
Dam methyltransferase catalyzes the transfer of a methyl group to the N6 position
of the adenine on both strands within its recognition sequence (Marinus and
Morris, 1974). All three sequences will be discussed in more detail.

In addition to these sequences, several promoters are located within and
flanking the minimal oriC (Figure 3) (Asai et al., 1992; Braun and Wright, 1986;
Lother and Messer, 1981; Schauzu et al., 1987). Transcripts from the promoter
PoriL originate from just right of DnaA box R2 within oriC and proceed toward
the left end of oriC (Figure 3). This promoter appears to be negatively regulated
by the binding of DnaA protein to boxes R2 and R4 (Asai et al., 1992).
Transcription from Porle, which begins near the 13-mers and proceeds
rightward, appears to be positively regulated by DnaA protein (Asai et al., 1992).
An additional promotor, PorzR, is located just to the right of R4 and directs
transcription to the right (Schauzu et al., 1987). From a promoter located left of
the AT-rich 13-mers, gidA (glucose inhibition of division) transcription is directed
away from oriC (Kolling et al., 1988). mioC (modulation of initiation at oriC) is

located several hundred base-pairs to the right of oriC and is transcribed into

Figure 2. Consensus sequence of the minimal chromosomal origin (Zyskind et
al., 1983). The sequences from six bacterial chromosomal origins are compared.
They were aligned to minimize the number of changes of the consensus
sequence. The large box indicates the boundaries of the minimal oriC sequence
required to confer autonomous replication. A large capital letter indicate that the
residue is found in all six bacteria; a small capital letter indicates that the residue is
found in five of the six origins; a lower case letter indicates that it is found in
three or four origins; and 11 indicates that all four nucleotides are found at that
position. Dashes indicate deletions. The bold letters indicate residues where
single base substitutions produce a oriC- phenotype (Oka et al., 1984). The
AT-rich 13-mers are indicated with the arrows at the left border of oriC. The
DnaA boxes and their orientations are indicated by arrows and are labelled R1,
R2, R3, and R4. The Dam methylation sites (GATC) are underlined.

 

 

 

 

 

 

 

 

 

 

 

338.83.33.53 . 3.33333333.333 3.33 3.333.33.333 .3333. 33 . 3. 3 33333.3333.
288983 352.3 . .3-33333.3333 3.3. 3.3333333333 333:3 3. 3 3. 3 3333:3333..
83:83:... 33333333 . ...333333:333 3.3. 3.333.33.333 .33... 3. . 33 3 333.3333
323388 38382333 . ..333333..33 333. 3.333.33.333 .33... 3. . 3. 3 3333:3333.
53.3.2333 332.8333 . ...333333:333 3.3. 3.33.33.333 .33... 3. . 3. 3 3333:3333.
338333338333 . ..-33333..333 3.3. 3333.33.33 .3... 3. as . 3.. 3 3333:3333.
3
E
3538.3. 3553 . h<wocccccc<ocwc 59—h c< ”(92:63.33vac-303...:3humcu<hu<<h§.=o3hutwuccau<cr acc<<<
33 333 ..:333 .33
3 333 3 3.3333. 3. 3 333 3 3333333 --3 33 333333 ..33333 333333 . 3333
. 333 3 3.333.. 33 . ... . 3.333.3 ... -- .33333 333333 33333. . 3.33
. 333 3 3.333.. 3. . ... .. 3.333.3 ... -- .33333 .33333 33333. 3 3.33
. 333 3 3.333.. 3. . ... . 3.333.3 ... .- .33333 .33333 33333. . 3.33
O PU‘ ‘ U‘Pu90u ‘0 O .0. 0 ”Chan." 0.. -- Co‘FU‘ .=‘”” ””9””. O ‘0:
. 333 3 3. 333.. 3. . ... . 3.333. 3' ... .. .33333l .33333 33333. _ . 3. 33
<33..3<333.3<33¢33 3c3333333333333333333333333.333333333333333-.333333333433333333¢¢¢3¢¢c¢¢3<u3<u3<c33c
33.3 «3 ...23 8.. .3.
333333.33333 33333333 3 -. - - -3 3 3.. 3.3 3. 333333333333
333333333333 33333333 . .3 . 3 .3 . ..3 33. .3 3..3333.....
333333.33333 33333333 . 3. 3 3 .3 . ..- 3.. .. 3..3333......
333333.33333 33333333 . 3. 3 3 .3 . ..- 3.. .. 3..3333.....
333333.33333 33333333 . 3. 3 3 .3 . ..3 3.. .. 3..3333.q...
333333.33333 .3 33333333 . 3. 3 3 .3 . ..3 3.. .. 3..3333.....
3E II: A .

 

n........... 333333333........333333333333333333.33333.33.

oo.

 

.353

333 l3c<3333333c3333

  

.33<333333«33333333

3.

 

...-a

10

Figure 3. A schematic representation of relevant sequences in and flanking
oriC. The top figure represents oriC between mioC and gidA. The directions of
transcription of these genes are indicated by the arrows. The lower figure
illustrates the location of important sequences involved in initiation. The
promoters and their orientations are indicated by the open arrows; the three 13-
mers by the slashed boxes; the DnaA boxes and their orientations by the filled
triangles; an IHF binding site by the vertical lined box; and the FIS binding site
by the checkered box. The astericks indicate the Dam methylation sites.

 

 

 

 

 

 

 

E E RS
3m 3m 3.". mm 3:. E l.\
3305-33
. .
83 83 333 ..3 an

 

 

 

“6251—.— 9:3 _._<3..3 i“

 

 

12

without transcribing through oriC. The function of the gene products encoded
by gidA and mioC are not known.

The difficulty of studying replication of the entire chromosome is
overcome by the cloning of oriC on plasmids, called minichromosomes (Messer et
al., 1978). These plasmids are dependent on oriC for replication and retain many
of the same characteristics as chromosomal replication including bidirectional fork
movement (Meijer and Messer, 1980), synchronous initiation (Helrnstetter and
Leonard, 1987b; Leonard and Helrnstetter, 1986), dependence on protein and
RNA synthesis (Lark and Renger, 1969; Messer, 1972), and dependence on
growth rate (Lobner-Olesen et al., 1987).

Initiation of replication from oriC

The development of in vitro oriC dependent replication assays using
crude enzyme extracts (Fuller et al., 1981) or purified proteins (Kaguni and
Kornberg, 1984), led to a model by which DNA replication is initiated from oriC.
Like in vivo, in vitro replication from oriC proceeds bidirectionally (Kaguni et al.,
1982; Meijer and Messer, 1980), requires a negatively supercoiled template
(Kowalski and Eddy, 1989; von Freiesleben and Rasmussen, 1992), and is
dependent on DnaA protein (Carl, 1970; Wechsler and Gross, 1971), DnaB
protein (Zyskind and Smith, 1977b), and DnaC protein (Wechsler, 1975).

The molecular model of initiation of chromosomal replication
Biochemical studies have indicated that the first step in the replication of

the E. coli chromosome involves recognition and topological alteration of the

l3

oriC region by DnaA protein (Figure 4) (Fuller et al., 1984). The initial binding of
DnaA protein to the DnaA boxes is proposed to enhance the binding of
additional DnaA molecules. The final DnaA-oriC complex that is thought to be
active in promoting initiation of DNA replication contains approximately 30 to 40
DnaA monomers (Fuller et al., 1984). oriC appears to be wrapped around a core
of DnaA molecules. In the presence of HU or integration host factor (IHF)
(Hwang and Kornberg, 1992a; Skarstad et al., 1990), this nucleoprotein complex
is proposed to distort the topology of oriC resulting in unwinding of the AT-rich
13-mers, forming the open complex (Bramhill and Kornberg, 1988a). In the
absence of HU or IHF, unwinding of the 13-mers can be facilitated by RNA
polymerase (Baker and Kornberg, 1988). Although DnaA protein binds to oriC
equally well in the presence or absence of nucleotide (Bramhill and Kornberg,
1988a), unwinding of oriC requires the ATP-bound form of DnaA protein
(Sekimizu et al., 1988a; Yung et al., 1990).

Following unwinding in the presence of ATP, six DnaC monomers form a
complex with a hexameric DnaB helicase (termed DnaBC complex) (Kobori and
Kornberg, 1982; Wickner and Hurwitz, 1975). DnaA protein is proposed to
guide the DnaBC complex into the open complex thorough a direct DnaA-DnaB
interaction to form the prepriming complex (Marszalek and Kaguni, 1994). DnaB
helicase then is proposed to unwind the strands of DNA bidirectionally (Baker et
al., 1986), (Baker et al., 1987). In this model, the presence of DnaC protein in the
preinitiation complex inhibits DnaB helicase activity (Allen and Kornberg, 1991).
DnaC protein is thought to dissociate from the complex of DnaA protein and

DnaB protein bound to the origin sequence (Allen and Kornberg, 1993). Once

 

 

14

Figure 4. Model of initiation from oriC (Messer, 1996). Initiation occurs in 6
steps: 1) DnaA binds to oriC to form the initial complex; 2) DnaA unwinds the
13-mers to form the open complex; 3) DnaBC complex enters the unwound oriC
to form the prepriming complex I; 4) DnaC dissociates from the prepriming
complex; 5) primers are formed; and 6) DNA polymerase III holoenzyme uses
these primers to initiate DNA synthesis.

 

15

3.33.33 3.833. B M§§§3§ E 3 3.323336 «3% 23.23.33?

00.3.0 c

E3532.

33th 0335

MI :. _0n_

 

 

 

 

 

 

 

 

 

_ onEoo
oEEtaoE

 

 

 

3633.033933ch
333

3.322 a 35383 .3335

 

 

 

 

__ onEoo V xanoo V _.
. :53. no.
mfiEtama mEEta ‘ o .._
n.-<zm
mi .3...: .3:
.33
J
meEoo 38.93.03 to
:30 j REE J 0.
333 .253 338-333
.2352 \o M53353§ comuusfixflmfioo

 

+ 03.93

N3 33:33.8 3.33% 26.33.33.333;

 

 

 

 

 

 

 

 

 

 

 

 

 

16

DnaB helicase has separated a portion of the origin in both directions, primase
enters the initiation complex to synthesize short RNAs (van der Ende et al., 1985)
which act as primers for DNA polymerase III holoenzyme (Maki and Kornberg,
1988). Elongation of these primers ultimately results in the formation of two

daughter chromosomes.

Other factors required for oriC-dependent replication

In addition to DnaA protein, DnaB protein, DnaC protein, HU, primase and
DNA polymerase III holoenzyme, several other proteins are required for in vitro
replication. Several of these proteins, such as RNA polymerase, topoisomerase I
and DNA gyrase are involved in changing the topology of DNA. Others factors,
such as RNase H, are involved as oriC specificity factors (Kogoma et al., 1985).
Lastly, single stranded DNA binding protein (SSB), is involved in stabilizing
single-stranded DNA (Sigal et al., 1972) which is exposed during DNA
replication. Though DnaA protein can unwind the origin in the absence of SSB
(Bramhill and Kornberg, 1988a), SSB stimulates DnaB helicase activity (LeBowitz
and McMacken, 1986) and the processivity of DNA polymerase III holoenzyme
(Fay et al., 1981; Fay et al., 1982; LaDuca et al., 1983).

Topoisomerase I and DNA gyrase are responsible for maintaining the
negative superhelicity of the chromosome (Pruss, et al., 1982; Wang, 1996).
Mutations in gyrA or gyrB which encode the A and B subunits of DNA gyrase,
respectively, suppress deletion mutations in topA, encoding topoisomerase I
(DiNardo et al., 1982; Pruss et al., 1982). Topoisomerase I and DNA gyrase have

opposite effects; topoisomerase I relaxes negative supercoils (Pruss et al., 1982),

17

whereas DNA gyrase introduces negative supercoils (Marians et al., 1977).

DNA Gyrase plays an important role in maintaining the negative
superhelicity required for initiation of chromosomal replication (Hiasa and
Marians, 1994b; Kowalski and Eddy, 1989) and releasing positive supercoils that
form ahead of the replication forks (Filutowicz and Jonczyk, 1983; Kreuzer and
Cozzarelli, 1979). Both DNA gyrase and topoisomerase I, are involved in
modulating the changes in supercoiling due to chain elongation by RNA
polymerase (Liu and Wang, 1987). DNA Gyrase acts ahead of transcribing RNA
polymerase removing positive supercoils whereas topoisomerase I acts behind

RNA polymerase relaxing negative supercoils.

oriC-independent replication

Chromosomal replication can occur in the absence of oriC and dnaA via
three different mechanisms. The first mechanism is by integrating a plasmid or
phage origin into the chromosome (Koppes and Nordstrom, 1986). Replication
can be also driven from other origins (collectively known as oriK) already located
on the E. coli chromosome via induction of the SOS response (Magee et al.,
1992). This type of replication is termed damage-induced stable DNA replication
(iSDR) and is thought to enable the cell to meet various environmental stresses
(Hong, et al., 1995; Kogoma et al., 1979). In addition, mutations in mhA can
suppress oriC and dnaA deletions (de Massy et al., 1984; Kogoma et al., 1985).
However, these strains grow poorly. mhA encodes RNase H which specifically
degrades RNA-DNA hybrids (Arendes, et al., 1982; Miller et al., 1973) which

could potentially could act as primers for DNA polymerase III holoenzyme.

18

Mutations in mhA are thought to allow RNA primers other than those located at
oriC to serve as primers for DNA polymerase 1H (Horiuchi et al., 1984). In
support, in vitro replication remains dependent on oriC in the presence of RNA

polymerase only with the addition of RNase H (Ogawa et al., 1984).

DnaA protein
The dnaA Gene

The dnaA gene was originally identified by the identification of
temperature sensitive mutants that exhibited a slow stop phenotype at elevated
temperatures (Kohiyama et al., 1966). Employing a temperature sensitive mutant,
the dnaA gene was mapped and cloned by complementation (Hansen and von
Meyenburg, 1979; Miki et al., 1979). The E. coli dnaA gene is located at 83
minutes on the revised E. coli genetic map, close to oriC. From the deduced
amino acid sequence, the gene encodes a basic polypeptide of 467 amino acids
with a calculated mass of 52.5 kDa (Ohmori et al., 1984). The dnaA gene is the
first gene in an operon containing dnaN (Sakakibara et al., 1981), encoding the B

sliding clamp of DNA polymerase HI holoenzyme (Burgers et al., 1981), recF,
encoding a protein involved in recombination (Ream et al., 1980) and gyrB,
encoding the B subunit of DNA gyrase (Hansen and von Meyenburg, 1979)
(Figure 5).

DnaA protein appears to be found in all bacteria from E. coli and Bacillus
subtilis to Mycoplasma and cyanobacteria (reviewed in Messer, 1996). Both the
conservation of the arrangement of the dnaA operon and the similarity of the

DnaA amino acid sequence in a large variety of bacteria suggests that DnaA

 

 

19

Figure 5. A schematic of the dnaA operon and its promoter region. The genes
and the direction of transcription are indicated by the open boxes. Promoters are
indicated by the filled arrowheads. The dnaA promotor region is expanded
below. The -10 and -35 sequences are indicated by the filled boxes, the DnaA
boxes and their orientation by the shaded triangles; the FIS binding site with the
slashed box; and the Dam methylation sites by the asterisks.

20

 

F... cm. 09—...

 

 

 

 

 

 

 

   
 

3. mm- 3- mm-
I / «Q32... mE <55 <25 2:125
/
/
/
/
/
/
min to?

 

 

 

 

 

 

 

 

 

21

protein shares a common function in these bacteria (Ogasawara and Yoshikawa,

1992; Yoshikawa and Ogasawara, 1991).

The promoter and regulation of the dnaA gene

The dnaA gene contains two promoters, dnaApI and dnaApZ, located 250
and 160 nucleotides, respectively, from the translational start site (Hansen et al.,
1982) (Figure 5). A DnaA box identical in sequence to boxes R1 and R4 of oriC
is located between the two promoters. Protection experiments reveal another
potential DnaA box, also located between the promoters (Wang and Kaguni,
1987). This second site contains the sequence 5’-T'I'I'I‘CCACA-3’ which fits the
more relaxed definition of the DnaA box (see below). Binding of DnaA protein
to these sites inhibits transcription from both promoters (Braun et al., 1985; Lee
and Hwang, 1997; Wang and Kaguni, 1987). Like oriC, the promotor region also
contains numerous Dam-methylation sites. Transcription from dnaApZ is more
efficient when the promoter region is fully methylated (Braun and Wright, 1986;
Kucherer et al., 1986). Factor for inversion stimulation (FIS), a histone-like
protein involved in regulating transcription from some promoters in relation to the
rate of growth (Finkel and Johnson, 1992), binds to sequences that overlap the
-35 box of dnaApZ (Froelich, 1996). F18 appears to negatively regulate dnaA
expression as fis null mutants have a 2.5 fold increase in the level of DnaA
protein (Froelich, 1996). The dnaAZp promoter, the DnaA box and several of the
GATC sites are conserved between E. coli, S. typhimurium and S. marcesces
suggesting that the dnaA gene in these different bacteria is regulated in a similar

manner (Skovgaard and Hansen, 1987).

22

Biochemical properties of DnaA protein

DnaA protein was first purified by a DNA binding assay then by
complementing crude extracts from a dnaA(ts) strain in in vitro oriC replication
assays (Chakraborty et al., 1982; Fuller and Kornberg, 1983). DnaA protein has
several biochemical activities: DNA binding (Fuller et al., 1984); ATP binding
(Sekimizu et al., 1987); ATP hydrolysis; cAMP binding (Hughes et al., 1987);
interaction with acidic phospholipids (Sekimizu et al., 1988b), and self
aggregation (Sekimizu et al., 1988b).

DNase I protection and filter binding were used to demonstrate the ability
of DnaA protein to bind the four DnaA boxes within oriC (Fuller et al., 1984).
DnaA protein binds to an oligonucleotide containing the sequence of oriC DnaA

box R4 in its native context with a KD of 1 nM (Schaper and Messer, 1995).

Using gel mobility shift assays with 21-mer oligonucleotides, the DnaA protein
recognition sequence is rigidly defined as 5’- TTATNCACA-3’ (Schaper and
Messer, 1995). However, flanking sequences adjacent to DnaA boxes are also
important in determining the efficiency with which DnaA protein will bind as its
affinity for a 21-base pair oligonucleotide containing the oriC DnaA box R4 is
reduced 50 fold by changing the flanking sequences. Surprisingly, DnaA protein
binds an oligonucleotide containing oriC DnaA box R3 with an affinity no
greater than an oligonucleotide containing random sequences. The ability of
DnaA protein to protect R3 contained within oriC is thought to be the result of
cooperativity. The binding of highly divergent sequences in the presence of an
adjacent strong consensus site is common for DnaA protein and suggests that

protein-protein interactions facilitate the binding to weaker sites (Stenzel et al.,

23

1991; Wang and Kaguni, 1987). The combination of the effect of flanking

sequences and cooperative binding may explain why DnaA protein appears to

also recognize a more relaxed definition of the DnaA box, 5’-

(T /C)(T /C)(Aff /C)T(A/C)C(A/G)(A/C/T )(A/C)-3’ (Schaefer and Messer, 1991).
As expected from the conserved ATP binding motif, DnaA protein binds

ATP and ADP with high affinity (KD of 0.03 uM and 0.1 uM, respectively)

(Sekimizu et al., 1987). DnaA protein is speculated to have a second lower
affinity site based on the requirement of 5 mM ATP to unwind the AT-rich 13-
mers (Sekimizu et al., 1987). Hydrolysis of ATP to ADP occurs slowly; 50% of
bound ATP is hydrolysed in 15 minutes. This ATPase activity is dependent on
DNA. ATP hydrolysis is not required for DnaA protein dependent unwinding of
oriC as the nonhydrolyzable ATP analog, ATPyS, can substitute for ATP in
unwinding oriC. Instead, the binding of ATP is thought to produce a
conformational change which is important for unwinding (Yung and Kornberg,
1989).

DnaA protein also binds cAMP with high affinity (KD 1 uM) (Hughes et

al., 1988). Although ATP inhibits cAMP binding, this inhibition is
noncompetitive. Dissociation of ADP from DnaA protein in the presence of ATP
is slow (Sekimizu et al., 1987). cAMP increases the rate of dissociation of bound
ADP but not of bound ATP (Hughes et al., 1988).

Typically 50% of DnaA protein expressed from its natural promoter is
found in the membrane fraction, suggesting a DnaA protein-membrane interaction
(Sekimizu, et al., 1988b). Mutants of phospholipid metabolism which limit the

levels of anionic phospholipids exhibit a slow stop phenotype (Heacock and

24

Dowhan, 1989; Xia and Dowhan, 1995). This phenotype is suppressed by
mutations in rnhA (encodes RNase H) (Xia and Dowhan, 1995) which
circumvents the requirement for oriC and dnaA (Ogawa et al., 1984; von
Meyenburg et al., 1987). Acidic phospholipids inhibit the reassociation of DnaA
protein with ATP and ADP. However, in the presence of oriC, DnaA protein can
rebind both ATP and ADP, even in the presence of phospholipids (Castuma et al.,
1993; Crooke et al., 1992; Sekimizu and Kornberg, 1988; Yung and Kornberg,
1988). This phospholipid effect does not discriminate between ATP and ADP;
however, the cellular concentration of ATP is approximately 10 times higher than
that of ADP (Mathis and Brown, 1976), suggesting a mechanism for regeneration
of the ADP bound form of DnaA protein.

DnaA protein is isolated as a monomer and an aggregate (Hwang et al.,
1990). The aggregate is poorly characterized. This multimerization is thought to
be important in DnaA protein function when bound at oriC (Fuller et al., 1984)
and even at promoters (Lee and Hwang, 1997). In electron micrographs,
DnaA-oriC complexes appear to contain 20 to 30 monomers. Furthermore, a
mutant of DnaA protein, which is defective in specific DNA binding and is
inactive in oriC replication, is able to augment the limiting levels of wild type in

vitro oriC replication (Sutton, 1996).

Structural organization of DnaA protein
Until recently, little was known about structure function relationships of
DnaA protein. Only sequence comparisons gave any information of the DnaA

protein structure (Figure 6). This comparison divided the protein into a Figure 6.

25

E. coli DnaA amino acid deduced sequence compared with the DnaA
consensus sequence (Messer, 1996). The deduced amino acid sequence of E. coli
is indicated relative to the consensus sequence obtained from comparing fifteen
different dnaA homologs (Escherichia coli, Salmonella typhimurium, Serratia
marcescens, Proteus mirabilis, Buchnera aphidicola, Pseudomonas putida,
Bacillus subtilis, Streptomyces coelicolor, Micrococcu luteus, Caulobacter
crescentus, Rhizobium meliloti, Synechocys sp., Borrelia burgdorferi,
Spiroplasma citri, Mycoplasma capricolum). An underlined letter in the

conserved sequence indicates a residue conserved in 12 or more of the 15
homologs. A letter represents a residue conserved in 9 or more. A dot (')

indicates a residue conserved (either identical residue or conservative
replacement) in less than 9. See Messer, 1996 for a description of the method

used for the sequence comparison.

26

1 60
E. coli MSLSLWQQCLARLQDELPATEFSMWIRPLQAELSDNTLALYAPNRFVLDWVRDKYLNNIN
consensus MSL.Lfl.Q.LA.L..EL....E..HIR.LQ.EL...TL.L.APN.FVLDflM..KYL..;.

61 120
E. coli GLLTSFCGRIAPQLRFEVGTKPVTQTPQAAVTSNVAAPAQVAQTQPQRAAPSTRSGWDNV
consensus .LL..F ....... L.£.y ...................................... w...

121 180
E. coli PAPAEPTYRSNVNVKHTFDNFVEGKSNQLARAAARQVADNPGGAYNPLFLYGGTGLGKTH
consensus ......... S.yN.K.T£DN£yE§.SN.LA.AAAR.MADNPG.AZNELELXQG.QL§KIH

181 240

E. coli LLHAVGNGIMARKPNAKVVYMHSERFVQDMVKALQNNAIEEFKRYYRSVDALLIDDIQFF
consensus LLHAMGN..M...PNAKMVXM.SEREV.DMM.ALQ.N.IEEEK.YZRSVD.LLIQDIQEF

241 300
E. coli ANKERSQEEFFHTFNALLEGNQQIILTSDRYPKEINGVEDRLKSRFGWGLTVAIEPPELE
consensus A.KE..QEEEIHIENALLE...QIILTEDBYEKEI.GMEDBLKSRE.WQL.VAIEPEELE

301 360

E. coli TRVAILMKKADENDIRLPGEVAFFIAKRLRSNVRELEGALNRVIANANFTGRAITIDFVR
consensus IBEAIL-KKADE- -l-LP-EV-FElA-RL-SNIBELE§ALNBEIA-A-E- . . -IT.I_QFY-

361 420
E. coli EALRDLLALQEKLVTIDNIQKTVAEYYKIKVADLLSKRRSRSVARPRQMAMALAKELTNH
consensus E.LRDLL...E.LyTIDNIQK.MAEYXKIKy.DLLSKRBSRSMARPEQMAM.L.KELINH

421 467
E. coli SLPEIGDAFGGRDHTTVLHACRKIEQLREESHDIKEDFSNLIRTLSS
consensus ELEELQD.EQQBDHIIKLHACRKIE.LR.E..DlK.DE..LIR.L..

27

conserved N-terminal segment, a less conserved linker segment and the larger
conserved C-terminal segment which contains the conserved ATP-binding motif

(G-X-X-G-X—G-K-T- XS-V) between residues 172-185 (Fujita et al. , 1990;

Ogasawara et al., 1985; Skovgaard and Hansen, 1987; Yoshikawa and
Ogasawara, 1991). There is little direct evidendence that ATP actually binds this
site. Suggestive evidence is provided from a mutation of amino acid 184 from an
alanine to a valine which results in a DnaA protein defective in binding ATP (Carr
and Kaguni, 1996).

The C—terminal 94 amino acids of DnaA protein are responsible for DNA
binding (Roth and Messer, 1995) (Figure 7). The region from residues 372-381
appears to be involved in phospholipid mediated turnover of bound nucleotide.
A fragment containing residues 115 to 381 releases ATP in the presence of acidic
phospholipids, whereas a peptide of residues 115 to 372 binds ATP, fails to
release the nucleotide in the presence of phospholipids (Garner and Crooke,
1996).

Sequences near the N-terminus are speculated to be involved in
DnaA-DnaA interactions (Mark Sutton, personal communication). Residues
111-148 near the N-terminus are involved in the interaction with DnaB protein

(Marszalek and Kaguni, 1994; Marszalek et al., 1996).

Roles in the cell other than initiation of chromosomal replication
Replication of plasmids
DnaA protein is also involved in the replication of other replicons such as

phage P1 (Mukhopadhyay et al., 1993) and plasmids pSClOl (Murakami et al.,

28

Figure 7. Structure-function of DnaA protein. The amino acid sequences
which are highly conserved among the dnaA homologs are indicated by the filled
sequences. The less conserved residues are indicated by a line. The residue
numbers are indicated to the left.

29

N-terminus
amino acid 1 -

DnaA Multimerization ?

DnaB interaction
130 -

180 _ ] ATP binding motif

 

373 - - ] Acidic Phospholipid interaction

DNA binding

 

467 - .
C-terminus

 

30

1987), R1 (Masai and Arai, 1987), R6K (Wu et al., 1992), and F (Hansen and
Yarmolinsky, 1986). All replicons contain one or more DnaA boxes. We are only
beginning to understand the role of DnaA protein in the initiation of these
replicons.

These replicons have a structure similar to oriC. They contain several
repeats, called interons, which are bound by a plasmid-encoded initiator protein,
and an AT-rich region. Until recently, all dnaA mutants defective in oriC
replication was found to be inactive for pSClOl replication (Felton and Wright,
1979; Frey et al., 1979; Hasunuma and Sekiguchi, 1977). However, recently C-
terminal point and deletion mutants that were active in pSClOl and inactive in
oriC repication were isolated (Sutton, 1996). The other replicons (P1, F, RK2,
R6K) can be maintained by various dnaA(ts) but could not be maintained in
dnaA null mutants (Hansen and Yarmolinsky, 1986; Gaylo et al., 1987;
MacAllister, T. W. et al., 1991; Wu, F. et al., 1992). Although DnaA protein is
required for R1 replication in vitro (Masai and Arai, 1987; Ortega et al., 1986), R1
can be maintained in vivo though less efficiently without DnaA protein
(Bernander et al., 1991).

Although the role of DnaA protein at these origins is thought to be similar
to that which it plays at oriC, primary recognition of the origin appears to be the
role of a plasmid encoded initiator. DnaA protein is thought to be involved
primarily in unwinding and/or loading of DnaB protein. However, evidence for
DnaA protein actually unwinding or loading DnaB protein onto these origins is
lacking except for the requirement of DnaA protein for unwinding the Pl origin

(Mukhopadhyay et al., 1993). In contrast to in vitro requirements, in vitro

 

31

replication of R1 and R6K plasmids proceeds independently of DnaA protein
(Masai and Arai, 1987; Wu et al., 1992).

Regulation of Transcription

DnaA protein regulates its own expression (Braun et al., 1985; Wang and
Kaguni, 1987) and the expression of other genes (Wang and Kaguni, 1987).
Transcription of mioC, rpoH and dnaA is negatively regulated by the binding of
DnaA protein to DnaA boxes located within promoters of these genes (Braun et
al., 1985; Hansen et al., 1987; Lother, et al., 1985; Schauzu et al., 1987; Stuitje et
al., 1986; Wang and Kaguni, 1987; Wang and Kaguni, 1989). The binding of
DnaA protein to a dnaA promoter fragment has been demonstrated to occlude
binding of RNA polymerase (Lee and Hwang, 1997). Although many genes have
been reported to contain DnaA boxes within their promoters, not all have been
shown to be regulated by DnaA protein (Garrido et al., 1993).

DnaA protein is also been reported to function as a transcriptional
terminator (Gielow, et al., 1988; Schaefer and Messer, 1988). This termination
activity is dependent upon the orientation of the DnaA box (Schaefer and
Messer, 1989). Flanking sequences may also be required as not all DnaA boxes in
the correct orientation are capable of terminating transcription (Wang and
Kaguni, 1989).

In contrast to its more common role as a negative regulator of transcription,
at least one operon, the nrd operon encoding the subunits of ribonucleoside
diphosphate reductase, appears to be stimulated by the binding of DnaA protein

(Augustin et al., 1994). Though the mechanism of this stimulation is not known,

32

DnaA protein might change the topology of the promoter to make RNA

polymerase binding more efficient.

Regulation of initiation at oriC

It is well documented that DNA replication in vivo is regulated at initiation
and not at elongation or termination (Helrnstetter, 1994). Little is known about
the mechanism of its regulation. Models include the accumulation of the initiator
protein (Donachie, 1968) or the dilution of repressor proteins (Pritchard, 1969).
Any model must account for the physiological observations that the rate of
initiation corresponds with the rate of cell growth, and that all origins are initiated
synchronously.

Although DnaA protein is central to most models, there is a period of time
in the cell cycle during which oriC is refractory to further initiation, even when
activators, such as DnaA protein, are present in excess. This refractory period is
now referred to as the eclipse period. For example, when some dnaA(ts) mutants
are held at non-permissive temperature for several mass doublings and then
returned to permissive temperatures, the first two rounds of replication are
initiated 25 to 30 minutes apart, which defines the eclipse (Hanna and Carl, 1975;
Helrnstetter and Krajewski, 1982). The eclipse period presumably involves

negative regulatory factors.

DnaA protein in regulation of chromosomal replication
Because DnaA protein acts in initiation of chromosomal replication and

regulates the expression of a number of genes, it is an obvious candidate for

 

33

regulating the time of initiation. Both biochemical and genetic evidence indicates
that DnaA protein plays a role in triggering initiation at the appropriate time.
First, dnaA mutants result in a slow stop phenotype, indicating that DnaA protein
plays a role in initiation (Kohiyama, et al., 1966; Wechsler and Gross, 1971).
Second, mutants that reduce the ability of DnaA protein to bind ATP result in
asynchronous initiation (Skarstad et al., 1986; Skarstad et al., 1988). Third,
temperature sensitive mutants, dnaA46 and dnaA5, retain the potential to initiate
even when incubated at nonpermissive temperatures (Evans and Eberle, 1975;
Hanna and Carl, 1975). When returned to the permissive temperatures the missed
initiation events proceed. These initiations are independent of de novo protein
synthesis but still dependent on RNA synthesis (Lark, 1972). Fourth, in a strain
containing a temperature sensitive suppressor, initiation is rapidly inhibited when
amber mutants of dnaA are shifted to the non-permissive temperature. This
suggests that continuous synthesis of DnaA protein is required for initiation
(Schaus et al., 1981).

If the concentration of DnaA protein is important in determining the
initiation mass, changes in the amount of DnaA protein expressed should change
the initiation mass. In earlier studies, no change in DNA content is detected when
wild type DnaA protein is overproduced (Churchward et al., 1983). More
recently, several groups using more sensitive techniques have observed that
initiation is shifted to an earlier time in the cell cycle, when DnaA protein was
overproduced (Atlung et al., 1987; Lobner-Olesen et al., 1989; Pierucci et al.,
1987; Skarstad et al., 1989; Xu and Bremer, 1988). This led to the speculation

that initiation was controlled by de novo DnaA protein synthesis and that the

34

concentration of DnaA protein would increase with the age of the cell. Although
growth rate and cell cycle-dependent changes in the concentration of dnaA
transcripts are observed (Campbell and Kleckner, 1990; Chiaramello and Zyskind,
1990; Polaczek and Wright, 1990), the concentration of DnaA protein does not
appear to change (Hansen et al., 1991; Sakakibara and Yuasa, 1982). This issue
should be revisited using more sensitive techniques.

It may not be the concentration of DnaA protein but its activity in
replication that is regulated (Hupp and Kaguni, 1993a). Several studies have led
to suggestions that the levels of DnaA protein need not vary during the cell cycle
but it may be modulated by interactions with nucleotides, acidic phospholipids,
CAMP or possibly other proteins. The high affinity of DnaA protein for
nucleotides may be significant for regulating DnaA protein in vivo. Tightly
bound ADP can be released by DnaA protein interaction with phospholipids
(Sekimizu and Kornberg, 1988) or cAMP (Hughes et al., 1988). Another possible
mechanism may involve a specific inactivator protein (Katayama and Crooke,

1995), or activators (Hupp and Kaguni, 1993b; Hwang et al., 1990).

Other proteins which bind oriC

oriC binding proteins other than DnaA protein may also be involved in the
regulation of initiation. These proteins could indirectly regulate DnaA protein
activity by binding oriC, thereby stimulating or inhibiting the binding of DnaA

protein or unwinding of oriC.

35

Inhibitor of chromosomal initiation (IciA)

IciA, originally identified for its ability to bind specifically to the AT-rich
l3-mers of oriC (Hwang and Kornberg, 1990), is able to inhibit oriC dependent
in vitro replication when added to the reaction prior to DnaA protein (Hwang
and Kornberg, 1992b). IciA inhibits replication by blocking the
DnaA-dependent unwinding of the AT-rich region (Hwang and Kornberg, 1990;
Hwang and Kornberg, 1992b). However, no phenotype is observed in iciA null
mutants, suggesting that it is not a key regulator of the timing of initiation in vivo

(Thony et al., 1991).

Histone-like proteins

Histone-like proteins are thought to organize the bacterial chromosome,
absorbing superhelical density induced by the negative superhelical nature of the
bacterial chromosome, or in some circumstances, increasing superhelical tension to
facilitate the melting of DNA necessary for replication and transcription
(Pettijohn, 1988; Tanaka et al., 1995). Histone-like proteins HU, inversion host
factor (IHF) and factor for inversion stimulation (FIS) act in DNA replication
(Dixon and Kornberg, 1984; Filutowic, et al., 1992; Skarstad et al., 1990),
site-specific recombination (Kano et al., 1989; Mertens et al., 1986; Nash and
Robertson, 1981), and regulation of transcription (Kohno et al., 1994; Molina et
al., 1994; Ninnemann et al., 1992; Rouviere and Gros, 1975) by changing the
topology of the DNA and acting as architectural elements by stabilizing
protein-protein and protein-DNA interactions (Finkel and Johnson, 1992; Segall,

1994). At oriC, HU or IHF facilitate the activity of DnaA protein in initiation.

 

36

HU binds DNA with little sequence specificity but has a preference for dA
tracts (Shimizu et al., 1995), bent DNA (Tanaka, 1984), and kinked DNA
(Pontiggia et al., 1993). On the binding of several dimers of HU, HU bends DNA
(Shimizu et al., 1995). Furthermore, HU can induce supercoiling in relaxed
plasmids in the presence of topoisomerase I (Rouviere et al., 1979).

Unlike HU, both IHF and FIS bind to specific DNA sequences (Finkel and
Johnson, 1992; Wang et al., 1995) to induce a bend. IHF binds bent DNA with a
higher affinity than unbent DNA (Bonnefoy and Rouviere, 1991). Although IHF
can induce supercoiling in relaxed DNA similar to HU, this occurs only at
concentrations 10 times that required by HU (Tanaka et al., 1995). Both IHF and
FIS have a binding site in oriC (Filutowicz and Roll, 1990; Gille, et al., 1991).

HU is required for efficient in vitro replication of an oriC minichromosome
(Dixon and Kornberg, 1984). HU facilitates DnaA protein-dependent unwinding
of the AT—rich sequences within oriC (Bramhill and Kornberg, 1988a). HU is
thought to bend oriC by wrapping DNA around several heterodimers (Broyles
and Pettijohn, 1986). This is thought to aid DnaA protein in inducing torsional
stress that is required to separate the two strands at the AT-rich region. HU may
bind specifically to a naturally bent sequence, or a sequence bent by the binding
of other proteins, and further bends oriC in a manner similar to IHF. In support of
this idea, HU appears to bind preferentially to oriC in prepriming complexes
visualized in electron micrographs and probed with anti-HU antibodies (Funnell
et al., 1987). The bending introduced by HU may aid DnaA-DnaA and/or
DnaA-oriC interactions. IHF can replace HU in in vitro replication assays and in

facilitating DnaA protein dependent unwinding of the AT-rich sequences

 

37

(Skarstad et al., 1990). Although IHF and HU have different DNA binding
characteristics, the mechanism by which they aid DnaA protein in unwinding the
origin may be similar.

Ample evidence exists for the role of IHF in in vivo replication. oriC
contains an IHF site located between DnaA boxes R1 and R2 (Filutowicz and
Roll, 1990). When several point mutations are introduced at this site, the mutant
minichromosomes fail to replicate (Roth et al., 1994). In vivo, footprinting in
synchronized cells suggests that IHF binds just prior to initiation (Cassler, 1995).
Mutations in himA or hip, encoding the on and the B IHF subunits, respectively,
fail to replicate minichromosomes in a manner characteristic of oriC dependent
replication (Filutowicz and Roll, 1990). In addition mutations in him result in
asynchronous initiation (Boye et al., 1993). IHF is not generally thought to be a
candidate for the regulation of initiation as it is found at high cellular

concentrations, 8,500 to 17,000 dimers per cell, levels well above the KD for

binding oriC (Ditto et al., 1994). However, this conclusion appears to contradict
the in vivo footprinting results that indicate that the IHF binding site in oriC is
bound only just before initiation of replication (Cassler, 1995).

The role of FIS at oriC is not well understood. fis mutants have an
asynchronous phenotype (Boye et al., 1993), are inefficiently transformed by
minichromosmes, and are unable to stably maintain oriC plasmids (Gille et al.,
1991). Point mutations in the FIS binding site, located in oriC between DnaA
boxes R2 and R3, inactivate oriC in vivo (Roth et al., 1994). In vivo footprinting
studies suggest that FIS is bound to oriC until the time of initiation (Cassler,

1995). Binding of FIS to this site is proposed to occlude DnaA protein from

38

DnaA box R2 (Gille et al., 1991). Dissociation of FIS immediately prior to
initiation is thought to allow DnaA protein to bind. On the other hand, FIS has
also been speculated to facilitate unwinding the AT-rich region (Messer, 1996).

Interestingly, FIS levels vary with growth phase (Ball et al., 1992). In early
log phase, its cellular concentration reaches a maximum of 33,000 dimers per cell
then diminishes over 500-fold. A burst of FIS dependent transcription of rRN A
and tRNA operons occurs upon resumption of growth from stationary-phase
cultures. Nutritional shift-up to a faster growth rate is accompanied by an
increase of FIS levels (Ballet al., 1992; Xu and Johnson, 1995). Consequently, it
is speculated to serve as an element involved in the coupling of the growth rate

to nutritional and environmental conditions.

Dam Methylation

Proteins proposed to function in the control of replication during the
eclipse period include Dam methylase, and a number of membrane bound proteins
that bind hemimethylated DNA. Dam methylase is a methyltransferase that
modifies adenosine residues of the sequence GATC. Eleven Dam methylation
sites are located within oriC and are conserved throughout the enterobacterial
family (Zyskind et al., 1983). dam mutants are asynchronous (Boye et al., 1988;
Lobner-Olesen et al., 1994). Overexpressing Dam causes random timing of

initiation (Bakker and Smith, 1989; Boye and Lobner-Olesen, 1990). Finally, in

contrast to pBR322, minichromosomes isolated from dam+ strains have a lower

transformation efficiency into dam' strains than wild type strains (Landoulsi et al.,

1989; Messer et al., 1985). Since DNA is replicated semiconservatively, newly

39

replicated DNA is hemimethylated. Hemimethylated oriC is speculated to be
inactive for replication in vivo. In agreement with this hypotheses, Russel and
Zinder found that hemimethylated DNA accumulated in a dam mutant when
transformed with fully methylated DNA (Russell and Zinder, 1987).

Most Dam methylation sites on the E. coli chromosome are fully
methylated within 3 minutes after passage of the replication fork. Methylation at
oriC and at the dnaA promotor occurs more slowly, occurring approximately 13
minutes after replication (Campbell and Kleckner, 1990), suggesting that oriC is
sequestered from Dam methylase following replication. In support of this
hypothesis, overproducing Dam methylase shortens the time between initiation
events (Smith et al., 1985). Based on the observations that membrane fractions
bind specifically to hemimethylated oriC, Ogden et. al. speculated that the
cellular membrane is involved in sequestration (Ogden et al., 1988). This model is
supported by the observation that membrane fractions, which bind specifically to
hemimethylated oriC, inhibit replication specifically from hemimethylated oriC
(Boye, 1991; Landoulsi et al., 1990).

A number of different proteins isolated from membrane fractions are
implicated in oriC-membrane interactions (Chakraborti et al., 1992; Herrick et al.,
1994; Jacq et al., 1989; J acq and Kohiyama, 1980; J acq et al., 1983; Landoulsi et
al., 1990). Squ protein is the best studied of these and has characteristics

suggesting its involvement in sequestration. The squ gene was identified by its
ability to restore the transforrnablity of a dam' strain with methylated plasmids (Lu

et al., 1994; von Freiesleben et al., 1994). squ mutants exhibit an asynchronous

phenotype. In addition, the life time of hemimethylation of oriC is reduced in a

 

 

40

squ mutant. Squ binds hemimethylated DNA with an affinity 5 times greater
than that for methylated oriC (Slater et al., 1995). However, Squ binds
specifically to oriC only when it is fully methylated suggesting that there must be

other factors that are involved in specifically sequestering hemimethylated oriC.

Transcription around oriC

In vivo, the RNA polymerase inhibitor, rifampicin, inhibits replication from
oriC (Lark, 1972). This requirement for RNA synthesis is independent of
subsequent protein synthesis. As a result, RNA polymerase was identified as a
factor in initiation. Originally, RNA polymerase was thought to form the primer
that DNA polymerase III holoenzyme extends from oriC. However, omission of
primase and inclusion of RNA polymerase resulted in very inefficient in vitro
replication (van der Ende et al., 1985). In contrast, efficient in vitro replication
occurs when primase is included in the reaction and RNA polymerase omitted,
suggesting that primase fulfills the function of priming. Currently, RNA
polymerase is thought to involve changing the topology of oriC. Efficient in
vitro replication can be made dependent on RNA polymerase by non optimal
levels of the histone-like HU protein, low temperatures, or high levels of
topoisomerase I (Baker and Kornberg, 1988; Ogawa et al., 1985).

Changes in DNA topology by RNA polymerase can have a negative or a
positive effect on initiation. RNA polymerase forms positive supercoils ahead of
the transcriptional complex, which inhibit unwinding of oriC and forms negative
supercoils behind, which aid in unwinding (Liu and Wang, 1987). Transcription

away from oriC stimulates initiation (Asai et al., 1992), whereas transcription into

 

 

41
oriC inhibits initiation.

Sequestration of hemimethylated oriC appears to inhibit replication from
occurring in the first portion of the eclipse period. As oriC is fully methylated 10
to 20 minutes after the previous initiation event (Campbell and Kleckner, 1990;
Ogawa and Okazaki, 1994; von Freiesleben et al., 1994), inhibition by

sequestration is overcome one third through the eclipse period. However,

 

initiation induced by DnaA protein does not occur for about another 15 minutes .-
(Hanna and Carl, 1975; Helrnstetter and Krajewski, 1982; Leonard et al., 1982).
Recently, transcription from promoters flanking oriC has received attention
because of the proposal that transcription of mioC inhibits initiation after
sequestration, and transcription from gidA stimulates initiation (Figure 3).
However, transcription from these promoters is not required for replication of
oriC containing plasmids either in vivo (von Meyenburg, 1980) or in vitro
(Kaguni and Kornberg, 1984). However, several reports indicate that replication
of minichromosomes is more efficient in the presence of these promoters (Lobner-
Olesen et al., 1987; Ogawa and Okazaki, 1991; Stuitje et al., 1986; Stuitje and
Meijer, 1983).

Both mioC and gidA promoters are stringently controlled by ppGpp
(Ogawa and Okazaki, 1991; Rokeach and Zyskind, 1986). This signal molecule is
the mediator of the stringent response, varying in concentration following shifts
in growth rates or amino acid starvation (Svitil et al., 1993). Transcription from
these promoters is cell cycle dependent, indicating a link with the timing of
initiation (Bogan and Helrnstetter, 1996; Theisen et al., 1993). In synchronized

cells, at the time of initiation mioC transcription is down-regulated. Transcription

42

from an inducible promoter into oriC from the right strongly inhibited initiation,
consistent with transcription of mioC being inhibitory (Tanaka and Hiraga, 1985).
In contrast, transcription from gidA is highest at the time of initiation (Bogan and
Helrnstetter, 1996; Ogawa and Okazaki, 1994; Theisen et al., 1993). This is
consistent with the notion that the formation of negative superhelicity due to
gidA transcription would enhance unwinding of the AT-rich 13-mers.

Other properties of the mioC promotor add support to the model. The
promotor region contains a DnaA box and transcription is repressed by DnaA
protein (Bogan and Helrnstetter, 1996; Lother, et al., 1985; Nozaki, et al., 1988;
Schauzu et al., 1987) MGG 209,518). Maximal transcriptional activity requires
that the promotor is fully methylated (Schauzu et al., 1987). These observations
suggest the importance of transcription from these promoters in the proper timing
initiation from oriC. However, recent studies indicate that these promoters can be
deleted with no apparent effect on the timing or the synchrony of initiation

(Bogan and Helrnstetter, 1996).

VI. Thesis overview

My thesis project involves investigating the detailed mechanism of binding
of DnaA protein to oriC. This investigation, described in Chapter 2, resulted in
the observation that DnaA protein binds to oriC in a sequential manner, in which
DnaA protein recognizes the outer DnaA boxes with higher affinity than the
interior boxes. In addition, the formation of a complex in which DnaA protein
bound all four DnaA boxes correlated with intiation of DNA replication.

Furthermore, in vivo observations supportes the notion that binding of the

43

interior boxes may be important for the regulation of initiation at oriC (Cassler et
al., 1995; Samitt et al., 1989). Additional in vivo observations suggestes that
other oriC binding proteins, IHF and FIS, may play a role in binding of DnaA
protein to the interior boxes. As little is known of the role of FIS in replication
and the mechanism by which IHF stimulates replication, we investigated the
effect of both proteins on the binding of DnaA protein and on oriC-dependent

replication which is described in Chapters 3 and 4.

 

Chapter II

Ordered and Sequential Binding of DnaA Protein to oriC, the
Chromosomal Origin of Escherichia coli

44

Abstract

DnaA protein of Escherichia coli acts in initiation of chromosomal DNA
replication by binding specific sequences, termed DnaA boxes in the

chromosomal origin, oriC. On binding, it induces a localized unwinding to create

 

a structure recognized by other replication proteins that act subsequently in the
initiation process. In this report, we examined the binding of DnaA protein to
each of the DnaA boxes in oriC. By gel mobility-shift assays, DnaA protein
formed at least six discrete complexes. ATP or ADP included in the reaction
mixture prior to electrophoresis was required. Chemical cleavage of isolated
complexes with 1,10-phenanthroline-copper revealed that DnaA protein binds in
an ordered manner to the DnaA boxes in oriC. Preferential binding to one DnaA
box (R4) was confirmed by demonstration that a DNA fragment containing it was
bound with greater affinity than another DnaA box sequence (R1). In vitro
replication activity correlated with a complex formed at a ratio of 30 DnaA
monomers per oriC in which all DnaA boxes are occupied. The last site bound is
DnaA box R3. This event may be critical in promoting initiation from oriC as it
correlates with in vivo observations that binding of DnaA protein to box R3
occurs at the time of initiation of chromosomal replication whereas other DnaA
boxes are bound by DnaA protein throughout the cell cycle (M. R. Cassler, J. E.
Grimwade, and A. C. Leonard (1995) EMBO J. 14, 5833-5841).

45

 

Introduction

DnaA protein of Escherichia coli is a sequence-specific DNA binding
protein, proposed to recognize 9-mer sequences termed DnaA boxes, present in 4
copies within the chromosomal origin, oriC (Figure 1) (Fuller et al., 1984). At
oriC, its binding promotes an ordered series of events to result in the initiation of
chromosomal DNA replication (reviewed in Bramhill and Kornberg, 1988b). The
binding of DnaA protein to oriC has been examined by a variety of methods. By
electron microscopy (Crooke et al., 1993; Funnell et al., 1987) and DNase I
footprinting (Fuller et al., 1984), a large nucleoprotein structure containing 20-30
monomers of DnaA protein is formed at oriC. Experiments to examine its
interaction with individual DnaA boxes led to the conclusion that it bound to the
two centrally located DnaA boxes in oriC (R2 and R3) with greater affinity than
to the flanking DnaA boxes (R1 and R4) (Schaefer and Messer, 1991). This was
based on an indirect assay that assessed the activity of DnaA protein as a
transcriptional terminator in vivo. Expression from the lac promoter, located
upstream to the mutant DnaA box being examined, was measured.

By contrast, gel mobility shift assays with oligonucleotides of 21 base pairs
containing various DnaA boxes with natural flanking sequences indicated that
DnaA protein binds to DnaA boxes R1 and R4 of oriC with higher affinity than
R2 (Schaper and Messer, 1995). DnaA box R3 was bound as poorly as
non-specific oligonucleotides. In addition to these in vitro findings, in vivo
footprinting of oriC plasmids with dimethylsulfate in exponentially growing cells

revealed protection of DnaA boxes R1, R2, and R4 with little binding to R3

46

47

Figure l. The oriC region. DnaA boxes R1-R4 are the 9-mers recognized by
DnaA protein. DnaA box R5 (Matsui et al., 1985) is also shown. Binding sites
for IHF (Filutowicz and Roll, 1990; Polaczek, 1990), and Fis (Filutowicz et al.,
1992; Finkel and Johnson, 1992; Gille, et al., 1991), 13-mer motifs recognized by
IciA protein (Hwang and Kornberg, 1990), and restriction enzyme sites (S, Sma I;
Hf, Hinf 1; A, Ava H; H, HinD III; Ac, Acc I; X, Xho I; and P, Pst I) of pBSoriC are
indicated.

48

99‘

$3...”

8a
a: at «a

2.:
mm n—I- Pm— flLQE fl —.

 

 

g

 

 

(as.

49

(Cassler, 1995; Samitt et al., 1989). These observations, suggesting that the
binding of DnaA protein to R3 is critical for the initiation process, are supported
by the observation that occupancy of R3 occurs at the time of initiation of DNA
replication in synchronized cultures. Another study reported that mutations in
single DnaA boxes (R1 and R4) of oriC reduced binding of DnaA protein to
respective sites (Schaefer and Messer, 1991) but only reduced replication activity
when both mutant sequences were present together (Holz et al., 1992). The
replication activity of oriC may tolerate an alteration of one of the binding sites
(Holz et al., 1992), perhaps by the speculated cooperative binding of DnaA
protein, or occupancy of all four DnaA boxes is not required for replication.
Other proteins have been characterized to bind to specific regions of oriC.
IciA protein was isolated by its ability to bind specifically to 13-mer motifs near
the left boundary of oriC (Hwang and Kornberg, 1990) (Figure 1). Its binding
inhibits the initiation process. IHF, and Fis binding sites in oriC have been
described (Filutowicz and Roll, 1990; Filutowicz et al., 1992; Finkel and Johnson,
1992; Gille et al., 1991; Polaczek, 1990). Rob protein binds to a region near the
right boundary of oriC but its significance is unknown (Skarstad et al., 1993).
The studies summarized above do not provide a clear understanding of
whether DnaA protein binds to sites in oriC randomly, or in an ordered and
sequential manner in the process of initiation of chromosomal replication. If this
event is ordered, the binding of other proteins to oriC may inhibit or augment the
initiation process. In this report, we use gel mobility-shift and DNA footprinting
techniques to characterize complexes of DnaA protein bound to ori C. Results

indicate that DnaA protein binds to oriC in an ordered manner. DnaA box R4 is

50

bound first, then R1, and finally the two inner boxes. Formation of these discrete
complexes was dependent on ATP or ADP. Replication activity correlates with

binding to all four DnaA boxes.

Experimental Procedures

Reagents and chemicals--4-(2-hydroxyethyl)-1-piperazineethane-sulfonic
acid (HEPES), Tris, and dithiothreitol (DTT) were obtained from
Calbiochem-Behring. Cesium chloride was obtained from Gallar-Schlesinger
Industries, Inc.; triton X-100 from Research Products International, Co.; agarose
from Gibco BRL; acrylamide from Fisher Biotech; bisacrylamide from Boehringer
Manheirn; urea from Research Organics; (ethylenedinitrilo)tetracetic acid (EDTA)

and CuSO4 from JT Baker Chemical Co.; 1, 10-phenathroline and 2,9 dimethyl-
phenanthroline were obtained from Aldrich; [a-32P] dNTPs (3000 Ci/rnmol) from

DuPont-New England Nuclear Corp; [3H]'ITP obtained from ICN

Radiochemicals.

Proteins and plasmids—--SmaI was obtained from New England Biolabs:
Xhol from United States Biochemicals; the large fragment of DNA polymerase I
from Boehringer Manheim. All plasmids were purified by cesium chloride
gradient.

Gel mobility-shift assays (Fried and Crothers, 1981; Garner and Revzin,
1981)-—Unless noted, a Sma I-Xho 1 fragment containing oriC, gel—purified from
pBSoriC (Baker and Kornberg, 1988) with a Qiaex DNA extraction kit (Qiagen)

and quantified by absorbance at 260 nm, was 3’ end-labelled with the large

fragment of DNA polymerase I, and [a32P-dTI'P], then combined with the same

unlabelled fragment to adjust its specific radioactivity to 4 x 103 cpm per 25 fmol

of DNA. Reactions (10 [11) with the labelled oriC fragment (25 fmol) and

51

5 2
indicated amounts of DnaA protein were incubated in buffer containing 20 mM
HEPESI-KOH pH 8.0, 5 mM magnesium acetate, 1 mM EDTA, 4 mM D'I'T, 0.2%
Triton X-100, 5 mg/ml bovine serum albumin, and 0.5 [1M ATP (unless noted
otherwise) at 20 0C for 5 min (Parada and Marians, 1991). The samples were

electrophoresed in a 4% polyacrylamide gel (60 parts acrylamidezl part
bis-acrylamide) (13.5 x 13.5 x 0.15 cm) in 45 mM Tris-borate, and 1 mM EDTA at
80 V for 3 to 4 hours. Gels were dried and autoradiographed with Hyperfilm MP

(Arnersham) at -70 °C using a Cronex Quanta III intensifying screen, or

image-analyzed with a Molecular Dynamics Phosphorlmager.

In situ cleavage with 1,10 phenanthroline-copper (Kuwabara and
Sigman, 1987)--Gel mobility-shift assays were performed as above but scaled up
10-fold. After electrophoresis, the wet gel was immersed for 2.5 to 4.5 min in 200

ml of 42 mM Tris-HCl pH 8.0, 0.2 mM phenanthroline, 38 1.1M CuSO4, and 5 mM

3-mercaptopropionic acid at room temperature. To quench the reaction, 2,9
dimethyl-phenanthroline was added to 2.3 mM followed by incubation for 2 min.
The gel was quickly washed with water and autoradiographed for 1 h at room
temperature. The developed film was used to guide excision of the complexes.
DNA from the gel slices was eluted overnight at 37 °C in 500 til of elution buffer
(0.5 M ammonium acetate, 0.2% SDS, 1 mM EDTA, 10 jig/ml Proteinase K, and
100 jig/ml tRNA). After recovering the elution buffer, an additional 200 ul of
elution buffer was used to wash the gel slices, and both were pooled. The eluted
DNA was ethanol precipitated, washed with 70% ethanol, dried, and resuspended
in 5 1110f 80% (v/v) formarnide, 10 mM NaOH, 1 mM EDTA, 0.1% bromophenol

 

53

blue, and 0.1% xylene cyanol, heated to 95 °C for 2 min, and electrophoresed at

50 W on a pre-run 6% sequencing gel. After autoradiography, beta emission
scanning was with a Molecular Dynamics PhosphorImager. Graphed with Excel
(Microsoft), the radioactivity in each lane was normalized to the cleavage pattern
of unbound DNA that was isolated from the gel.

DNA replication assays--Reactions (25 1.11) were performed as described
(Hwang and Kaguni, 1988) with a crude protein fraction deficient in DnaA

protein activity, M13oriC26 DNA (25 fmol) as a template and the indicated

amounts of DnaA protein. Incubation to measure DNA synthesis was at 30 °C for

20 min. Acid-insoluble incorporation of [3H]TI'P was quantified by liquid

scintillation counting.

Rams

Six discrete complexes are formed on binding of DnaA protein to
oriC--By footprint analysis, and electron microscopy, the nucleoprotein complex
of DnaA protein bound to oriC is estimated to contain 20-30 monomers
organized to occupy the four DnaA boxes (Crooke et al., 1993; Fuller et al.,
1984; Funnell et al., 1987). Sequence comparison of these DnaA boxes reveal
that R1 and R4 are identical, whereas R2 and R3 differ at the fifth and seventh
positions, respectively (Table l). Inasmuch as other studies indicated that several
nucleotide changes at each of the positions only marginally affected the ability of
DnaA protein to bind to oriC, and that mutant DnaA boxes of oriC did not affect
in vivo replication activity when singly present (Holz et al., 1992), the binding of
DnaA protein to oriC may not show a strong preference to one site relative to
others. To investigate this, we examined the binding of DnaA protein to sites in
oriC by use of a gel mobility-shift assay. With increasing amounts of DnaA
protein, six discrete complexes were observed (Figure 2). Complex I was
observed at the lowest ratio of DnaA protein to oriC fragment. Complexes of
slower mobility were observed at higher ratios. Compared to other complexes,
the minor abundance of Complexes IV, and V suggests their lesser stability.
Complex VI was predominant at a ratio of 30 monomers of DnaA protein per
DNA fragment, consistent with electron microscopic measurements (Fuller et al.,
1984). In this lane, other material near the well of the gel corresponds to that

formed by a self-aggregate of DnaA protein (unpublished results).

54

55

Table 1. Sequences of DnaA boxes in oriC and in the dnaA promoter region

D_NA W

ori C RI CGGATCCIT G TI‘ATCCACA GGGCAGTGCG
ori C R2 GAATGAGGGG TTATACACA ACT CAAAAAC
ori C R3 CAACCGGTAG TI‘ATCCAAA GAACAACI‘ GT
ori C R4 CCTGACAGAG 'ITATCCACA GTAGATCGCA
DnaA box in the

dnaA promoter 'l'I'I'I'CCCGAT TI‘ATCCACA GGACI'ITCCA
region

DnaA box in ATCACI‘CGAG TIATCCACA Cl‘AGAGTCGA
M13mp18
Position -10 -1 123456789 10 19

core sequence

0The polarity of respective DnaA boxes is from left to right.

56

Figure 2. DnaA protein bound to oriC forms six complexes that are resolved
by native polyacrylamide gel electrophoresis. Gel mobility-shift assays were
performed as described in “Experimental Procedures” with the indicated
amounts of DnaA protein and a 32P-labelled DNA fragment containing oriC.
Unbound DNA is noted as “Free.”

57

3273

Complex VI ‘3 . "

Complex V
Complex IV

Complex lll
Complex II

Complex I

 

Free

 

DnaA(ng) 0 0.6 1.2 2.5 5 10 20 40
DnaA/orIC " 0.5 1 2 4 8 15 30

58

Formation of Complex VI correlates with formation of an active
replication complex--Quantitative analysis was done of complexes in an
experiment comparable to that of Figure 2 but with a larger range of added
protein (Figure 3). In parallel, the replication activity of DnaA protein was
measured. Replication activity correlated with the level of DnaA protein that
formed Complex VI. At this level, complexes of greater mobility were relatively
minor in abundance (as in Figure 2). Higher levels of DnaA protein resulted in
material that remained near the wells of the gel (denoted as “well” in Figure 3).
This material, whose appearance correlated with inhibition of replication activity,
could be resolved into two species on longer electrophoresis (data not shown).
For simplicity, quantitative analysis of Complexes II-V has not been shown in
Figure 3 as their abundance at lower amounts of DnaA protein are represented in
Fig. 2, and at higher DnaA protein levels, they were rare.

DnaA protein bound to ATP is active in unwinding the AT-rich l3-mers
located in oriC (Figure 1) whereas the ADP-bound form is relatively inert
(Bramhill and Kornberg, 1988a; Yung et al., 1990). The importance of the
nucleotide-bound form led us to investigate the effect of ATP and ADP on
complex formation. Formation of discrete complexes was dependent on inclusion
of ATP or ADP in the reaction prior to electrophoresis (Figure 4), although with
ADP, the amount of Complex VI was reduced. The minor species resolved
between complexes V and VI in Figure 4 were rarely observed in other
comparable experiments. In the absence of nucleotide, trace amounts of Complex
I and VI were observed. Instead, the labelled DNA remained near the wells of the

gel. The effect of nucleotide on formation of discrete complexes may relate to the

59

Figure 3. Formation of Complex VI correlates with DNA replication activity.
A gel mobility-shift assay was performed with the indicated levels of DnaA
protein. The relative amounts of Complex 1, VI, (as well as Complexes II-V, not
shown) and material near the well were quantified by beta emission scanning. In

a parallel experiment, replication activity of DnaA protein was measured (see
“Experimental Procedures”).

Fraction Bound

60

 

 

 

 

   
   

 

 

 

    

 

DnaA protein (ng)
0 10 20 30 40
I I l
\ complex I
0.2 L-
s
0.1
\ well __
complex VI
0.0
1.0 I
well _,
0.8
0.6 d
0.4 replication ‘
complex VI

 

 

 

 

100 200 300
DnaA protein (ng)

150

100

50

200

150

100

50

Replication Activity (pmol)

61

Figure 4. Discrete complexes are formed by the ATP- or ADP-bound form of
DnaA protein. Gel mobility-shift assays were performed as described in
“Experimental Procedures” with the indicated amounts of DnaA protein and a

32P—labelled DNA fragment containing oriC. ATP or ADP (2 mM each) were
included in the indicated reactions.

62

94‘ [fit It.
““1 Ni I
“new H

be -

d I L...

DnaA(ng) 0 816 3366 0 81633660 8 163366

ATP ADP No Nucleotide

63

observation that DNase I protection of DnaA boxes in oriC by the ATP-bound
form of DnaA protein was localized to the DnaA boxes whereas binding by the
nucleotide-free form was less specific (Hwang and Kornberg, 1992b) By electron
microscopy (Crooke et al., 1993), the classes of structures formed in the presence
of either nucleotide may correlate to some of the complexes resolved .here
(Figures 2, & 4).

Relative binding aflinities of DnaA protein to supercoiled and linear
DNA-~DNA replication activity of DnaA protein is dependent on a supercoiled
plasmid containing oriC (Funnell et al., 1986). By comparison, the gel
mobility-shift assays were with a linear DNA fragment. Nitrocellulose filter
binding assays showed that DnaA protein bound with about 3-fold higher
affinity to a supercoiled oriC plasmid than to the linearized or relaxed form (Fuller
and Kornberg, 1983). To examine this issue with the gel mobility-shift assay,
unlabelled competitor DNA was added to reactions containing a fixed level of
DnaA protein and radioactively labelled oriC fragment (Figure 5). Addition of
either supercoiled (estimated to be contaminated by ~10% nicked DNA by
resolution by agarose gel electrophoresis and quantitative densitometry),
linearized oriC plasmid, or the same unlabelled restriction fragment resulted in a
comparable reduction of DNA binding to the labelled DNA, measured by
densitometric analysis of the autoradiogram. By contrast, poly dIdC was an
ineffective competitor. These results indicate a comparable binding affinity of
DnaA protein to supercoiled or linear DNAs containing oriC. The apparent
discrepancy between these observations and the cited study may be due to the

absence of ATP in the filter binding assays (Fuller and Kornberg, 1983) whereas it

64

Figure 5. Both supercoiled and linear oriC plasmids are comparable in
reducing binding of DnaA protein to DnaA box R4. The indicated amounts of
unlabelled DNAs (supercoiled pBSoriC, it linearized with Pst I, or the Sma I-Xho I
oriC fragment) as competitor were combined with a Sma I-Xho I oriC-containing
fragment (25 fmol) radioactively labelled by 3’ end-filling of the Xho I site with
[a-32P] 'ITP. DnaA protein (18 ng) was then added. Poly dIdC was added in
equivalent mass to plasmid DNA. Other procedures were as described in
“Experimental Procedures”. The amount of free DNA in each lane was
quantified by beta emission scanning to calculate the amount of DNA bound.

P
on

P
m

l

P
a

l

Fraction Bound

P
N

O

65

 

        
 

"near
oriC fragment

supercoiled

 

 

 

l I l l
0 50 100 150 200 250

Competitior (fmol)

66

was present in the gel mobility-shift experiments (Figure 5). We have not
examined the effect of ATP on binding affinity to different topological forms of
oriC-containing plasmids nor compared the effect of supercoiled and linear oriC-
containing DNAs by each assay method.

Ordered binding of DnaA protein to oriC--To determine the sites bound
by DnaA protein in the separated complexes, in situ footprint analysis was
performed with 1,10 phenanthroline-copper (Kuwabara and Sigman, 1987).
Complexes formed with DNA labelled in the top or bottom strand were examined
(Figure 6 A, B). Regions protected from chemical cleavage were identified by
quantitative analysis of the resultant autoradiograms (Figure 6 C, D). Complex I
consisted of DnaA protein bound to R4. R1 was additionally protected in
Complex II. Complex III differed from Complex II by protection of R2 as well as
sequences to the left of R4 in vicinity of the Ace I site. Binding to the region
encompassing the Acc I site is likely not responsible for the electrophoretic
position of this and more slowly migrating complexes (described below).
Gel-mobility shift experiments with a DNA fragment lacking the Ace I site resulted
in a similar number and proportion of complexes (data not shown). The scarcity
of Complex IV and V relative to other complexes (Figure 2, lanes with 10-40 ng of
DnaA protein) suggests that these may be of lesser stability. Nonetheless, greater
protection of R2 was observed in Complex IV compared to Complex HI (as well
as protection of the region containing the Ace I site). Whereas some protection of
R3 was seen in Complex IV (Figure 6 D), it was enhanced in Complexes V and VI.
Complex VI consisted of protection of the 4 DnaA boxes as well as flanking

sequences in the region from R2 to R4. The altered mobility of Complex IV and V

67

Figure 6. In situ footprinting of complexes with 1,10-phenanthroline-copper.
In situ cleavage was performed as described in “Experimental Procedures” with
a Sma I-Xho I fragment labelled at the Xho I site with the large fragment of DNA
polymerase I and all 4 [a32P] deoxyribonucleotides (top strand, panel A), or at the
Hinfl site of a Hinfl -Pst I fragment by incorporation of all 4 [a32P]
deoxyribonucleotides and subsequently cleaved with Xho I (bottom strand, panel
B). The cleavage pattern of respective complexes was analyzed by beta emission
scanning and compared to the cleavage pattern of the corresponding unbound
fragment that was treated similarly (panels C and D). Maxam-Gilbert G and G+A
reactions performed on the appropriate oriC fragment served as size markers. The

sequence of R4 (panel C) and R1 (panel D) with sequences at -l and -2 positions
(Table l) is presented at the bottom.

68

 

a... g . 1 1‘
56.9.30 .33: ,3: 2 :22. z. i
tease gee , 1.; gal
>_xo_nEoo

é. 3:211:11

.ex. .x as
. . as Si
11.1 1:31

Exoacao
=xoa=ao
_ 63:50
3..".

533800 ,
> onEoo
>_ 63:50
_=x2oEoo
__ onEoo
_ onEoo

ooh".
A

 

 

Complex l

 

Complex II

Complex III

Complex IV

Complex V

Complex VI

 

IIIIII
IIIIII
......
......
......
......
OOOOOO
......
......
......

......
......
......
......
000000
IIIIII
......
nnnnnn
IIIIII
......
......
IIIIII
......
......
......
......

......
llllll
llllll
......
......
......
......
......
......
......
......
......
......
......
......
......

nnnnnn
IIIIII
......
llllll
000000
uuuuuu
oooooo
......
oooooo
......
......
......
000000
......
oooooo
......

 

 

 

R1 R5

 

AGTTATCCACA

Accl

 

70

 

 

Complexl Complex — 555555555

Free _ :::::::::

 

   

Complex VI

to he I52 .55

 

 

 

TG'ITATCCACA

 

71

relative to III may be due to more stable binding of DnaA protein to R2, and R3,
possibly by interaction among proteins bound to different DnaA boxes by
bending or looping of the DNA. In summary, these findings indicate that DnaA
protein binds, in order, to R4, R1, then R2 and R3. These methods do not
distinguish the possible contribution that binding to one site may have on
subsequent binding to another site due to cooperativity.

DnaA protein binds to DnaA box R4 with greater affinity than to
R1--Results from gel retardation and footprinting experiments indicated that
DnaA box R4 is bound with greater affinity than R1 by DnaA protein. To
determine the relative affinity of DnaA protein to restriction fragments containing
only box R1 or R4, gel shift assays were performed with unlabelled DNAs as
competitors. Addition of increasing amounts of unlabelled fragment containing
R4 resulted in proportional inhibition of binding to the labelled R4 fragment
present at a constant level (Figure 7). By comparison, addition of unlabelled R1
fragment was less effective. These results, the average of 3 independent
experiments, confirm that DnaA protein binds to R4 in oriC with greater affinity
than R1. As the 9-mer sequence of R4 is identical to that of R1, sequences that
flank the DnaA box appear to contribute to binding affinity. The protection of
residues at -1 and -2 positions (Table 1) outside the 9-mer sequence of R4 in
Complex I (Figure 6C) and of R1 in Complex II (Figure 6D) supports this
conclusion. Also, the binding affinity to R2 and R3 apparently is less and may be
due to sequence differences in respective 9-mers in addition to the influence of

flanking sequences (Table l).

72

Figure 7. DnaA protein binds to DnaA box R4 with greater affinity than to
R1. An EcoOlO91-Pst I restriction fragment (25 fmol) containing DnaA box R4
from pBSR4, radioactively labelled by end-filling at the EcoOlO91 site with
[a32P] dGTP, was incubated with the indicated amounts of the same unlabelled
fragment, or an unlabelled Sma I-Ava II restriction fragment containing DnaA box
R1. pBSR4 was constructed by insertion of the Hind III-Pst I fragment
(gel-purified) containing DnaA box R4 of oriC into corresponding sites of
pBluescript II SK+ (Stratagene). DnaA protein (3 ng) was added and complexes
resolved from free DNA as described in “Experimental Procedures.” After
autoradiography, the amount of free DNA remaining in each lane was quantified
by beta emission scanning, then the amount of DNA bound was calculated. The

amount of radiolabelled DNA bound in the absence of competitor was normalized
to l.

73

 

 

 

O ‘ T

 

 

Fraction of R4 fragment Bound

T l
O 50 1 00 1 50
Competitor (fmol)

Discussion

Ordered and sequential binding of DnaA protein to oriC--By use of a gel
retardation assay in conjunction with protection from cleavage by 1,10
phenanthroline-copper, the binding of DnaA protein to sequences in oriC was
found to be ordered and sequential. The 6 discrete complexes (and material near
the wells seen at higher levels of DnaA protein) may correlate with the seven
unique structures detected by electron microscopy (Crooke et al., 1993). At
higher levels of DnaA protein, the inhibition of replication activity correlated with
formation of material that entered the gel poorly (Figure 3). Complex VI (Fig. 2)
may correspond to structure 3 (Crooke et al., 1993) as the formation of both
correlated with optimal replication activity. Second, both structure 3 and
complex VI formed more efficiently with ATP than with ADP in the reaction
mixture. In the absence of nucleotide DnaA protein failed to form discrete
complexes. This apparently is due to aggregation of DnaA protein that occurs on
its incubation without ATP (J. Marszalek and J. M. Kaguni, unpublished results).

By comparison, similar experiments with a dnaA promoter fragment
containing a DnaA box identical to R4 of oriC (Table 1) and a weak DnaA box
have been performed (unpublished results). We observed two prominent
complexes and two minor, more slowly migrating species.

A supercoiled template containing oriC is required for in vitro replication
(Funnell et al., 1986). Relating this requirement to structure 3, it was observed
more frequently with supercoiled DNA than linear DNA (Crooke et al., 1993).

We have not determined whether complex VI forms more efficiently on

74

75

supercoiled DNA than linear or relaxed DNA, despite attempts to resolve
complexes formed on supercoiled DNA in low percentage agarose gels. In
addition, we have been unsuccessful in demonstrating ordered binding of DnaA
protein on a supercoiled oriC-containing plasmid. The method used was
quantitative footprint analysis with DNase I or 1,10 phenanthroline of complexes
formed in solution, followed by primer extension. Footprinting in solution
provides an averaged picture of complexes formed. At lower ratios of DnaA
protein to oriC where we expected to see preferential binding to DnaA box R4
then to R1, we presume that the amount of free DNA masks the protection pattern
resulting from ordered binding. Also, we presume that this reason explains why
ordered binding was not observed in previous reports (Fuller et al., 1984; Hwang
and Kornberg, 1992b). Although solution footprinting on supercoiled DNA
failed to detect ordered binding, competition experiments demonstrated that
DnaA protein binds with a similar affinity to supercoiled or linear DNAs
containing oriC (Figure 5). This suggests that DnaA protein binds to either
topological form by a similar mechanism.

Despite the identical 9-mer sequences of R1 and R4 (Table 1), DnaA
protein bound to R4 with about 3-fold higher affinity than to R1 (Figure 7).
Presumably, sequences that flank the 9—mer in R4 contribute to its higher binding
affinity. Indeed, the protection of flanking sequences at -1 and -2 positions of R4,
and R1, clearly seen in Complexes I and 11 (Figure 6C & 6D), indicates that DnaA
protein binds to residues outside of the core sequence. Whether the differences
in sequences at the -2 position of R4 compared to R1 is responsible for the

different binding affinities can be tested directly. Other evidence supports the

76

notion that flanking sequences contribute to binding affinity. With a
nitrocellulose filter binding assay, we found that DnaA protein bound 4-fold
greater to the DnaA box in a dnaA promoter-containing fragment than to a
synthetic DnaA box (9-mer) inserted into the multiple cloning site of M13mp18
(Table 1) (Q. Wang and J. M. Kaguni, unpublished results). These observations
are also supported by the 50-fold difference in binding affinity to a specific DnaA
box when flanking sequences were varied (Schaper and Messer, 1995).

The observations described here are in contradiction to the conclusion that
DnaA protein bound with higher affinity to central DnaA boxes (R2 and R3)
relative to the flanking sites (R1 and R4) (Schaefer and Messer, 1991). This
deduction was based on an indirect method that measured expression of galK
dependent on transcription from the lac promoter. The DnaA box sequence
being assessed was positioned between the promoter and the galK gene. The
relative ability of DnaA protein to function as a transcriptional terminator to affect
galK expression was the basis of the assay. It is possible that differences in
mRNA stability and/or translational efficiency may have influenced the results
obtained.

Four lines of evidence correlate the initiation of DNA replication to the
binding of DnaA protein to DnaA box R3. First, dimethylsulfate treatment of an
oriC plasmid carried in an exponentially growing strain revealed that DnaA box
R3 was not bound whereas the remaining sites were (Samitt et al., 1989). Such
minichromosomes in which plasmid replication occurs from oriC are duplicated
once per generation (Koppes and von Meyenburg, 1987), and synchronously

with the bacterial chromosome (Helrnstetter and Leonard, 1987a). Assuming that

77

replication fork movement is the same as that of the bacterial chromosome, oriC
plasmid replication should be completed within a few seconds. As
dimethylsulfate treatment was for 2 min, most plasmids should not be active in
replication. These observations suggest 2 possibilities. One is that the level of
DnaA protein throughout most of the cell cycle may be insufficient to bind to this
site, and that a critical level must be attained to promote initiation. Alternatively,
this site may be occluded (see below). Second, in synchronous cultures, initiation
of oriC plasmid replication correlated with the binding of DnaA protein to R3
(Cassler, 1995). Third, elevated expression of DnaA protein stimulated initiation
(Atlung et al., 1987; Lobner-Olesen et al., 1989; Skarstad et al., 1989), possibly
by increased occupancy of DnaA box R3. Fourth, formation of Complex VI in
which DnaA box R3 is occupied last correlated with replication activity (Figure 3
& 6).

The method used here does not provide a clear picture of the structure of
Complex VI except for additional protection of sequences flanking the DnaA

boxes. The protected site between R1 and R2 (Figure 6D) contains the sequence

TI‘ATACGGT that resembles the DnaA box sequences ('I'TATA/CCAA/CA) of ori C

and presumably is bound for this reason. The protected region between R2 and
R3 contains a Fis binding site (Figure 1) (Filutowicz et al., 1992; Finkel and
Johnson, 1992; Gille et al., 1991). That fis null mutants maintain poorly
oriC-dependent plasmids (Filutowicz et al., 1992; Gille et al., 1991), and are
asynchronous in initiation (Boye et al., 1993) suggests a positive role for Fis
binding. However, certain in vitro conditions demonstrate that Fis is inhibitory to

oriC plasmid replication (Hiasa and Marians, 1994a), a finding consistent with the

78

report that binding of DnaA protein to R3 is mutually exclusive to the binding of
Fis at its respective site (Gille et al., 1991). Indeed, footprinting studies of oriC
minichromosomes in synchronous cultures suggest that Fis blocks the binding of
DnaA protein to R3 (Cassler, 1995). At initiation, the protection pattern
attributed to Fis was not observed. Instead, DnaA box R3 was protected. Shown
here, occupancy of R3 by DnaA protein correlates with optimal replication
activity (Fig. 3 & 6). DnaA protein and Fis may compete for binding with
contrasting effects on replication activity.

HU protein or IHF act in initiation (Dixon and Kornberg, 1984; Skarstad et
al., 1990) to facilitate unwinding of the l3-mers near the left boundary of oriC
(Hwang and Kornberg, 1992a). A preferred IHF binding site between R1 and R2
(Filutowicz and Roll, 1990; Polaczek, 1990) suggests the possibility that IHF may
enhance one or more steps in formation of Complex VI as a prerequisite to
unwinding. In synchronously growing cells, IHF occupies this site just before
initiation (Cassler, 1995). IHF and DnaA protein were concluded to bind
independently to oriC, based on the lack of effect of IHF on the DNase I
protection pattern by DnaA protein (Fig. 5 of (Hwang and Kornberg, 1992a)).
However, the level of DnaA protein examined was in excess of the level optimal
for formation of Complex VI (Figure 2), and would have obscured detecting
enhancement of DnaA protein binding.

In addition to the 4 DnaA boxes described above, a fifth site, R5, has been
proposed as a site of binding of DnaA protein (Matsui et al., 1985). Little, if any,
protection was observed at this site. This may be partly due to the reduced ability

of phenanthroline-copper to cleave in this region.

79

Except in Complex 1 and II, all other complexes revealed a protected
region encompassing the Ace I site. DNase I footprinting with the nucleotide-free
form of DnaA protein showed that it bound to this region (Hwang and Kornberg,
1992a). However, this region was not protected by DnaA protein bound to ATP,

ADP, or ATP'yS. Although the studies presented here involved incubation of

DnaA protein with the oriC-containing restriction fragment and 0.5 uM ATP
prior to electrophoresis, protection of the region containing the Ace I site
suggests that dissociation of DnaA protein and rebinding of the nucleotide-free
form occurs during electrophoresis. The significance of binding to this site is

unclear as it is not part of the functional oriC sequences (Asai et al., 1990).

Chapter III

The influence of IHF on the binding of DnaA protein to oriC

80

Abstract

The role of integration host factor (IHF) in initiation of DNA replication at
the E. coli chromosomal origin (oriC) was investigated by studying the effect of
IHF on the binding of DnaA protein to oriC. In vitro, IHF can replace HU for
oriC dependent replication (K. Skarstad, T, A. Baker, A. Kornberg (1990) EMBO
J. 9, 2341-2348). HU or IHF is required in addition to DnaA protein for efficient
unwinding of the AT-rich 13-mers found within oriC (D. S. Hwang, A. Kornberg
(1992) J Biol. Chem. 276, 23083-23086). For initiation, DnaA protein binds four
sites, termed DnaA boxes, within oriC. Binding to the two outside DnaA boxes
R1 and R4, is followed by the binding to the two inner boxes, R2 and R3. As
IHF bends DNA on binding and as the IHF site is located between R1 and R2,
IHF has been proposed to enhance DnaA-DnaA interactions and to facilitate the
binding of DnaA protein to R2 and R3. Experiments described here indicate that

IHF does not facilitate DnaA binding to linear oriC.

81

Introduction

HU and integration host factor (IHF) are abundant, small basic DNA
binding proteins organize the bacterial nucleoid (Pettijohn, 1988). In addition,
these proteins have been implicated in site specific recombination, transcription
(Goosen and van de Putte, 1995), and the initiation of plasmid and chromosomal
replication (Dixon and Kornberg, 1984; Funnell et al., 1987; Gamas et al., 1986;
Skarstad et al., 1990). HU and IHF are often interchangeable in many (Goodman
et al., 1992; Hwang and Kornberg, 1992a; Surette et al., 1989) but for not all
processes (Segall, 1994). These bacterial processes are dependent on highly
ordered nucleoprotein complexes. HU and IHF are thought to play a role in
stabilizing these complexes.

Both IHF and HU are composed of two subunits and share 50% amino
acid similarity (Drlica and Rouviere, 1987). Based on mutational studies (Goshima
et al., 1992; Goshima et al., 1990) and protein crystallographic analysis (Rice et

al., 1996; Tanaka, 1984), each subunit contains an anti parallel B-ribbon “arm”
that contacts DNA. On binding DNA, the B-ribbon from each subunit inserts into
the minor grove with one B-ribbon positioned on one side of the DNA strand and
the other B-ribbon on the other side to encircle the DNA (Wang et al., 1995;
Yang and Nash, 1989).

Despite their homology, HU and IHF differ in their DNA binding
properties. HU binds DNA that contains bends, cruciform structures or single

stranded gaps with higher affinity, but it has little sequence specificity (Bonnefoy

et al., 1994; Castaing et al., 1995 ; Pontiggia et al., 1993; Shimizu et al., 1995). In

82

83

contrast, IHF is a sequence specific DNA binding protein (Craig and Nash, 1984;
Goodrich et al., 1990). Second, IHF bends DNA on binding of a single
heterodimer, whereas DNA bending by HU is thought to require the binding of 3
heterodimers (Tanaka, 1984). Furthermore, the models for their ability to bend
DNA are different. Besides interacting with the residues on the B-ribbon, DNA

interacts with the body of IHF to stabilize a bend. HU is proposed to introduce
bends in DNA through protein-protein interactions with HU dimers bound to
adjacent DNA sequences (Tanaka, 1984; Yang and Nash, 1989).

IHF is proposed to be a structural element in protein-DNA complexes by
bending DNA to draw distantly located DNA elements into close proximity
(Finkel and Johnson, 1992; Segall, 1994). In support of this model, lambda
integration reactions were made independent of IHF by replacing the IHF binding
site with either stably bent DNA or binding sites for other proteins that bend
DNA (Giese et al., 1992; Goodman and Nash, 1989; Goodman et al., 1992). In the
latter studies, site specific recombination became dependent on the sequence
specific DNA binding protein, cyclic AMP activator protein (CAP) or lymphoid
enhancer-binding protein (LEF-l), whose site replaced the IHF site (Giese et al.,
1992; Goodman and Nash, 1989).

HU may facilitate the formation of nucleoprotein complexes via a general
mechanism similar to IHF as HU can replace IHF in stimulating lambda excision
(Goodman et al., 1992), and Mu transposition (Surette et al., 1989). In lambda
excision, both proteins stabilize the binding of lambda recombinase, Int, to its
DNA substrate (attL) which contains an IHF binding site between two binding

sites for Int (Segall, 1994). As IHF inhibits HU binding to a fragment containing

84

this site (Segall, 1994), HU appears to bind to the IHF binding site when Int is
bound to attL. HU may bind specifically to the sequences between Int binding
sites because these sequences may be bent. Alternatively, the binding of Int may
bend attL to stimulate HU binding.

As in site specific recombination, IHF facilitates replication of the E. coli
plasmid pSClOl by stabilizing protein-DNA interactions. Replication of pSC101
requires IHF and DnaA protein as well as the plasmid encoded RepA protein
(Gamas et al., 1986). The pSClOl origin contains one consensus DnaA box,

called dnaAS (Stenzel et al., 1991) and other sites, dnaAw, that do not rigorously

match the consensus sequence and are bound weakly. In footprinting studies,

little binding of dnaAW is observed when the adjacent dnaAs site was deleted

(Stenzel et al., 1991). Binding to these weak sites is probably mediated through
protein-protein interactions by the bending of the intervening sequences. IHF

binds to a site between the dnaAS and the two dnaAW sites. The binding of IHF

enhances the binding of DnaA protein to both its sites, presumably by the ability
of IHF to bend the intervening sequences (Stenzel et al., 1991).

Replication from the E. coli chromosomal origin (oriC) also depends on
DnaA protein and IHF (Kaguni and Kornberg, 1984; Skarstad et al., 1990). oriC
contains four DnaA boxes, R1, R2, R3, and R4 (Fuller et al., 1984) (Figure 1).
DnaA protein binds to R1 and R4 with higher affinity than to R2 and R3
(Schaper and Messer, 1995). As in the pSClOl origin, oriC contains an IHF
binding site located between a strong site and a weak site; in the case of oriC the
IHF binding site is located between R1 and R2 (Filutowicz and Roll, 1990;
Polaczek, 1990). The binding of DnaA protein to all four DnaA boxes is thought

85

Figure 1. The oriC region. The 245 bp minimal oriC is indicated by the open
rectangle. The IHF consensus sequence is indicated by the vertically striped box.
DnaA boxes R1-R4 which are the 9-mers recognized by DnaA protein are
indicated by the filled in triangles. The l3-mer motifs and restriction enzyme sites
(S, Sma I; Acc I; and X, Xho I) of pBSoriC are indicated.

86

 

 

...—:—

 

WE

E IL\
EOE-mp

8..

87

to be required for unwinding of the AT-rich l3-mers adjacent to the DnaA boxes
(Margulies and Kaguni, 1996; Oka et al.., 1984). Either HU or IHF facilitates
unwinding of the 13-mers (Hwang and Kornberg, 1992a). The mechanism by
which IHF facilitates unwinding has not been determined. In a mechanism similar
to the function of these proteins in replication of pSC101, IHF may act in early
events at oriC by stimulating the binding of DnaA protein. Alternatively, it may
act at the subsequent step of unwinding the AT-rich region. Here, we investigate

whether IHF promotes the binding of DnaA protein to the DnaA boxes within

oriC.

Experimental Procedures

Reagents and chemicals--3-(cyclohexylamino)-1-propanesulfonic acid
(CAPS) was obtained from United States Biochemical Corporation (USB); sodium
pyrophosphate and trichloroacetic acid (TCA) from J .T. Baker

Proteins and enzymes--The monoclonal DnaA antibody, M43, was
obtained as described in (Marszalek et al., 1996). The polyclonal IHF antibody
was obtain as a gift from Dr. Howard Nash at the National Cancer Institute, NIH.
Horseradish peroxidase-conjugated goat anti-mouse IgG and goat anti-rabbit IgG

were obtained from Bio Rad. Highly purified replication proteins were obtained
as described (Hwang and Kaguni, 1988), DnaB protein (Fraction V, 6 x 105
units/mg); DnaC protein (Fraction VI, 6 x 105 units/mg); primase (Fraction V, 2 x
106 units/mg); single stranded DNA binding protein (SSB) (Fraction V, 4 x 105
units/mg); DNA polymerase III holoenzyme (Fraction V, 2 x 105 units/mg); DNA
gyrase A subunit (Fraction III, 2 x 105 units/mg); DNA gyrase B subunits
(Fraction V, 1 x 105 units/mg); RNA polymerase (4 mg/ml); RNase H (fraction IV, 8
x 105 units/mg), topoisomerase I (fraction IV, 5 x 104 units/mg). HU was purified

as described (Dixon and Kornberg, 1984). IHF was a gift from Dr. Filutowiz at the
University of Wisconsin, Madison. DnaA protein was purified as described
(Sekimizu et al., 1988b).

DNA replication assays-DNA replication reactions using purified
enzymes were performed as previously described (Hwang and Kaguni, 1988).

The reactions (25 [11) contained: HEPES-KOH, pH 7.8, 25 mM; Tris-HCl, pH 7.5,

88

89
20 mM; sucrose 4% (w/v); ATP, 2 mM; CTP, GTP, and UTP, each at 0.5 mM;
dATP, dCT P, dGTP and (3H)'ITP (30 cpm/pmol) each at 100 M; magnesium
acetate, 11 mM; phosphocreatine, 2 mM; DTI‘, 5 mM; creatine kinase, 100 jig/ml;
bovine serum albumin, 0.08 mg/ml; SSB, 160 ng; gyrase A subunit, 500 ng; gyrase
B subunit, 600 ng; primase, 10 ng; DnaB protein, 50 ng; DnaC protein, 40 ng;

DnaA protein, 100 ng; DNA polymerase III holoenzyme, 300 ng; pBSoriC
supercoiled DNA, 25 frnol; and the indicated amounts of HU or IHF. The

reactions were assembled at 0 °C. After incubating at 30 0C for 30 minutes, the
reactions were stopped and the DNA precipitated with 1.0 ml 10% (wt/vol)
trichloroacetic acid and 0.1 M sodium pyrophosphate. The incorporated (3H)TTP

was measured by collecting the precipitated DNA by filtration through glass-fiber
filters (Whatrnan GF/C). The filters were dried and the radioactivity collected on
the filters was quantified by liquid scintillation counting.

DNA binding assays--The gel mobility shift assays were performed as
described (Chapter 2).

DNA protection assay--In situ cleavage with 1,10-phenanthroline-copper
was performed as described (Chapter 2).

Probing complexes with antibodies--Gel mobility shift assays were
performed as described in Chapter 2 except that the reactions were scaled up
5-fold. After electrophoresis, the complexes were transferred from the wet gel to
a PVDF membrane (Schleicher & Schuell) in 9.9 mM 3-(cyclohexylamino)-l-
propanesulfonic acid (CAPS) pH 11, and 10% (vol/vol) methanol for 9 hours at
150 mAmp. The membrane was incubated with 2% (w/vol) milk then probed with

anti-DnaA monoclonal antibody (M43) followed by a horseradish peroxidase-

90

conjugated goat anti-mouse IgG. Detection was by chemiluminescence (ECL,
Amersharn). The membrane was stripped with 0.2 M glycine (pH 2.8) and 1 mM
EGTA, reblocked and reprobed for IHF with anti-IHF polyclonal antibody

followed by horseradish peroxidase conjugated goat anti-rabbit IgG. Detection

was as above. To locate the 32P labelled oriC fragment, the blot was exposed to

film after the ECL signal decayed.

Determination of the dissociation rate constant of the IHF Complex 1. A

Sma I-Xho I oriC fragment 3’-end labelled at the Xho I site with [a32P] TIP was

preincubated with IHF for 5 minutes under standard gel mobility shift conditions

(Chapter 2) in a total of volume of 120 [.11. After preincubation, a 20 ul aliquot
was removed to a separate tube and 10 ul of this was loaded onto a native 4%

polyacrylamide gel. A 100-fold molar excess of pBSoriC was added to the

remaining reaction mixture and 10 ul was immediately loaded onto the gel
electrophoresing at 90 V. Additional 10 ul aliquots of the mixture were then

loaded onto the gel at various times after the addition of the competitor DNA. At

the end of the time course the remaining, 10 ul of the mixture with IHF not
containing the competitor and another 10 ul of the mixture containing just the

labeled DNA were loaded. An additional control in which a 100 fold molar
excess competitor was added before IHF was loaded onto the gel at the
beginning of the time course. Electrophoresis continued for 4 hours at 90 V after
the last sample was loaded. The amount of DNA bound at each time point was

quantitated using [3 emission scanning. The data was plotted and the curve fitted

(-k 0

off

according to the equation Bound,/Boundo = e , where Boundt is the

91

amount of DNA bound at time t, Bound o is the amount of DNA bound
immediately prior to the addition of competitor, and k off is the dissociation rate

constant.

Results

IHF substitutes for H U in purified oriC-dependent replication--In the
absence of RNA polymerase, HU or IHF is required for optimal replication (Figure
2A) (Skarstad et al., 1990). In vitro replication can be made to be dependent on
RNA polymerase by including topoisomerase I and RNase H. RNA polymerase is
not involved in forming primers but is more likely involved in altering the
topology of oriC making it more susceptible to unwinding. Either protein
facilitates DNA replication in the presence and absence of RNA polymerase
(Figure 2B). HU and IHF promote replication by altering the topology of oriC to
facilitate DnaA protein unwinding the AT-rich 13-mer of oriC (Hwang and
Kornberg, 1992a).

IHF forms a single major complex with oriC whereas H U forms multiple
complexes--To investigate whether these proteins affected the initial binding of
DnaA protein to the four DnaA boxes within oriC, gel mobility shift assays were
performed. Although HU and IHF are similar at the amino acid and the structural
level (Drlica and Rouviere, 1987), they are strikingly different in their DNA
binding characteristics. These differences were also demonstrated on binding to
oriC (Figure 3A and B). Three complexes were observed in proportion to the
amount of IHF added (Figure 3A). A major and minor complex (Complex 1 and
Complex 2, respectively) were formed at lower levels of IHF (0.6-10 ng). The third
complex, Complex 3, with lower mobility was observed only when at least 10 ng
of IHF was added.

In similar assays, HU formed as many as eight complexes with oriC (Figure

92

93

Figure 2. IHF can replace HU in in vitro oriC-dependent replication in the
presence or absence of RNA polymerase. IHF (I) or HU (O) were titrated in
reconstituted replication assays which were independent (A) or dependent (B)
on RNA polymerase as described in “Experimental Procedures”.

00
O
O

Replication Activity (pmol)

as
as

N
O
O

'2‘

O

94

 

 

 

B
400
‘ 300
200
1 00

 

 

50

100

, 0
150 200 100 200 300 400 500
Protein (ng)

Replication Activity (pmol)

95

Figure 3. Both IHF and HU bind oriC. Gel mobility shift assays were performed
as described in“Experimental Procedures” with the indicated amounts of IHF (A)
and HU (B) protein and 25 fmol of a Sma I-Xho I oriC fragment labelled at the

Xho I site with the large fragment of DNA polymerase I and [a32P] 'ITP.
Unbound DNA is noted as “Free”.

96

Complex 3
Complex 2
Complex 1

 

 
 
  

Free , _
IHF/oriC 0 1 3 5 10 20 40
IHF (ng) 0 0.6 1.3 2.7 5.4 10 22

 

“

Bound

 

Free ‘7 . .,
HUNG o 0.6 1 3 5 6 12 26 52 100200400
HU(ng) o 0.3 0.6 1 2 3 6 12 24 50 100200

 

97

3B). The HU-oriC complexes decreased in mobility proportional to the amount
of HU added. Only one complex was resolved when HU was added at levels
above 24 ng (Figure 3B). As the IHF binding site was located near the center of
the oriC fragment (Figure 1), the large retardation of the IHF complexes relative
to the unbound DNA suggested that the fragment was highly bent by the
binding of IHF. In contrast, the small change in mobility of the first HU complex
suggested that less bending of the fragment occured on the binding of a single
HU dimer.

IHF binds oriC at a single site--To investigate the DNA sequences bound,
the IHF complexes were footprinted using an in situ method with
phenanthroline-copper. In each of the complexes, the IHF binding site between
DnaA boxes R1 and R2 was protected (Figure 4). Complex 2 included additional
protection of flanking sequences compared with Complexes 1 and 3.

Novel complexes with oriC form in the presence of IHF and DnaA
protein--As the binding of IHF to oriC resulted primarily in a single complex
when IHF was added at lower amounts, gel mobility shift assays were used to
investigate the effect of IHF on binding of DnaA protein to oriC. In the absence
of IHF, DnaA protein formed at least 6 complexes with oriC (Figure 5).
Complexes with slower mobilities were observed with higher levels of protein
added (Figure 5 lanes 2-7, see Chapter 2). At the highest levels tested, the
majority of the fragment was in Complex VI and at the wells of the gel (lane 7).

With a level of IHF sufficient to form IHF Complex 1, addition of DnaA
protein resulted in complexes with mobilities intermediate to that of IHF Complex

1 (lane 8) and that of DnaA Complex VI (lane 7). Based on the relative mobilities

98

Figure 4. In situ footprinting of IHF complexes with 1,10-phenanthroline-
copper. In situ cleavage was performed as described (“Experimental
Procedures”) with IHF and a Sma I-Xho I fragment labelled at the Xho I site with
the large fragment of DNA polymerase I and all 4 [a32P] deoxyribonucleotides.
The IHF complexes were resolved on a standard native gel. The entire gel was
immersed in a phenanthroline-copper solution. The reaction was stopped. Film
was exposed to the gel and was used to guide the excision of the complexes.

The DNA was eluted, precipitated, resuspended in formamide buffer then loaded
on a standard 6% sequencing gel. The resulting cleavage patterns are shown in
panel A. The gel was quantified by [3 emission scanning. The quantified cleavage

pattern of each complex was compared with the cleavage of unbound and shown
in panel B.

99

nxfloEoU

 

NxQQEoo. .
wxgoEoo . -
not -
tl v A 14.1
m W m m R

100

 

 

 

IHF Complex 1 ‘Free
. ', l q 1 —Complex
1.. i 31 '
[M
l
IHF Complex2
"1 1
II '1: I '
U1 ,1 1 ,
t » . '
IHF Complex 3
‘ I
5 ~ 1
it
i
R1 IHF R2 R3 R4

 

101

Figure 5. Novel complexes form in the presence of both IHF and DnaA
protein. A) Gel mobility-shift assays with DnaA protein, IHF or both and a Sma
I-Xho I oriC fragment labelled at the Xho I site with the large fragment of DNA
polymerase I and [0132P] TTP were performed essentially as described in
“Experimental Procedures” of Chapter 2. IHF was added to the reaction before
DnaA protein. B) The gel was quantified by B emission scanning. The total
amount bound in each lane (1 though 7) was quantified and expressed as the
fraction bound of oriC (I). The fraction of oriC bound in complexes containing
DnaA protein in lanes 8 through 14 was determined by subtracting the amount of

oriC bound in the position of IHF Complex 1 from the total oriC bound in each
lane (0).

102

A
Lanes 12 34 56 7 8 91011121314

.73'E':|”.d4-n‘ ' ' ' fl :1 *5“;

  
 
  
  
  
  

Complex Vl'

Complex IV'
.Complex III'
Complex II'
Complex I'

; IHF complex1
Complex II

Complex I

_ , . Free

DnaA/oriC 0‘" 1112 4 8 16 20 0 1 2 4 81620
IHF/oriC 0 ———> 2 ———->

 

Fraction of oriC bound In
DnaA protein complexes

 

 

 

 

DnaA/oriC

103

of these complexes (Complex 1’ through Complex VI’) to the mobility of IHF
complex, they presumably were the result of binding of DnaA protein to IHF-
bound oriC.

Western blot analysis was performed to determine whether the novel

complexes contained both IHF and DnaA protein (Figure 6). Autoradiography
of the membrane was also performed to locate the 32P-labelled oriC fragment

(Figure 6 A). To minimize the complexity of this experiment, a ratio of 3
molecules of DnaA protein to oriC was used so that only Complexes I and II
formed. In the lane in which no DNA was added, DnaA protein runs as a large
smear from the top of the gel (Figure 6B, lane 5). In reactions with DnaA protein
and an oriC fragment, DnaA protein was detected in Complexes I and II, as well
as Complex 1’ and II’, as expected (lane 3). Likewise, IHF was located in IHF
Complex 1 formed with IHF alone and Complex 1’ (Figure 6C). Complex II’ also
cross reacts with anti-IHF antibody (data not shown). These observations
supported the conclusion that Complexes 1’ through VI’ each contain both DnaA
protein and IHF bound to oriC. The ratio of DnaA protein to oriC appears higher
in Complex 1’ than in Complex I (compare lanes 3 and 4 in Panel A and B)
suggesting that IHF facilitated the binding of DnaA protein to oriC. However,
the total amount of oriC fragment bound in DnaA-oriC complexes was similar in
the presence and absence of IHF (Figure 5 B) indicating that IHF did not alter the
affinity of DnaA protein for an oriC fragment. Results may indicate that IHF

increased DnaA-DnaA interactions.

104

Figure 6. Novel complexes contain both DnaA protein and IHF. Gel mobility
shift assays were performed as in Figure 5 (“Experimental Procedures” of
Chapter 2) except that reactions were scaled-up 5-fold. In all reactions
containing IHF and DnaA protein, IHF was added first. The complexes were
electrophoretically transferred to a membrane then probed for DnaA protein and
IHF as described in “Experimental Procedures”. The 32P-labelled oriC fragment
was detected by autoradiography. No oriC was added to lane 5. The position of
unbound oriC is noted as “Free”. The positions of the various complexes are
marked.

105

 

5 Lane
0 IHF/on'C
3 DnaA/oriC

00"
OO'IN

3 4
0 5

Complex II'
Complex |'

IHF Complex 1
DnaA Complex II

DnaA Complex I

 

Free

 

106

IHF does not aid the binding of DnaA protein to the weaker inside DnaA
boxes within oriC--DnaA protein bound to oriC in an ordered manner as
demonstrated by in situ footprinting (Chapter 2). At lower levels, DnaA protein
formed Complex I and 11 (Figure 5A, lanes 2 and 3) by binding to the outside
DnaA boxes, R1 and R4 (Margulies and Kaguni, 1996). At higher concentrations,
DnaA protein formed higher ordered complexes by the additional binding to the
interior boxes, R2 and R3.

More DnaA protein appeared to bind in Complex 1’ than Complex I
indicating additional sites in oriC may be bound by DnaA protein in the presence
of IHF (Figure 6A and B lane 4). To investigate this possibility, in situ
phenanthroline-copper footprinting was performed (Figure 7). As expected, oriC
was protected from cleavage at the IHF binding site in the complex which had
the same mobility as IHF Complex 1. In Complex 1’, both the IHF site and DnaA
box R4 were protected. In Complex 11, additional protection of DnaA box R1
was observed. Only in the lowest mobility complexes was protection of R2 and
R3 observed. The same order of binding was observed in complexes with or
without IHF bound. This suggests that IHF does not facilitate the binding of
DnaA protein to sequences within an oriC fragment.

IHF does not stabilize DnaA-oriC complexes--Although the order of
binding of DnaA protein was not altered by binding of IHF, the kinetics of
binding of DnaA protein to oriC might be altered in the presence of IHF.
Competition gel shifts were used to investigate whether IHF might stabilize DnaA
protein binding by decreasing the dissociation rate of DnaA protein bound to a

radiolabelled oriC fragment. These assays were performed by incubating the

107

Figure 7. The order of DnaA protein binding to oriC is not altered by IHF.
Gel mobility shift assays were performed as described (“Experimental
Procedures” of Chapter 2) except that reactions were scaled-up 10-fold. In all
reactions containing IHF and DnaA protein, IHF was added first. In situ
cleavage with 1,10-phenanthroline-copper was performed as described
(“Experimental Procedures” Chapter 2) with a Sma I-Xho I fragment labelled at
the Xho I site with the large fragment of DNA polymerase I and all 4 [0132P]
deoxyribonucleotides. The cleavage pattern of respective complexes was
analyzed by B emission scanning and compared to the cleavage pattern of the
corresponding unbound fragment that was treated similarly.

108

 

 

 

 

 

109

appropriate proteins with an oriC fragment under standard conditions as
described (see“Experimental Procedures”) except that the complexes were
challenged with unlabelled supercoiled oriC plasmid as a competitor. The
half-life of IHF Complex 1 was approximately 1 minute on the addition of 100-
fold excess of supercoiled oriC plasmid (Figure 8). To avoid competing IHF from
oriC, a 20 base pair oligonucleotide containing one DnaA box sequence was
used as a competitor to investigate whether IHF affected the dissociation of
DnaA protein form oriC. On the addition of the competitor, DnaA—oriC
Complexes I and II dissociated quickly (Figure 9). When DnaA-IHF—oriC
complexes were challenged with an excess of the oligonucleotide containing a
DnaA box, Complexes I and II have comparable half-lives as Complexes 1’ and
II’. These results suggested that IHF did not stabilize DnaA protein bound to
oriC. DnaA protein complexes of lesser mobility formed with or without IHF

dissociated at similar rates on the addition of competitor DNA (data not shown,

Chapter 4).

 

110

Figure 8. Determination of the half-er of the IHF Complex 1. Dissociation of
IHF/oriC Complex 1 was measured by preincubating IHF with an end-labelled
Sma I-Xho I oriC fragment under gel mobility shift assay conditions for 5 minutes
at 20 0C (described in “Experimental Procedures”), then 100-fold molar excess of
supercoiled pBSoriC to the labelled oriC fragment was added. At various times
aliquots were removed and electrophoresed on a native 4% polyacrylamide gel.
A) This graph represents the data derived from the experiment below. Boundo
represents the amount of DNA bound by IHF prior to the addition of unlabeled
DNA. Boundt represents the amount of DNA in complex with IHF at time t. The
resulting line is described by the equation f(t) = 0.91 e-0-63t. The first order
dissociation rate constant, k off, is 0.63 min-1 and corresponds to a half-life of 1
minute for Complex 1. B) Autoradiograph of the gel mobility shift assay. The

asterisks indicate that the challenge DNA was added before the protein. Time 0
has no competitor DNA added.

111

 

Boundt/Bound0
P

 

 

 

0.01

 

012345678

Time (minutes)

 

B
IHF - + + + + + + + + + -
Competitor - + - + + + + + + - ..
Time (min) 0 * O 1I2 1 2 4 8 30 30 30

112

Figure 9. The dissociation of DnaA protein from oriC was not altered by IHF.
DnaA protein and/or IHF as noted was incubated with an end-labelled Sma I-Xho
I oriC fragment in a scaled-up gel mobility shift assay. The complexes were
challenged with a 1000-fold molar excess of an unlabelled double stranded
oligonucleotide containing a DnaA box. At various times aliquiots were removed
and separated on a 4% polyacrylarnide gel. The asterisks indicate that the

‘ challenge DNA was added before the protein.

113

 

DnaA - + + + + + + + - + + + + +
IHF - - - - - - - + + + + + + +
Competitor - + - + + + + + - + + + + + ..
Time (min) * 0 1I2 2 5 10 * 0 1I2 2 5 1010

 

 

Discussion

Although IHF and H U bind to oriC difierently, they can replace each
other in oriC replication--Eight complexes were formed on the 464 base pair
oriC fragment on binding of HU, suggesting that at least 8 HU dimers could bind
a single oriC fragment. However, Bonnefoy et al. (Bonnefoy and Rouviere,
1991) found that two complexes were formed with 21- and 29-mer double
stranded oligonucleotides, and four complexes with a 42-mer oligonucleotide,
suggesting that a HU dimer could bind approximately every 11 bp. Based on one
dimer every 11 bp, as many as 40 dimers may bind to the oriC fragment used. At
the highest level of HU tested (a ratio of 100 dimers per oriC fragment), the
complex formed was much more resistant to phenanthroline-copper cleavage
than free DNA or the complexes with greater electrophoretic mobility (data not
shown) suggesting that oriC in this complex was coated with a large amount of
protein. As expected for a DNA binding protein with little sequence specificity,
no distinct protection pattern of oriC was seen in any of the HU complexes (data
not shown). Because of the large number of complexes formed, gel shift assays
investigating the effect of HU on DnaA protein binding was not pursued.

Addition of IHF to the oriC fragment resulted in three complexes by gel
mobility shift analysis. IHF is thought to bind as a heterodimer composed of IHF-

01 and IHF-B subunits (Nash and Robertson, 1981). However, the homodimer
IHF-B was reported to bind specifically to the same IHF site as the heterodimer,

albeit with lower affinity (Zulianello et al., 1994). Cu-phenanthroline footprint
analysis of the IHF complexes with oriC revealed protection patterns that were

essentially identical. As crosslinking studies suggested that IHF forms

114

115

homodimers and trimers and tetramers (Bonnefoy and Rouviere, 1991). A
possible explanation for the differing mobilities of these complexes is the binding
of homodimers or possibly the binding of trimers or tetramers to oriC.

As HU can replace IHF in some processes (Goodman et al., 1992; Surette et
al., 1989), HU has been speculated to bind specifically to DNA sequences
involved in forming nucleoprotein complexes. Results from immunoelectron
microscopy localizing HU to oriC in replicative complexes with minichromosomes
(Funnell et al., 1987) and footprinting of HU when bound in a Mu invertasome
complex, (Lavoie and Chaconas, 1993; Lavoie and Chaconas, 1994) indicated
that HU could be targeted by the binding of other proteins.

HU can replace IHF in oriC-dependent replication (Figure 1). Both act
early in initiation to facilitate DnaA protein-dependent unwinding of oriC. They
have been speculated to produce a bend in oriC thereby facilitating the
cooperative binding of DnaA protein. Although HU has no sequence-specific
DNA binding activity, it might bind specifically to some bent or kinked DNA to
facilitate DnaA protein function at oriC. This model was supported by the
observations that a relatively low ratio of HU to DNA was required for stimulation
of replication (Figure 2) and that HU has been localized by immunoelectron
microscopy to a complex involved in initiation at oriC (Funnell et al., 1987).

IHF does not afiect the afifinity of DnaA protein for DnaA boxes within
oriC--Based on crude estimates by electron microscopy, 20-40 monomers of
DnaA protein were bound to oriC to form an active complex (Fuller, etal., 1984).
Because the spatial arrangement of individual DnaA boxes in oriC is important

(Oka et al., 1984; Zyskind et al., 1983), it is likely that this nucleoprotein complex

116

has a specific structure.

Results by Fuller et al. (Fuller et al., 1984) suggested that the binding of
DnaA protein to oriC is cooperative, possibly involving interactions among DnaA
protein monomers. The high affinity DnaA boxes, R1 and R4, probably could be
filled without any protein-protein interactions, as a single monomer binds to an
oligonucleotide containing these DnaA box sequences (Schaper and Messer,
1995). Binding to the other two sites, R2 and R3, was weaker (Schaper and
Messer, 1995) and may be stabilized by protein-protein interactions. Whereas the
severe bend introduced by IHF between DnaA boxes R1 and R2 (Figure 3A)
might aid in the binding of DnaA protein to R2 and R3, the studies presented
here indicated that IHF did not aid in the formation or stabilization of DnaA
complexes with a linear oriC fragment. One interpretation of these results is that
the bent structure induced by IHF is not required to form the DnaA-oriC complex
that is active in replication. Alternatively, the proposed interaction among DnaA
protein monomers may be sufficient in formation of a large nucleoprotein
complex. A third possibility is that a supercoiled oriC plasmid is required to
observe an effect of IHF on DnaA protein binding. The studies described here

were with a linear DNA containing oriC.

Chapter IV

The Influence of FIS on the Binding of DnaA
Protein to oriC and on Replication from oriC

117

 

Abstract

A model of factor for inversion stimulation (FIS) protein function in initiation of
replication from oriC is based largely on observations from in viva footprinting of
oriC in synchronized cells (M. R. Cassler, J .E. Grimwade, and A. C. Leonard
(1995) EMBO J. 14, 5833-5841). From these experiments, the FIS protein binding
site appears to be occupied throughout much of the cell cycle but is vacant just
prior to the time of initiation. Furthermore, dissociation of FIS is coincident with
binding of DnaA protein to DnaA box R3. As binding of DnaA protein to R3
correlates with initiation of chromosomal replication in viva and in vitro, this
event appears to be a key step in the initiation process. Because FIS may
antagonize the binding of DnaA protein to DnaA box R3 due to their
overlapping binding sites (H. Gille, J. B. Egan, A. Roth, W. Messer (1991) Nucleic
Acids Res. 15, 4167), we investigated the effect of FIS in reconstituted in vitro
replication, gel mobility shift and footprinting assays. Contrary to our
expectations, our results suggest that FIS does not block the binding of DnaA
protein to the two DnaA boxes R2 and R3; nor does it inhibit in vitro replication.
In light of these results, other possible mechanisms in which FIS might play a role

in initiation of replication are discussed.

118

Introduction

The factor for inversion stimulation (FIS), a small basic histone-like protein,
is a member of a group of DNA-binding proteins that are proposed as major
constituents of nucleoid (Pettijohn, 1988). FIS is a homodimer composed of 11.5
kDa subunits. X-ray diffration studies indicate that each subunit contains four

a-helices (Kostrewa et al., 1991; Kostrewa et al., 1992) with the two N-terminal

helices of each subunit interdigitated to form the homodimer. The C-terminal
helices form a helix-turn-helix DNA binding motif. The N-terminal 26 amino acids
are disordered in the crystal structure and are thought to be involved
protein-protein interactions (Heichman and Johnson, 1990; Koch et al., 1991;
Osuna, et al., 1991). FIS is thought to interact physically with proteins, such as
0'70, and Hin recombinase (Muskhelishvili et al., 1995; Newlands et al., 1992;
Osuna et al., 1991; Ross et al., 1990).

FIS was originally identified by virtue of its ability to stimulate DNA
inversion by a family of site specific recombinases, Hin, Gin and Cin (Haffter and
Bickle, 1987; Johnson et al., 1988; Koch and Kahmann, 1986). Hin controls
flagellar phase variation in Salmonella typhimurium; Gin and Cin control
tail-fiber expression for bacteria phage Mu and P1, respectively. FIS also
stimulates lambda excision (Thompson et al., 1987) and integration (Ball and
Johnson, 1991). Finally, FIS acts both as a positive and a negative transcriptional
regulator. FIS operates via several different mechanisms in these processes. In
some processes, DNA binding of FIS sterically inhibits the binding of another
protein to an overlapping DNA sequence (Ball and Johnson, 1991; Ball et al.,

119

120

1992; Froelich, 1996; Ninnemann et al., 1992; Numrych et al., 1991; Thompson
and Landy, 1988). In other processes, FIS stabilizes nucleoprotein complexes
through bending DNA and possibly via protein-protein interactions (Koch et al.,
1991; Muskhelishvili, et al., 1995; Numrych, et al., 1992; Osuna, et al., 1991; Ross,
et al., 1990).

Although FIS is considered a sequence specific DNA binding protein,
recognizing the highly degenerate sequence
(G/I‘)NNN(A/G)NN(T/A)NNTNNN(C/A), DNA topology is as important as
sequence in determining FIS affinity (Bailly et al., 1995; Finkel and Johnson,
1992). Like the other histone-like proteins, HU and integration host factor (IHF),

FIS bends DNA upon binding. Bent angles of 40° to 90° have been measured for

different FIS-DNA complexes (Gille et al., 1991; Thompson and Landy, 1988).

Expression of FIS protein is growth-rate dependent (Ball et al., 1992;
Thompson et al., 1987). The expression of FIS is greatest in early log phase. FIS
concentrations increase rapidly when cells move from stationary to log phase
growth and decrease through mid to late log phase. Consequently, it is
speculated to serve as an element involved in the coupling of the growth rate to
nutritional and environmental conditions.

Initiation at oriC, the E. coli chromosomal origin, is dependent on the
binding of DnaA protein to four DnaA boxes (Fuller et al., l984)(referred to as
R1, R2, R3 and R4; see Figure 1) within oriC. On binding its four sites in oriC,
DnaA protein is proposed to unwind the AT-rich 13-mers allowing other
replication proteins access to single stranded DNA to form primers which are then

elongated by DNA polymerase HI holoenzyme (Bramhill and Kornberg, 1988a;

121

Bramhill and Kornberg, 1988b). Although the general mechanism for initiation of
replication is well understood, its regulation is not.

The binding of DnaA protein to DnaA boxes within oriC is cell-cycle
dependent (Cassler, 1995; Samitt et al., 1989). This could be due to either an
increase of DnaA protein concentration or its activity. However, there is little
evidence for variations in DnaA protein concentrations as a function of cell cycle
(Hansen et al., 1991; Sakakibara and Yuasa, 1982). Other models for the
regulation of chromosomal replication involve the regulation of DnaA protein 1
activity by its interaction with ATP (Sekimizu et al., 1988a), acidic phospholipids
(Sekimizu and Kornberg, 1988) or possibly other proteins (Hupp and Kaguni,
1993b; Katayama and Crooke, 1995). Another model involves other oriC
binding proteins that either facilitate or inhibit DnaA binding to oriC. It has been
suggested previously that FIS is one of these factors (Gille et al., 1991).

Four lines of evidence suggest that FIS is involved in initiation of
chromosomal replication at oriC: 1) mutants of FIS are asynchronous in initiation
from oriC (Boye et al., 1993); 2) fis mutants inefficiently maintain plasmids
dependent on oriC for replication (Filutowicz et al., 1992); 3) mutations (Figure
1) in the FIS binding site within oriC result in an inactive origin ; and 4) in viva
footprinting of oriC suggests that FIS is bound throughout the cell cycle until
just prior to the time of initiation (Cassler, 1995). Based primarily on the latter
observation, a model was proposed in which the binding of FIS to a site
overlapping with DnaA box R3 occludes the binding of DnaA protein to this site
(Gille et al., 1991). Under this model, dissociation of FIS is required for the
binding of DnaA protein to box R3. Previously, we determined that DnaA

122

Figure 1. The oriC region. The 245 bp minimal oriC is indicated by the open
rectangle. The arrows indicate promoters and their direction of transcription.
DnaA boxes Rl-R4, the 9-mers recognized by DnaA protein, are indicated by
filled triangles also indicating their orientation. The AT-rich 13-mer motifs are
indicated by cross-hatched squares. Restriction enzyme sites (S, Sma I; Acc I; and
X, Xho I) of oriC are indicated. The sequences that match the FIS consensus
sequence (Finkel and Johnson, 1993) are indicated by the checkered boxes. The
sequences that diverge from the consensus sequence are indicated by the shaded
box. The sequence of the putative FIS binding sites between DnaA boxes R2
and R3 are indicated. A set of point mutations that destroy binding of FIS
between R2 and R3 and inactivate oriC for in viva replication is indicated.

123

 

e < 5 o.— .55:
wtoto<o<<w<<aeo<<<<<opom

 

 

 

 

I
E E E E
0< #1 MN— Nm Pm hw-Fhl
Au 3 flue. 2
.56.. 2:1 _ .56..
can CON co _.

 

 

”—4

124

protein binds to DnaA boxes in oriC in an ordered manner (Margulies and
Kaguni, 1996). Its binding to DnaA boxes R2 and R3 correlates with optimal
replication activity. Because of the possibility that FIS binding may modulate the
initiation process, we investigated the effect of FIS on oriC replication using in
vitro assays, gel mobility shift, footprinting and reconstituted oriC dependent
replication. Our results indicate that FIS does not occlude binding of DnaA

protein to box R3 within oriC.

 

Experimental Procedures

Proteins and plasmids--FIS protein was a generous gift of Reid Johnson
(Department of Biological Chemistry , University of California, Los Angeles).

DNA binding assays-- The procedure used for the binding reactions was
described in “Experimental Procedures” in Chapter 2.

In situ Cleavage with 1, 10-Phenanthroline -Copper--The procedure used
for the protection experiments was described in “Experimental Procedures” in
Chapter 2.

Dissociation of protein-oriC complexes--The procedure used for
comparing the dissociation of DnaA-oriC complexes and FIS-oriC complexes
were performed as described in “Experimental Procedures” in Chapter 3.

Replication assays-~The reconstituted replication assays were performed
as described in “Experimental Procedures” in Chapter 3.

Supercoiling assay-Reactions (20 ul) containing 90 fmol of supercoiled

pBSoriC, the indicated amounts of FIS and 20 mM Tris-HCl (pH 7.5) 10 mM
MgC12, 2 mM DDT, 5% glycerol and 5 mM NaCl were preincubated for 15

minutes, at 30 °C. E. coli topoisomerase I (15 U) was added. Reactions were

incubated for 30 minutes at 30 oC and then quenched with 5 til 30% glycerol, 10

mM EDTA, 6% SDS, and heated to 85 °C for 3 minutes prior to electrophoreses

through a 0.8% alkaline agarose gel containing 1 ug/mL chloroquine.

125

Results

F is farms multiple complexes with a DNA fragment containing oriC--The
consensus DNA binding site recognized by FIS is highly degenerate
[(G/T)NNN(A/G)NN(T/A)NNTNNN(C/A)] (Finkel and Johnson, 1992). Although
oriC contains several exact and close matches to the FIS consensus sequence
(Figure 1), the consensus is a poor indicator of FIS binding. FIS binding is in a
large part dependent on DNA topology (Bailly et al., 1995). To determine
whether FIS bound specifically to a DNA fragment containing oriC, gel mobility
shift assays were performed (Figure 2). Multiple complexes were resolved by the
binding of FIS to oriC. At levels as low as 0.1 ng, FIS formed a single complex
(Complex 1). At higher levels (0.4 ng), Complex 1, as well as a second complex
(Complex 2) were observed. At levels greater than 0.8 ng of FIS, complexes with
slower electrophoretic mobilities were observed. The resolution of multiple
complexes suggested the binding of FIS to several sites in oriC.

F is binds several sites within oriC. To determine which sites were bound
in each complex, FIS-oriC complexes were footprinted by cleaving the DNA in
situ with phenanthroline-copper and analyzed quantitatively. The only site
protected in Complex 1 was located between DnaA boxes R2 and R3 (Figure 3A
and 3B). Two FIS sites are predicted between R2 and R3, with the site bound as
the more degenerate of the two (Figure 1). Protection of sequences between R2
and R3 was seen in all FIS-oriC complexes. In Complex 2 and the other
complexes of lower mobility, additional sequences to the right of DnaA box R4

were protected. The protection pattern of Complexes 3, 4 and 5 was similar to

126

 

127

Figure 2. FIS bound to oriC forms multiple complexes that are resolved by
native polyacrylamide gel electrophoresis. Gel mobility shift assays were
performed as described in “Experimental Procedures” with the indicated
amounts of FIS protein and 25 fmol of a 32P labelled Sma I-Xha I oriC fragment.
Unbound DNA is noted as “Free.”

128

HSComphx5
HSCompbx4
._HSCompbx3

iHSCompbe

HSComphx1
Free

 

FIS (ng) 0 0.1 0.2 0.4 0.81.6 3.2
FIS/oriC 0 0.2 0.3 0.7 1.4 2.8 6

129

Figure 3. In situ footprinting of complexes with 1,10-phenanthroline-copper.
In situ cleavage was performed as described (“Experimental Procedures”). The
FIS complexes were resolved on a standard native gel. The entire gel was
immersed in a phenanthroline-copper solution. The reaction was stopped. Film
was exposed to the gel and was used to guide the excision of the complexes.
The DNA was eluted, precipitated, resuspended in formamide buffer and loaded
on a standard 6% sequencing gel. The resultin cleavage patterns are shown in
panel A. The gel in panel A was quantified by emission scanning. The

quantified cleavage pattern of each complex was compared with the cleavage of
free DNA in panel B.

O
3
1

                

        

a 63:50 E. HES

 

h erEoo ..me

m 63:80 .. 3:1
monEoo . s. .
eonEoo . . , . . 1:: .5.
a .6328 an. games 35!: . ......» s... .

N onEoo
_. onEoo

 

#5:: s§§._
e 3. .. .1 1.13.2. _ .3......3
......eau. age;

can“.

 

S
mnm m

131

 

 

Complex 1

    

Complex 4

// 7///‘ 7? // :1, ,,//

[Am/n. :
, /// /

/

/ ”7 ,1; I).
77/54/71; :1

7.
'//

:21! A 41 47/

M/I/I/Illtfllflavv"
» / ,/ ,/ / .'

{I/M
k.
/ M 9; '1’)“. ,

3‘
m;
.\ ~_
§

3

\I

§

 

W

/
a;
//

//

/ V/Mfllflllttrw“ . .. ,:

//

#0

y flay/”mu"...
//il‘ 'A$,,///m [/1/ //
x

W

 

 

— Free

- Complex

 

82 FIS R3

 

Fi9Ure 38 continued

132

 

J Complexs 1;?$

1
1' '1 1 1

«l: _
a.

Complex 6

1

Ad—
...
‘ 1
III».
/

l

1 Complex 8

I ' {
._ q 1
. l :

 

 

 

.,/// 47/A4//;%7,/ 77% % ,0}?

III.

 

%

 

— Free

— Complex

1‘1‘11'

 

 

“1 R2FIS R3

 

 

133

that of Complex 2 except that protection between R2 and R3 extended into R2.
In Complexes 6 and 7, the protected sequences between R2 and R3 extend into
boxes R2 and R3. In addition, sequences to the right of R1 were protected. In
Complex 8, protection by FIS became more pronounced and was seen within
DnaA boxes R1, R2, and R3. These observations suggest that FIS at high levels
may inhibit binding of DnaA protein to oriC to inhibit the initiation of DNA
replication.

FIS dissociates from oriC more slowly than DnaA protein. DnaA protein
formed several complexes with oriC in gel mobility shift assays (Margulies and
Kaguni, 1996) (Figure 5, lane 2 and 9). In order to compare the dissociation rates
of DnaA-oriC complexes with the dissociation rate of FIS-oriC complexes, the
appropriate protein was preincubated with radiolabelled oriC, and then
challenged with a 100-fold molar excess of unlabelled oriC plasmid. At the
indicated times, reactions were electrophoresed in native polyacrylarnide gels to
separate bound from unbound DNA.

Without competitor, DnaA protein (at a molar ratio of protein to DNA
equal to 30) formed a specific complex termed Complex VI (Figure 4A) that
involveed the binding of DnaA protein to all four DnaA boxes in oriC (Margulies
and Kaguni, 1996). At the earliest time point (30 seconds) after addition of the
unlabelled oriC, most of the radiolabelled oriC fragment migrated to the position
of “free” fragment indicating the half-life of Complex VI was less than 30
seconds. The complex with the greatest half-life, Complex I, had a half-life of
approximately 30 seconds (data not shown, see Chapter 3, Figure 9).

At a molar ratio of 1.5 of FIS to oriC fragment that resulted in the

 

134

formation of three discrete complexes, the addition of the unlabelled competitor
did not result in the dissociation of FIS from the radiolabelled oriC fragment until
5-10 minutes had elapsed (Figure 4B, C). These results suggest that FIS
dissociates from its respective binding sites at a rate slower than DnaA protein
(Figure 4C).

Novel oriC complexes form in the presence of both FIS and DnaA
protein. To determine whether FIS-DnaA-oriC complexes could be resolved
from DnaA-oriC and FIS-oriC complexes, binding reactions with both proteins
were compared to reactions with the individual proteins. At a fixed level of F18
and the lowest level of DnaA protein a complex (labelled A) was resolved with an
intermediate mobility to DnaA Complexes H and 111 (Figure 5). Two other novel
complexes, labelled B and C, were resolved at higher levels of DnaA protein.
These intermediates complexes suggested that both FIS and DnaA protein were
bound in a single complex.

Binding of FIS and DnaA protein are not mutually exclusive--We were
interested in confirming the report that the binding of DnaA protein to DnaA box
R3, and that of FIS to the site between R2 and R3 were mutually exclusive (Gille,
et al., 1991). Gel mobility shift assays similar to that shown in Figure 5 were
performed, except that the level of FIS used was sufficient for forming only FIS
Complex 1 in which FIS was bound only to the site between R2 and R3. To
determine the sites bound in these novel complexes, in situ footprinting with Cu—
phenanthroline was performed. Complex A contained protection at DnaA box
R1 and R4, and the FIS site between R2 and R3 (Figure 6). Complex C was

protected at all four DnaA boxes and the FIS site between R2 and R3. These

135

Figure 4. The dissociation of DnaA-oriC and FIS-oriC complexes. The gel
mobility shift assay as described in “Materials and Methods”was scaled-up
lO-fold. After incubating the appropriate protein with a 32P-labelled DNA
fragment containing oriC, pBSoriC (lOO-fold molar excess over the oriC
fragment) was added. Aliquots of the binding reactions were loaded on an
already electrophoresing nondenaturing polyacrylamide gel at the indicated times
following addition of the challenge DNA. A) DnaA protein was added (at a
protein to oriC fragment ratio of 30) and incubated for 5 minutes at 20 0C prior to
adding the challenge DNA. B) FIS was added (at a protein to oriC fragment ratio
of 1.5) and incubated for 90 minutes at 20 0C prior to adding the challenge DNA.
The asterisks in lane 2 indicates that the challenge DNA was added prior to FIS.
C) The resulting gels in panels A and B were quantified by B emission scanning.
The total oriC bound in each lane was expressed as a fraction of the amount
bound prior to the addition of challenge DNA.

136

Complex VI .

Complex II
Complex I

 

+++++++++

U
-

Free
DnaA

+++++++
0 01I212510203030

1

Competitor
Time (min)

Lane

2345678910

‘3
p

++++++++++

Complex 3
Complex 2

  

1
x

b
n.
m
o

C

Free

FIS

+
+

++++++++-

Competitor
Time (min)

Lane

2040606060
910111213

10
8

5
7

137

 

 

C
1
'a
g 0.8 + DnaA
o -o— FIS
.a
r: 0.6
.2
§ 0.4
'6
E 0.2
0

 

 

010 20 30 40 50 60

Time (minutes)

138

Figure 5. Novel complexes with oriC form in the presence of both DnaA
protein and FIS. Examples of two gel mobility shift experiments with DnaA
protein or FIS or both proteins were performed essentially as described in
“Materials and Methods” except that reactions were incubated for 30 minutes
before loading on a polyacrylamide gel. Lanes 1 through 8 represent results from
one experiment and lanes 9 and 10 from another.

139

 

Free

DnaA/oriC -
FIS/oriC -

Lane 1

140

results indicate that FIS bound to the site between R2 and R3 does not
apparently block the binding of DnaA protein to these respective sites.

FIS does not afiect DNA replication on binding oriC--The influence of
FIS on oriC plasmid replication was examined in a reconstituted DNA replication
assay. This assay is dependent on a supercoiled template containing oriC and
purified DnaA, DnaB, and DnaC proteins, HU, primase, gyrase, DNA polymerase
III holoenzyme, and single stranded DNA binding protein (SSB). The same molar
amount (25 fmol) of oriC plasmid was used in the replication assays as in the gel
shift assays described above in Figure 1. When FIS was included at levels (0.1 to
3 ng) tested in gel mobility shift assays (Figure 2), its effect was negligible (Figure
7A). Only at elevated levels (greater than 100 ng) was inhibition seen (Figure
7B).

To investigate the possibility that the vector sequences included on the
oriC plasmid were competing FIS from binding the oriC sequences in replication
assays, unlabelled supercoiled plasmid containing only the vector sequences were
included in of gel mobility shifts with a labelled oriC fragment (Figure 8). Even at
the lowest level tested (0.5 ng), FIS bound oriC in the presence of the vector
sequences. At levels (160 ng) which were inhibitory for replication, a complex
with low mobility formed. These results verified that levels of FIS required to
bind sequences within oriC did not inhibit replication.

We investigated the biochemical basis of this inhibition, suspecting that
these high levels of FIS may result in overwinding of the plasmid, as HU does
when added at inhibitory levels for oriC replication (Skarstad et al., 1990). The

topological alteration in oriC plasmids bound by FIS could be fixed with E. coli

141

Figure 6. In situ footprinting of the DnaA-FIS-oriC complexes with
phenanthroline-copper. In situ cleavage was performed as described in
“Experimental Procedures” with a Sma I-Xho I fragment labelled at the Xho I site
with the large fragment of DNA polymerase I and all 4 [a32P]
deoxyribonucleotides. The cleavage pattern of respective complexes was
analyzed by [3 emission scanning and compared to the cleavage pattern of the
corresponding unbound fragment that was treated similarly.

142

 

w\\\\\\s\\\\\\\\\\\ 1 \\\\\\

 
 

 

a“. ll .. ..
...... .
J , in. 1m V a
_ F
N.‘ ..l r _ fl
.1. x .. l , x i - . l1 ...
w... 4 mm m mm x w
um. i .H m ,. i H .H m .H- H 11h
m i Mma. mmm .... mm pmmm -.l
S h M - l h M m. . l w a w a m . l

 

 

 

 

143

Figure 7. At high levels, FIS inhibits in vitro oriC dependent replication.
Reconstituted replication assays were performed with pBSoriC as a template as
described in “Experimental Procedures”. FIS was added prior to DnaA protein.

The FIS/oriC molar ratio is indicated in parenthesis for the different levels of FIS
added to each reaction.

500

DNA replication (pmol)
.. M w a
8 8 8 8

O

144

 

 

A B 500
7 1
3) 130)
(27) (53) (67) 40°
300
—o— Buffer
100
290) 530)

 

 

'10 20 30 400

FIS (I19)

0
100 200 300 400

DNA replication (pmol)

145

Figure 8. FIS binds oriC in the presence of supercoiled vector DNA. Gel
mobility shift assays were performed as described in “Experimental Procedures”
with the indicated amounts of FIS protein and 25 fmols of a 32P labelled Sma

I-Xho I oriC fragment and 25 fmols of unlabelled supercoiled pBluescript.
Unbound DNA is noted as “Free”.

146

Complex 3

 

Complex 2

Complex 1

  

Free

FlS(ng) 0 0.5 1 2 4 8 16 3240 80 160320
FllorI'C 0 1 2 3 7 1327 5367130270530

147

topoisomerase I and measured by electrophoresing the deproteinated plasmid on
an agarose gel containing chloroquine (Figure 9). Levels of FIS that are
inhibitory for replication form a highly negatively supercoiled topoisomer of the
oriC plasmid.

FIS can not substitute for H U in in vitro replication assays. FIS could
potentially functionally substitute for HU and IHF in oriC plasmid replication.
Both proteins aid in DnaA-dependent unwinding of the AT-rich region. HU and
IHF appear to perform similar functions. When HU was absent in oriC dependent
replication assays, IHF could stimulate oriC plasmid replication (Skarstad et al.,
1990). FIS was tested in the reconstituted oriC replication assay lacking both HU
and IHF. In the absence of HU and IHF, replication occured, albeit less efficiently
(Figure 10). Again, no effect was observed at levels at which FIS was known to
bind to linear oriC (Figure 10A). Only at an BOO—fold excess was FIS inhibitory
(Figure 103).

The effect of FIS at non-optimal levels of HU was examined to explore the
possibility that FIS might be stimulatory. At various levels of HU, no effect of FIS
was seen when added at levels known to bind linear oriC (Figure 10A). At
higher levels, FIS was able to inhibit (Figure 10B). Similar experiments were
performed in which non-optimal levels of IHF replaced HU with comparable
results (data not shown). These experiments indicated that FIS cannot substitute
for HU or IHF nor stimulated when the latter proteins were present at non optimal
levels in oriC plasmid DNA replication.

Binding of FIS to oriC does not affect initiation from oriC in RNA

polymerase dependent initiation--The sequences flanking and within oriC

148

Figure 9. Inhibitory levels of FIS induce a highly negatively supercoiled
topoisomer of pBSoriC. E. coli topoisomerase I was add after FIS was
preincubated with the supercoiled template DNA (lanes 1-9). The position of
nicked and relaxed plasmid DNA migrates is indicated. Negatively supercoiled
pBSoriC which is active for replication migrates to positions intermediate to the
nicked and relaxed DNA (lane 10).

149

Nicked

Negatively
Supercoiled

 

150

Figure 10. Only high levels of FIS inhibit oriC replication in the presence of
various amounts of HU. Reconstituted replication assays were performed as
described in “Experimental Procedures”. The amount of HU added is indicated.
FIS was added prior to DnaA protein.

151

 

   
 
 
  

 

 

 

 

 

 

 

 

500 A B 500
I6 {N d
g 400 + HU 0 I19 400
V HU 1 n
5 300 + 9 300
a; a + HU 29 ng
% 200 + HU 98 ng 200
2
< 100 100
Z
a o H -——- \ o

0 1o 20 30 o 100 200 300 400

FIS (ng)

DNA replication (pmol)

152

contain several promoters (Figure 1). Transcription from promoters in and around
oriC may influence, positively or negatively, replication initiation events that
occur at this site (Asai et al., 1992). As FIS acts as a transcription factor
(reviewed in (Finkel and Johnson, 1992)), FIS could modify transcription from
promoters within or flanking oriC, thereby affecting replication. To investigate
the effect of FIS in a reconstituted system containing RNA polymerase, it was
titrated in assays in the presence and absence of either HU or IHF (Figure 11). As
in the other replication experiments (Figures 7, and 10) FIS had little effect at low
levels and was inhibitory at much higher levels. These findings suggest that FIS

bound to oriC has no stimulatory effect under these in vitro conditions.

153

Figure 11. The binding of FIS to oriC does not affect initiation in RNA
polymerase dependent initiation. Reconstituted replication assays were
performed as described in “Experimental Procedures”. IHF (11 ng) or HU (10 ng)
were added as indicated. FIS was added prior to DnaA protein.

154

 

 

 

 

 

 

 

 

 

A 300
'6
S
‘g 200
.2 .
*3? l
fii, 1' 100
a:
<
E + without HU or IHF

0 1 1 0

0 20 40 100 200 300 400

FIS (ng)

DNA replication (pmol)

Discussion

FIS binds specifically to oriC. Here we showed that FIS bound to sequences
near the right boundary of DnaA box R2, and did not bind to a proposed site
(Gille et al., 1991) located nearer to R3 that more closely resembles the FIS
consensus sequence (Figure 3). This finding confirmed those of Filutowicz et al.
(Filutowicz et al., 1992). In complexes of greater mobility, protection by FIS
extending into DnaA boxes R2 or R3 was not observed, as previously reported
(Filutowicz et al., 1992; Gille et al., 1991). The discrepancies may be due to the
procedures used. The previous footprinting studies used DNaseI, whereas we
used phenanthroline-copper as a cleavage agent which is subject to less steric
hindrance. Another procedural difference was that the DNaseI protection
experiments were performed in solution with a mixture of FIS-oriC complexes,
whereas we used an in situ method that analyzes separated complexes. Indeed,
weak protection of the R2 and R3 boxes was observed in complexes of lower
mobility. This protection may be due to FIS binding elsewhere on the oriC
fragment and stabilizing weaker interactions to flanking sequences by the FIS
dimer bound at the site between R2 and R3.

Specific binding of FIS to oriC has no affect on initiation of
chromosomal replication in vitro. The binding of DnaA protein to DnaA box R3
was reported to be mutually exclusive to the binding of FIS (Gille et al., 1991).
Not only was FIS able to inhibit the binding of DnaA protein when FIS was
bound first, DnaA protein was able to inhibit FIS binding when DnaA protein was

bound first. This observation is unexpected in light of the additive protection

155

156

pattern seen in the presence of DnaA protein and FIS (Figure 6), and the relative
dissociation rates of DnaA protein and FIS when bound to oriC (Figure 4). The
rapid dissociation of DnaA protein from oriC would allow the binding of FIS. A
plausible explanation for these discrepancies is that DNaseI footprinting was
performed when binding had not reached equilibrium, as FIS has a slow rate of
association relative to DnaA protein (data not shown).

When added at a level lOO-fold over that sufficient to fill the highest
affinity site in oriC, FIS neither inhibited nor stimulated DNA replication from
oriC (Figure 7). Inclusion of FIS did not alter replication in the presence of HU or
IHF, or replication that was dependent on RNA polymerase (Figures 10 and 11).
Only at high concentrations (greater than 80 ng) was FIS inhibitory. The
conclusion that the inhibitory effect of FIS was not dependent on FIS binding to
the site between R2 and R3 was supported by the observation that a mutant
oriC plasmid (Mutant Figure 1) deficient in FIS binding to the site between R2
and R3 was equally inhibited by FIS as wild type oriC in vitro replication (Wold
et al., 1996). Instead, inhibition correlated with an alteration in superhelical
density of the DNA template (Figure 9). The in vitro inhibition of replication by
high concentrations of FIS may be relevant in vivo as in early log phase the
cellular concentrations of FIS are approximently lOO-fold greater than required to
inhibit in vitro replication.

Roles for FIS in initiation at oriC. The results presented here do not
support a model in which FIS plays a direct role in initiation at oriC. However, as
we used an in vitro system, some component critical for FIS function may be

missing. For example, the cellular membrane has been speculated to play a role in

157

regulation of initiation by associating with oriC. In vitro replication is inhibited
in the presence of cellular membrane fractions (Landoulsi et al., 1990). Several
membrane proteins that bind oriC are speculated to play role in this inhibition

(J acq et al., 1983; Lu et al., 1994). The role of FIS may be to alter the binding of
an inhibitor of initiation which was not included in these reconstituted replication
assays.

Alternatively, FIS may not have a specific role in initiation. In vivo
observations with fis mutants may be attributable to pleiotropic effects on
supercoiling and/or transcriptional regulation of initiation factors. FIS is proposed
to help organize the bacterial chromosome as fis mutants have aberrant nucleoid
structure. fis mutants may have an altered chromosome that affects chromosome
replication (Filutowicz et al., 1992).

Furthermore, as FIS is also a transcription factor, it may indirectly affect
initiation by changing the expression of replication factors. Recently, FIS was
reported to change the expression of DnaA protein (Froelich, 1996). In fis null
mutants, the level of DnaA protein increased approximately 2- to 3-fold relative to
a wild type fis strain. A 2-fold increase in the level of DnaA protein was sufficient
to cause asynchronous initiation (Atlung and Hansen, 1993; Lobner—Olesen et al.,
1989). Lastly, FIS may only play a role under specific growth conditions, such as
at elevated temperatures or at the transition from stationary phase cell to

exponential growth (Ball et al., 1992; Filutowicz et al., 1992).

Chapter V

Summary and Perspectives

158

This thesis demonstrates that DnaA protein binds to the DnaA boxes
within oriC in an ordered manner, and that the binding to all four DnaA boxes
correlates with replication of an oriC plasmid. These findings not only reveal
more detail into the mechanism of initiation but also provide insight into how
initiation from oriC may be regulated. The binding of the interior boxes at
higher concentrations of DnaA protein after the outer boxes are bound suggests
that binding of DnaA protein to the interior boxes triggers initiation. This model
is supported by in vivo footprinting which suggests that DnaA boxes R1, R2
and R4 are bound throughout the cell cycle and that only at the time of
initiation is the R3 bound. Recently, mutational analysis of oriC plasmids, in
which exchanging R3 and R4 sequences reduces oriC activity in vivo, also
supports the model (Langer et al., 1996). It would be of interest to determine the
order of binding of DnaA protein to this mutant and whether initiation is
synchronous from this at other mutant origins which alter the binding of DnaA
protein.

As little evidence exists for changes in DnaA protein concentration in a
cell-cycle dependent manner, other modes of DnaA protein regulation have
received attention. These include the regulation of DnaA protein activity
through its ability to bind ATP or through its ability to bind oriC (Katayama and
Crooke, 1995). The latter mechanism involves proteins which bind oriC and
therefore altering the binding of DnaA protein to oriC. FIS has been speculated
to be a regulatory factor as fis mutants have phenotypes that are consistent with

this function (Boye et al., 1993, Gille et al., 1991) and mutations in the FIS

159

160

binding site inactivate oriC in vivo (Roth et al., 1994). In addition, binding of its
site in oriC appears to be altered immediately prior to initiation (Cassler et al.,
1995). Furthermore, FIS cellular concentrations vary with different growth
conditions as might be expected for a regulatory protein (Ball et al., 1992).
However, in vitro studies presented here do not support the model in which the
binding of FIS to the site between R2 and R3 plays a direct role in initiation.
Binding of FIS to its site between boxes R2 and R3 does not inhibit the binding
of DnaA protein to the interior boxes as originally speculated.

These results do not exclude the possibility of FIS playing a role in
initiation. The in vitro inhibition of replication by high concentrations of FIS
may be relevant in vivo as in early log phase the cellular concentrations of FIS
are approximately lOO-fold greater than required to inhibit in vitro replication. It
would be of interest to measure the rate and the synchrony of initiation and
footprint oriC in vivo when FIS is present at various cellular concentrations.
The conditions for in vitro replication vary from those in vivo. As these
experiments were performed with highly purified proteins, factors important in
FIS function may be lacking. Experiments adding inhibitors of oriC replication
(i.e. membrane fractions, or possibly Squ) to the purified system or using
extracts deficient in FIS may reveal a direct effect of FIS on replication.
However, FIS may not play a direct role in initiation. Initiation defective
phenotype of fis mutants may be due to increased levels of DnaA protein in fis
mutants rather than any direct interaction at oriC. Studies altering the levels of
DnaA protein in a fis background may reveal that the fis mutant phenotype is

the result of pleiotropic effects.

161

Another oriC binding protein which could affect DnaA protein activity at
oriC is IHF. This protein is not thought to be a regulator of initiation as it is
found at high cellular concentrations (Ditto et al., 1994). Although IHF can
stimulate replication from oriC in the absence of HU, no effect of IHF on the
binding of DnaA protein to a linear oriC fragment was observed. Because the
DNA binding experiments were performed with linear DNA, and DnaA protein
dependent unwinding of oriC only occurs on a supercoiled template, we can not
exclude the possibility that IHF aids initial binding of DnaA protein only on a
supercoiled template. Additional footprinting experiments using a supercoiled
template may show that IHF stimulates the binding of DnaA protein to
supercoiled oriC as was seen for the binding of Int protein to lambda attP
involved in lambda integration (Richet et al., 1986). However, the results
presented here suggest that IHF facilitates DnaA protein-dependent unwinding
by promoting steps subsequent to the initial binding of DnaA protein to oriC.

Though my thesis has concentrated on three proteins involved in
initiation, other factors (i.e. Dam methylase and Squ) have been demonstrated
to play a role in regulating replication, and other factors probably have yet to be
identified. Squ may remain bound to oriC even when fully methylated,
inhibiting DnaA protein from interacting with oriC. As of now Squ has not
been tested in vitro for its ability to inhibit replication of hemimethylated or fully
methylated oriC. Finally, genetic approaches have been useful in identifying
factors involved in initiation in the past and may prove useful in identifying
other factors that are important in regulating initiation. Hyperactive initiation

mutants may be identified by selecting for an increased copy number of an oriC

162

rninichoromosome carrying an antibiotic resistance marker.

Bibilography

163

Bibliography

Allen, G. C., Jr and Kornberg, A. (1993) Assembly of the primosome of DNA
replication in Escherichia coli. J. Biol. Chem. 268: 19204-19209.

Allen, G. J. and Kornberg, A. (1991) Fine balance in the regulation of DnaB
helicase by DnaC protein in replication in Escherichia coli. J. Biol. Chem.
266: 22096-22101.

Arendes, J., Carl, P. L. and Sugino, A. (1982) A mutation in the mh-locus of
Escherichia coli affects the structural gene for RNase H. Examination of the
mutant and wild type protein. J. Biol. Chem. 257: 4719-4722.

Asai, T., Chen, C. P., Nagata, T., Takanami, M. and Imai, M. (1992) Transcription in
vivo within the replication origin of the Escherichia coli chromosome: a

mechanism for activating initiation of replication. Mol. Gen. Genet. 231: 169-
178.

Asai, T., Takanami, M. and Imai, M. (1990) The AT richness and gid transcription
determine the left border of the replication origin of the E. coli chromosome.
EMBO J. 9: 4065-4072.

Atlung, T. and Hansen, F. G. (1993) Three distinct chromosome replication states

are induced by increasing concentrations of DnaA protein in Escherichia
coli. J. Bacteriol. 175: 6537-6545.

Atlung, T., Lobner Olesen, A. and Hansen, F. G. (1987) Overproduction of DnaA
protein stimulates initiation of chromosome and minichromosome replication in
Escherichia coli. Mol. Gen. Genet. 206: 51-59.

Augustin, L. B., Jacobson, B. A. and Fuchs, J. A. (1994) Escherichia coli Fis and
DnaA proteins bind specifically to the nrd promoter region and affect
expression of an nrd-lac fusion. J. Bacteriol. 176: 378-387.

Bailly, C., Waring, M. J. and Travers, A. A. (1995) Effects of base substitutions on
the binding of a DNA-bending protein. J. Mol. Biol. 253: 1-7.

Baker, T. A., Funnell, B. E. and Kornberg, A. (1987) Helicase action of DnaB
protein during replication from the Escherichia coli chromosomal origin in
vitro. J. Biol. Chem. 262: 6877-6885.

Baker, T. A. and Kornberg, A. (1988) Transcriptional activation of initiation of
replication from the E. coli chromosomal origin: an RN A-DN A hybrid near
oriC. Cell 55: 113-123.

Baker, T. A., Sekimizu, K., Funnell, B. E. and Kornberg, A. (1986) Extensive
unwinding of the plasmid template during staged enzymatic initiation of DNA

164

165

replication from the origin of the Escherichia coli chromosome. Cell 45: 53-64.

Bakker, A. and Smith, D. W. (1989) Methylation of GATC sites is required for
precise timing between rounds of DNA replication in Escherichia coli. J.
Bacteriol. 171: 5738-5742.

Ball, C. A. and Johnson, R. C. (1991) Efficient excision of phage lambda from the
Escherichia coli chromosome requires the Fis protein. J. Bacteriol. 173: 4027-
403 1 .

Ball, C. A., Osuna, R., Ferguson, K. C. and Johnson, R. C. (1992) Dramatic
changes in Fis levels upon nutrient upshift in Escherichia coli. J. Bacteriol.
174: 8043-8056.

Bemander, R., Dasgupta, S. and Nordstrom, K. (1991) The E. coli cell cycle and

the plasmid R1 replication cycle in the absence of the DnaA protein. Cell 64:
1145-1 153.

Bird, R. E., Louam, J ., Martuscelli, J. and Caro, L. (1972) Origin and sequence of
chromosome replication in Escherichia coli. J. Mol. Biol. 70: 549-566.

Bogan, J. A. and Hehnstetter, C. E. (1996) mioC transcription, initiation of
replication, and the eclipse in Escherichia coli. J. Bacteriol. 178: 3201-3206.

Bonnefoy, E. and Rouviere, Y. J. (1991) HU and IHF, two homologous histone-
1ike proteins of Escherichia coli, form different protein-DNA complexes with
short DNA fragments. EMBO J. 10: 687-696.

Bonnefoy, E., Takahashi, M. and Yaniv, J. R. (1994) DNA-binding parameters of
the HU protein of Escherichia coli to cruciform DNA. J. Mol. Biol. 242: 116-
129. ‘

Boye, E. (1991) The hemimethylated replication origin of Escherichia coli can be
initiated in vitro. J. Bacteriol. 173: 4537-4539.

Boye, E. and Lobner-Olesen, A. (1990) The role of Dam methyltransferase in the
control of DNA replication in E. coli. Cell 62: 981-989.

Boye, E., Lobner-Olesen, A. and Skarstad, K. (1988) Timing of chromosomal
replication in Escherichia coli. Biochim. Biophys. Acta. 951: 359-364.

Boye, E., Lyngstadaas, A., Loebner-Olesen, A., Skarstad, K. and Wold, S. (1993)
Regulation of DNA replication in Escherichia coli. DNA replication and the
cell cycle. DNA replication and the cell cycle, p. 15-26. E. Fanning, R.
Knippers, E. L. Winnacker (ed.) DNA Replication and the Cell Cycle. 43.
Mosbacher Kolloquium. Springer, Berlin.

 

166

Bramhill, D. and Kornberg, A. (1988a) Duplex opening by DnaA protein at novel
sequences in initiation of replication at the origin of the E. coli chromosome.
Cell 52: 743-755.

Bramhill, D. and Kornberg, A. (1988b) A model for initiation at origins of DNA
replication. Cell 54: 915-918.

Braun, R. E., O'Day, K. and Wright, A. (1985) Autoregulation of the DNA
replication gene dnaA in E. coli K-12. Cell 40: 159-169.

Braun, R. E. and Wright, A. (1986) DNA methylation differentially enhances the
expression of one of the two E. coli dnaA promoters in vivo and in vitro.
Mol. Gen. Genet. 202: 246-250.

Broyles, S. S. and Pettijohn, D. E. (1986) Interaction of the Escherichia coli HU
protein with DNA. Evidence for formation of nucleosome-like structures with
altered DNA helical pitch. J. Mol. Biol. 187: 47-60.

Burgers, P. M., Kornberg, A. and Sakakibara, Y. (1981) The dnaN gene codes for
the beta subunit of DNA polymerase III holoenzyme of Escherichia coli.
Proc. Natl. Acad. Sci., USA. 78: 5391-5395.

Campbell, J. L. and Kleckner, N. (1990) E. coli oriC and the dnaA gene promoter
are sequestered from Dam methyltransferase following the passage of the
chromosomal replication fork. Cell 62: 967-979.

Carl, P. L. (1970) Escherichia coli mutants with temperature-sensitive synthesis
of DNA. Mol. Gen. Genet. 109: 107-122.

Carr, K. M. and Kaguni, J. M. (1996) The A184V missense mutation of the dnaA5
and dnaA46 alleles confers a defect in ATP binding and therrnolability in
initiation of Escherichia coli DNA replication. Mol. Micro. 20: 1307-1318.

Cassler, M. R., Grimwade, J .E. and Leonard, A. C. (1995) Cell cycle-specific

changes in nucleoprotien complexes at a chromosomal replication origin.
EMBO J. 14: 101-109.

Castaing, B., Zelwer, C., Laval, J. and Boiteux, S. (1995) HU protein of
Escherichia coli binds specifically to DNA that contains single-strand breaks
or gaps. J. Biol. Chem. 270: 10291-10296.

Castuma, C. E., Crooke, E. and Kornberg, A. (1993) Fluid membranes with acidic

domains activate DnaA, the initiator protein of replication in Escherichia coli.
J. Biol. Chem. 268: 24665-24668.

Chakraborti, A., Gunji, S., Shakibai, N., Cubeddu, J. and Rothfield, L. (1992)
Characterization of the Escherichia coli membrane domain responsible for
binding oriC DNA. J. Bacteriol 174: 7202-7206.

167

Chakraborty, T., Yoshinaga, K., Lother, H. and Messer, W. (1982) Purification of
the E. coli dnaA gene product. EMBO J. 1: 1545-1549.

Chiaramello, A. E. and Zyskind, J. W. (1990) Coupling of DNA replication to
growth rate in Escherichia coli: a possible role for guanosine tetraphosphate.
J. Bacteriol. 172: 2013-2019.

Churchward, G., Holmans, P. and Bremer, H. (1983) Increased expression of the
dnaA gene has no effect on DNA replication in a dnaA+ strain of Escherichia
coli. Mol. Gen. Genet. 192: 506-508.

Cooper, S. and Helrnstetter, C. E. (1968) Chromosome replication and the division
cycle of Escherichia coli B/r. J. Mol. Biol. 31: 519-540.

Craig, N. L. and Nash, H. A. (1984) E. coli integration host factor binds to specific
sites in DNA. Cell 39: 707-716.

Crooke, E., Castuma, C. E. and Kornberg, A. (1992) The chromosome origin of
Escherichia coli stabilizes DnaA protein during rejuvenation by
phospholipids. J. Biol. Chem. 267: 16779-82.

Crooke, E., Thresher, R., Hwang, D. S., Griffith, J. and Kornberg, A. (1993)
Replicatively active complexes of DnaA protein and the Escherichia coli

chromosomal origin observed in the electron microscope. J. Mol. Biol. 233:
16-24.

de Massy, B., Fayet, O. and Kogoma, T. (1984) Multiple origin usage for DNA
replication in sdrA(mh) mutants of Escherichia coli K-12. Initiation in the
absence of oriC. J. Mol. Biol. 178: 227-236.

DiNardo, S., Voelkel, K. A., Sternglanz, R., Reynolds, A. E. and Wright, A. (1982)
Escherichia coli DNA topoisomerase I mutants have compensatory mutations
in DNA gyrase genes. Cell 31: 43-51.

Ditto, M. D., Roberts, D. and Weisberg, R. A. (1994) Growth phase variation of
integration host factor level in Escherichia coli. J. Bacteriol. 176: 3738-3748.

Dixon, N. E. and Kornberg, A. (1984) Protein HU in the enzymatic replication of

the chromosomal origin of Escherichia coli. Proc. Natl. Acad. Sci., USA. 81:
424-428.

Donachie, W. D. (1968) Relationship between cell size and time of initiation of
DNA replication. Nature 219: 1077-1079.

Drlica, K. and Rouviere, Y. J. (1987) Histonelike proteins of bacteria. Microbiol.
Rev. 51: 301-319.

168

Evans, 1. M. and Eberle, H. (1975) Accumulation of the capacity of initiation of
deoxyribonucleic acid replication in Escherichia coli. J. Bacteriol. 121: 883-
891.

Fay, P. J ., Johanson, K. O., McHenry, C. S. and Bambara, R. A. (1981) Size classes
of products synthesized processively by DNA polymerase 1H and DNA
polymerase III holoenzyme of Escherichia coli. J. Biol. Chem. 256: 976-83.

Fay, P. J., Johanson, K. O., McHenry, C. S. and Bambara, R. A. (1982) Size classes
of products synthesized processively by two subassemblies of Escherichia
coli DNA polymerase HI holoenzyme. J. Biol. Chem. 257 : 5692-9.

Felton, J. and Wright, A. (1979) Plasmid pSClOl replication in integratively
suppressed cells requires dnaA function. Mol. Gen. Genet. 175: 231-233.

Filutowicz, M. and Jonczyk, P. (1983) The gyrB gene product functions in both
initiation and chain polymerization of Escherichia coli chromosome
replication: suppression of the initiation deficiency in gyrB-ts mutants by a
class of rpoB mutations. Mol. Gen. Genet. 191: 282-287.

Filutowicz, M. and Roll, J. (1990) The requirement of IHF protein for
extrachromosomal replication of the Escherichia coli oriC in a mutant
deficient in DNA polymerase I activity. New Biol. 2: 818-827.

Filutowicz, M., Ross, W., Wild, J. and Gourse, R. L. (1992) Involvement of Fis

protein in replication of the Escherichia coli chromosome. J. Bacteriol. 174:
398-407.

Finkel, S. E. and Johnson, R. C. (1992) The Fis protein: it's not just for DNA
inversion anymore. Mol. Micro. 6: 3257-3265.

Frey, J ., Chandler, M. and Caro, L. (1979) The effects of an Escherichia coli
dnaAts mutation on the replication of the plasmids ColEl pSClOl, R1001 and
RTF-TC. Mol. Gen. Genet. 174: 1 17-126.

Fried, M. and Crothers, D. M. (1981) Equilibria and kinetics of lac repressor-

operator interactions by polyacrylarnide gel electrophoresis. Nucleic Acids
Res. 9: 6505-6525.

Froelich, J. M., Phuong, T. K. and Zyskind, J. W. (1996) FIS binding in the dnaA
operon promoter region. J. Bacteriol. 178: 6006-6012.

Fujita, M. Q., Yoshikawa, H. and Ogasawara, N. (1990) Structure of the dnaA

region of Micrococcus luteus: conservation and variations among eubacteria.
Gene 93: 73-78.

169

Fuller, R. S., Funnell, B. E. and Kornberg, A. (1984) The DnaA protein complex
with the E. coli chromosomal replication origin (oriC) and other DNA sites.
Cell 38: 889-900.

Fuller, R. S., Kaguni, J. M. and Kornberg, A. (1981) Enzymatic replication of the
origin of the Escherichia coli chromosome. Proc. Natl. Acad. Sci., USA. 78:
7370-7374.

Fuller, R. S. and Kornberg, A. (1983) Purified DnaA protein in initiation of
replication at the Escherichia coli chromosomal origin of replication. Proc.
Natl. Acad. Sci., USA. 80: 5817-5821.

Funnell, B. E., Baker, T. A. and Kornberg, A. (1986) Complete enzymatic
replication of plasmids containing the origin of the Escherichia coli
chromosome. J. Biol. Chem. 261: 5616-5624.

Funnell, B. E., Baker, T. A. and Kornberg, A. (1987) In vitro assembly of a

prepriming complex at the origin of the Escherichia coli chromosome. J. Biol.
Chem. 262: 10327-10334.

Gamas, P., Burger, A. C., Churchward, G., Caro, L., Galas, D. and Chandler, M.
(1986) Replication of pSClOl: effects of mutations in the E. coli DNA binding
protein IHF. Mol. Gen. Genet. 204: 85-89.

Garner, J. and Crooke, E. (1996) Membrane regulation of the chromosomal

replication activity of E. coli DnaA requires a discrete site on the protein.
EMBO J. 15: 2313-2321.

Garner, M. M. and Revzin, A. (1981) A gel electrophoresis method for quantifying
the binding of proteins to specific DNA regions: application to components of

the Escherichia coli lactose operon regulatory system. Nucleic Acids Res. 9:
3047-3060.

Garrido, T., Sanchez, M., Palacios, P., Aldea, M. and Vicente, M. (1993)
Transcription of ftsZ oscillates during the cell cycle of Escherichia coli.
EMBO J. 12: 3957-3965.

Gaylo, P. J ., Turjman, N., and Bastia, D. (1987) DnaA protein is required for
replication of the minimal replicon of the broad-host-range plasmid RK2 in
Escherichia coli. J. Bacteriol. 169: 4703-4709.

Gielow, A., Kucherer, C., Kolling, R. and Messer, W. (1988) Transcription in the
region of the replication origin, oriC, of Escherichia coli: termination of aan
transcripts. Mol. Gen. Genet. 214: 474-481.

Giese, K., Cox, J. and Grosschedl, R. (1992) The HMG domain of lymphoid
enhancer factor 1 bends DNA and facilitates assembly of functional
nucleoprotein structures. Cell 69: 185-195.

 

170

Gille, H., Egan, J. B., Roth, A. and Messer, W. (1991) The FIS protein binds and
bends the origin of chromosomal DNA replication, oriC, of Escherichia coli.
Nucleic Acids Res. 19: 4167-4172.

Goodman, S. D. and Nash, H. A. (1989) Functional replacement of a protein-
induced bend in a DNA recombination site. Nature 341: 251-254.

Goodman, S. D., Nicholson, S. C. and Nash, H. A. (1992) Deformation of DNA
during site-specific recombination of bacteriophage lambda: replacement of
IHF protein by HU protein or sequence-directed bends. Proc. Natl. Acad. Sci.,
U.S.A. 89: 11910-11914.

Goodrich, J. A., Schwartz, M. L. and McClure, W. R. (1990) Searching for and
predicting the activity of sites for DNA binding proteins: compilation and

analysis of the binding sites for Escherichia coli integration host factor (IHF).
Nucleic Acids Res. 18: 4993-5000.

Goosen, N. and van de Putte, P. (1995) The regulation of transcription initiation
by integration host factor. Mol. Microbiol. 16: 1-7.

Goshima, N ., Kano, Y., Tanaka, H., Tanaka, H., Kohno, K., Yasuzawa, K. and
Imamoto, F. (1992) Amino acid substitution in the C-terminal arm domain of
HU-2 results in an enhanced affinity for DNA. Gene 121: 121-126.

Goshima, N ., Kohno, K., Imamoto, F. and Kano, Y. (1990)HU-1 mutants of
Escherichia coli deficient in DNA binding. Gene 96: 141-145.

Haffter, P. and Bickle, T. A. (1987) Purification and DNA-binding properties of
FIS and Cin, two proteins required for the bacteriophage P1 site-specific
recombination system, cin. J. Mol. Biol. 198: 579-587.

Hanna, M. H. and Carl, P. L. (1975) Reinitiation of deoxyribonucleic acid
synthesis by deoxyribonucleic acid initiation mutants of Escherichia coli: role

of ribonucleic acid synthesis, protein synthesis, and cell division. J. Bacteriol.
121: 219-26.

Hansen, E. B. and Yarmolinsky, M. B. (1986) Host participation in plasmid
maintainance: Dependence upon dnaA of replicons derived from P1 and F.
Proc. Natl. Acad. Sci., U.S.A. 83: 4423-4427.

Hansen, F. G., Atlung, T., Braun, R. E., Wright, A., Hughes, P. and Kohiyama, M.
(1991) Initiator (DnaA) protein concentration as a function of growth rate in
Escherichia coli and Salmonella typhimurium. J. Bacteriol. 173: 5194-5199.

Hansen, F. G., Hansen, E. B. and Atlung, T. (1982) The nucleotide sequence of the
dnaA gene promoter and of the adjacent rpmH gene, coding for the ribosomal
protein L34, of Escherichia coli. EMBO J. 1: 1043-1048.

171

Hansen, F. G., Koefoed, S., Sorensen, L. and Atlung, T. (1987) Titration of DnaA
protein by oriC DnaA-boxes increases dnaA gene expression in Escherichia
coli. EMBO J. 6: 255-258.

Hansen, F. G. and von Meyenburg, K. (1979) Characterization of the dnaA, gyrB
and other genes in the dnaA region of the Escherichia coli chromsome on
specialized transducing phages ktna. Mol. Gen. Genet. 175: 135-144.

Hasunuma, K. and Sekiguchi, M. (1977) Replication of plasmid pSClOl in
Escherichia coli K12: requirement for dnaA function. Mol. Gen. Genet. 154:
225-30.

Heacock, P. N. and Dowhan, W. (1989) Alteration of the phospholipid
composition of Escherichia coli through genetic manipulation. J. Biol. Chem.
264: 14972-14977.

Heichman, K. A. and Johnson, R. C. (1990) The Hin invertasome: protein-
mediated joining of distant recombination sites at the enhancer. Science 249:
51 1-5 17 .

Helrnstetter, C.-B. (1994) Timing of Synthetic Activities in the Cell Cycle. p. 1594-
1605 in Neidhardt, F. C., Ingraham, J. L., Low, K. B., Magasanik, B.,
Schaechter, M. and Umbarger, H. E. (eds.), Escherichia coli and Salmonella
typhimurium. Cellular and Molecular Biology. American Society for
Microbiology, Washington, D. C.

Helrnstetter, C. E. (1968) DNA synthesis during the division cycle of rapidly
growing Escherichia coli B/r. J. Mol. Biol. 31: 507-518.

Helrnstetter, C. E. and Krajewski, C. A. (1982) Initiation of chromosome

replication in dnaA and dnaC mutants of Escherichia coli B/r F. J. Bacteriol.
149: 685-693.

Helrnstetter, C. E. and Leonard, A. C. (1987a) Coordinate initiation of
chromosome and minichromosome replication in Escherichia coli. J. Bacteriol.
169: 3489-3494.

Hehnstetter, C. E. and Leonard, A. C. (1987b) Mechanism for chromosome and
minichromosome segregation in Escherichia coli. J. Mol. Biol. 197: 195-204.

Herrick, J ., Kern, R., Guha, S., Landoulsi, A., Fayet, O., Malki, A. and Kohiyama, M.
(1994) Parental strand recognition of the DNA replication origin by the outer
membrane in Escherichia coli. EMBO J. 13: 4695-4703.

Herrick, J., Kohiyama, M., Atlung, T. and Hansen, F. G. (1996) The initiation mess?
Mol. Micro. 19: 659-666.

 

172

Hiasa, H. and Marians, K. J. (1994a) Fis cannot support oriC DNA replication in
vitro. J. Biol. Chem. 269: 24999-25003.

Hiasa, H. and Marians, K. J. (1994b) Topoisomerase IV can support oriC DNA
replication in vitro. J. Biol. Chem. 269: 16371-16375.

Holz, A., Schaefer, C., Gille, H., Jueterbock, W. R. and Messer, W. (1992)
Mutations in the DnaA binding sites of the replication origin of Escherichia
coli. Mol. Gen. Genet. 233: 81-88.

Hong, X., Cadwell, G. W. and Kogoma, T. (1995) Escherichia coli RecG and
RecA proteins in R-loop formation. EMBO J 14: 2385-2392.

Horiuchi, T., Maki, H. and Sekiguchi, M. (1984) RNase H-defective mutants of
Escherichia coli: a possible discriminatory role of RNase H in initiation of
DNA replication. Mol. Gen. Genet. 195: 17-22.

Hughes, P., Landoulsi, A. and Kohiyama, M. (1988) A novel role for CAMP in the
control of the activity of the E. coli chromosome replication initiator protein,
DnaA. Cell 55: 343-350.

Hupp, T. R. and Kaguni, J. M. (1993a) Activation of DnaA5 protein by GrpE and
DnaK heat shock proteins in initiation of DNA replication in Escherichia coli.
J. Biol. Chem. 268: 13137-13142.

Hupp, T. R. and Kaguni, J. M. (1993b) Activation of mutant forms of DnaA
protein of Escherichia coli by DnaK and GrpE proteins occurs prior to DNA
replication. J. Biol. Chem. 268: 13143-13150.

Hwang, D. S., Crooke, E. and Kornberg, A. (1990) Aggregated DnaA protein is
dissociated and activated for DNA replication by phospholipase or DnaK
protein. J. Biol. Chem. 265: 19244-19248.

Hwang, D. S. and Kaguni, J. M. (1988) Purification and characterization of the
dnaA46 gene product. J. Biol. Chem. 263: 10625-10632.

Hwang, D. S. and Kornberg, A. (1990) A novel protein binds a key origin
sequence to block replication of an E. coli minichromosome. Cell 63: 325-
33 l .

Hwang, D. S. and Kornberg, A. (1992a) Opening of the replication origin of
Escherichia coli by DnaA protein with protein HU or IHF. J. Biol. Chem. 267 :
23083-23086.

Hwang, D. S. and Kornberg, A. (1992b) Opposed actions of regulatory proteins,
DnaA and IciA, in opening the replication origin of Escherichia coli. J. Biol.
Chem. 267: 23087-23091.

 

173

Jacob, F., Brenner, S. and Cusin, F. (1963) On the regulation of DNA replication in
bacteria. Cold Spring Harbor Symp. Quant. Biol. 28: 329-348.

Jacq, A., Kern, R., Tsugita, A. and Kohiyama, M. (1989) Purification and .
characterization of a low-molecular-weight membrane protein w1th affimty for
the Escherichia coli origin of replication. J. Bacteriol. 171: 1409-1416.

J acq, A. and Kohiyama, M. (1980) A DNA-binding protein specific for the early
replicated region of the chromosome obtained from Escherichia coli
membrane fractions. Eur. J. Biochem. 105: 25-31.

J acq, A., Kohiyama, M., Lother, H. and Messer, W. (1983) Recognition sites for a
membrane-derived DNA binding protein preparation in the E. coli replication
origin. Mol. Gen. Genet. 191: 460-465.

Johnson, R. C., Ball, C. A., Pfeffer, D. and Simon, M. I. (1988) Isolation of the gene
encoding the Hin recombinational enhancer binding protein. Proc. Natl. Acad.
Sci., U.S.A. 85: 3484-3488.

Kaguni, J. M., Fuller, R. S. and Kornberg, A. (1982) Enzymatic replication of E.
coli chromosomal origin is bidirectional. Nature 296: 623-627.

Kaguni, J. M. and Kornberg, A. (1984) Replication initiated at the origin (oriC) of
the E. coli chromosome reconstituted with purified enzymes. Cell 38: 183-
190.

Kano, Y., Goshima, N., Wada, M. and Irnamoto, F. (1989) Participation of hup gene

product in replicative transposition of Mu phage in Escherichia coli. Gene
76: 353-358.

Katayama, T. and Crooke, E. (1995) DnaA protein is sensitive to a soluble factor
and is specifically inactivated for initiation of in vitro replication of the
Escherichia coli minichromosome. J. Biol. Chem. 270: 9265-9271.

Kobori, J. A. and Kornberg, A. (1982) The Escherichia coli dnaC gene product.

111. Properties of the DnaB- DnaC protein complex. J. Biol. Chem. 257 : 13770-
13775.

Koch, C. and Kahmann, R. (1986) Purification and properties of the Escherichia

coli host factor required for inversion of the G segment in bacteriophage Mu.
J. Biol. Chem. 261: 15673-15678.

Koch, C., Ninnemann, O., Fuss, H. and Kahmann, R. (1991) The N-terminal part of
the E. coli DNA binding protein FIS is essential for stimulating site-specific
DNA inversion but is not required for specific DNA binding. Nucleic Acids
Res. 19: 5915-5922.

 

174

Kogoma, T., Subia, N. L. and von Meyenburg, K. (1985) Function of ribonuclease
H in initiation of DNA replication in Escherichia coli K-12. Mol. Gen. Genet.
200: 103-109.

Kogoma, T., Torrey, T. A. and Connaughton, M. J. (1979) Induction of UV-
resistant DNA replication in Escherichia coli: induced stable DNA replication
as an SOS function. Mol. Gen. Genet. 176: 1-9.

Kohara, Y., Akiyama, K. and Isono, K. (1987) The physical map of the whole E.
coli chromosome: application of a new strategy for rapid analysis and sorting
of a large genomic library. Cell 50: 495-508.

Kohiyama, M., Cousin, D., Ryter, A. and Jacob, F. (1966) [Thermosensitive
mutants of Escherichia coli K 12. 1. Isolation and rapid characterization]. Ann.
Inst. Pasteur. (Paris) 110: 465-486.

Kohno, K., Yasuzawa, K., Hirose, M., Kano, Y., Goshima, N., Tanaka, H. and
Irnamoto, F. (1994) Autoregulation of transcription of the hupA gene in

Escherichia coli: evidence for steric hindrance of the functional promoter
domains induced by HU. J. Biochem. (Tokyo) 115: 1113-1118.

Kolling, R., Gielow, A., Seufert, W., Kucherer, C. and Messer, W. (1988) Aan, a
multifunctional regulator of genes located around the replication origin of
Escherichia coli, oriC. Mol. Gen. Genet. 212: 99-104.

Koppes, L. and Nordstrom, K. (1986) Insertion of an R1 plasmid into the origin of

replication of the E. coli chromosome: random timing of replication of the
hybrid chromosome. Cell 44: 117-124.

Koppes, L. J. and von Meyenburg, K. (1987) Nonrandom minichromosome
replication in Escherichia coli K-12. J. Bacteriol. 169: 430-433.

Kostrewa, D., Granzin, J ., Koch, C., Choe, H. W., Raghunathan, S., Wolf, W.,
Labahn, J ., Kahmann, R. and Saenger, W. (1991) Three-dimensional structure
of the E. coli DNA-binding protein FIS. Nature 349: 178-180.

Kostrewa, D., Granzin, J ., Stock, D., Choe, H. W., Labahn, J. and Saenger, W.
(1992) Crystal structure of the factor for inversion stimulation FIS at 2.0 A
resolution. J. Mol. Biol. 226: 209-226.

Kowalski, D. and Eddy, M. J. (1989) The DNA unwinding element: a novel, cis-

acting component that facilitates opening of the Escherichia coli replication
origin. EMBO J. 8: 4335-4344.

Kreuzer, K. N. and Cozzarelli, N. R. (1979) Escherichia coli mutants
thermosensitive for deoxyribonucleic acid gyrase subunit A: effects on

deoxyribonucleic acid replication, transcription, and bacteriophage growth. J.
Bacteriol. 140: 424-435.

 

175

Kucherer, C., Lother, H., Kolling, R., Schauzu, M. A. and Messer, W. (1986)
Regulation of transcription of the chromosomal dnaA gene of Escherichia
coli. Mol. Gen. Genet. 205: 115-121.

Kuwabara, M. D. and Sigman, D. S. (1987) Footprinting DNA-protein complexes
in situ following gel retardation assays using 1,10-phenanthroline-copper ion:

Escherichia coli RNA polymerase-lac promoter complexes. Biochem. 26:
7234-7238.

LaDuca, R. J ., Fay, P. J ., Chuang, C., McHenry, C. S. and Bambara, R. A. (1983)
Site-specific pausing of deoxyribonucleic acid synthesis catalyzed by four
forms of Escherichia coli DNA polymerase IH. Biochem. 22: 5177-5188.

Landoulsi, A., Hughes, P., Kern, R. and Kohiyama, M. (1989) Dam methylation
and the initiation of DNA replication on oriC plasmids. Mol. Gen. Genet. 216:
2 17-223.

Landoulsi, A., Malki, A., Kern, R., Kohiyama, M. and Hughes, P. (1990) The E. coli
cell surface specifically prevents the initiation of DNA replication at oriC on
hemimethylated DNA templates. Cell 63: 1053-1060.

Langer, U., Richter, S., Roth, A., Weigel, C. and Messer, W. (1996) A
comprehensive set of DnaA-box mutations in the replication origin, oriC, of
Escherichia coli. Mol. Micro. 21: 301-311.

Lark, K. G. (1972) Evidence for the direct involvement of RNA in the initiation of
DNA replication in Escherichia coli 15T. J. Mol. Biol. 64: 47-60.

Lark, K. G. and Renger, H. (1969) Initiation of DNA replication in Escherichia
coli 15T-: chronological dissection of three physiological processes required
for initiation. J. Mol. Biol. 42: 221-235.

Lavoie, B. D. and Chaconas, G. (1993) Site-specific HU binding in the Mu
transpososome: conversion of a sequence-independent DNA—binding protein
into a chemical nuclease. Genes & Dev. 7: 2510-2519.

Lavoie, B. D. and Chaconas, G. (1994) A second high affinity HU binding site in
the phage Mu transpososome. J. Biol. Chem. 269: 15571-15576.

LeBowitz, J. H. and McMacken, R. (1986) The Escherichia coli DnaB replication
protein is a DNA helicase. J. Biol. Chem. 261: 4738-48.

Leonard, A. C. and Helrnstetter, C. E. (1986) Cell cycle-specific replication of
Escherichia coli minichromosomes. Proc. Natl. Acad. Sci., U.S.A. 83: 5101-
5 105.

 

176

Leonard, A. C., Hucul, J. A. and Helrnstetter, C. E. (1982) Kinetics of

minichromosome replication in Escherichia coli B/r. J. Bacteriol. 149: 499-
507.

Liu, L. F. and Wang, J. C. (1987) Supercoiling of the DNA template during
transcription. Proc. Natl. Acad. Sci., U.S.A. 84: 7024-7027.

Lobner-Olesen, A., Atlung, T. and Rasmussen, K. V. (1987) Stability and

replication control of Escherichia coli minichromosomes. J. Bacteriol. 169:
2835-2842.

Lobner-Olesen, A., Hansen, F. G., Rasmussen, K. V., Martin, B. and Kuempel, P. L.
(1994) The initiation cascade for chromosome replication in wild-type and
Dam methyltransferase deficient Escherichia coli cells. EMBO J. 13: 1856-

1 862.

Lobner-Olesen, A., Skarstad, K., Hansen, F. G., von Meyenburg, K. and Boye, E.

(1989) The DnaA protein determines the initiation mass of Escherichia coli K-
12. Cell 57: 881-889.

Lother, H., Kolling, R., Kucherer, C. and Schauzu, M. (1985) DnaA protein-
regulated transcription: effects on the in vitro replication of Escherichia coli
minichromosomes. EMBO J. 4: 555-560.

Lother, H. and Messer, W. (1981) Promoters in the E. coli replication origin.
Nature 294: 376-378.

Louarn, J ., Funderburgh, M. and Bird, R. E. (1974) More precise mapping of the
replication origin in Escherichia coli K-12. J. Bacteriol. 120: 1-5.

Lu, M., Campbell, J. L., Boye, E. and Kleckner, N. (1994) Squ: a negative
modulator of replication initiation in E. coli. Cell 77: 413-426.

MacAllister, T. W., Kelley, W. L., Miron, A., Stenzel, T. T., and Bastia, D. (1991)
Cooperativity at a distance promoted by the combined action of two

replication initiator proteins and a DNA bending protein at the replication
origin of pSClOl. J. Biol. Chem. 266: 16056-16062.

Magee, T. R., Asai, T., Malka, D. and Kogoma, T. (1992) DNA damage-inducible
origins of DNA replication in Escherichia coli. EMBO J. 11: 4219-4225.

Maki, S. and Kornberg, A. (1988) DNA polymerase III holoenzyme of
Escherichia coli. HI. Distinctive processive polymerases reconstituted from
purified subunits. J. Biol. Chem. 263: 6561-6569.

Margulies, C. and Kaguni, J. M. (1996) Ordered and sequential binding of DnaA
protein to oriC, the chromosomal origin of Escherichia coli. J. Biol. Chem.
271: 17035-17040.

 

177

Marians, K. J ., Ikeda, J. E., Schlagman, S. and Hurwitz, J. (1977) Role of DNA
gyrase in phiX replicative-form replication in vitro. Proc. Natl. Acad. Sci.,
U.S.A. 74: 1965-1968.

Marinus, M. G. and Morris, N. R. (1974) Biological function for 6-methyladenine
residues in the DNA of Escherichia coli K12. J. Mol. Biol. 85: 309-322.

Marsh, R. C. and Worcel, A. (1977) A DNA fragment containing the origin of
replication of the Escherichia coli chromosome. Proc. Natl. Acad. Sci., U.S.A.
74: 2720-2724.

Marszalek, J. and Kaguni, J. M. (1994) DnaA protein directs the binding of DnaB

protein in initiation of DNA replication in Escherichia coli. J. Biol. Chem.
269: 4883-4890.

Marszalek, J ., Zhang, W., Hupp, T. R., Margulies, C., Carr, K. M., Cherry, S. and
Kaguni, J. M. (1996) Domains of DnaA Protein Involved in interaction with

DnaB protein, and in unwinding the Escherichia coli chromosomal origin. J.
Biol. Chem. 271: 18535-18542.

Masai, H. and Arai, K. (1987) RepA and DnaA proteins are required for initiation
of R1 plasmid replication in vitro and interact with the oriR sequence. Proc.
Natl. Acad. Sci., U.S.A. 84: 4781-4785.

Mathis, R. R. and Brown, 0. R. (1976) ATP concentration in Escherichia coli
during oxygen toxicity. Biochim. Biophys. Acta. 440: 723-732.

Matsui, M., Oka, A., Takanami, M., Yasuda, S. and Hirota, Y. (1985) Sites of DnaA
protein-binding in the replication origin of the Escherichia coli K-12
chromosome. J. Mol. Biol. 184: 529-533.

Meijer, M. and Messer, W. (1980) Functional analysis of minichromosome
replication: bidirectional and unidirectional replication from the Escherichia
coli replication origin, oriC. J. Bacteriol. 143: 1049-1053.

Mertens, G., Fuss, H. and Kahmann, R. (1986) Purification and properties of the
DNA invertase gin encoded by bacteriophage Mu. J. Biol. Chem. 261: 15668-
15672.

Messelson, M. and Stahl, F. (1958) The replication of DNA in E. coli. Proc. Natl.
Acad. Sci., U.S.A. 44: 671-682.

Messer, W. (1972) Initiation of deoxyribonucleic acid replication in Escherichia
coli B- r: chronology of events and transcriptional control of initiation. J.
Bacteriol. 112: 7-12.

178

Messer, W., Weigel, C. (1996) Initiation of chromosome replication. p. 1579-1601
in Neidhardt, F. C. (eds.),Escherichia coli and Salmonella. ASM press,
Washington, DC.

Messer, W., Bellekes, U. and Lother, H. (1985) Effect of Dam methylation on the
activity of the E. coli replication origin, oriC. EMBO J. 4: 1327-1332.

Messer, W., Bergmans, H. E., Meijer, M., Womack, J. E., Hansen, F. G. and von
Meyenburg, K. (1978) Minichromosomes: plasmids which carry the E. coli
replication origin. Mol. Gen. Genet. 162: 269-275.

Miki, T., Kimura, M., Hiraga, S., Nagata, T. and Yura, T. (1979) Cloning and
physical mapping of the dnaA region of the Escherichia coli chromosome. J.
Bacteriol. 140: 817-824.

Miller, H. I., Riggs, A. D. and Gill, G. N. (1973) Ribonuclease H (hybrid) in
Escherichia coli. Identification and characterization. J. Biol. Chem. 248:
2621 -2624.

Molina, L. J ., Govantes, F. and Santero, E. (1994) Geometry of the process of
transcription activation at the sigma 54- dependent nifl-I promoter of
Klebsiella pneumoniae. J. Biol. Chem. 269: 25419-25425.

Mukhopadhyay, G., Carr, K. M., Kaguni, J. M. and Chattoraj, D. K. (1993) Open-
complex formation by the host initiator, DnaA, at the origin of P1 plasmid
replication. EMBO J. 12: 4547-4554.

Murakarni, Y., Ohmori, H., Yura, T. and Nagata, T. (1987) Requirement of the
Escherichia coli dnaA gene function for ori-2- dependent mini-F plasmid
replication. J. Bacteriol. 169: 1724-1730.

Muskhelishvili, G., Travers, A. A., Heumann, H. and Kahmann, R. (1995) FIS and
RNA polymerase holoenzyme form a specific nucleoprotein complex at a
stable RNA promoter. EMBO J. 14: 1446-1452.

Nash, H. A. and Robertson, C. A. (1981) Purification and properties of the
Escherichia coli protein factor required for lambda integrative recombination.
J. Biol. Chem. 256: 9246-9253.

Newlands, J. T., Josaitis, C. A., Ross, W. and Gourse, R. L. (1992) Both fis-
dependent and factor-independent upstream activation of the rrnB P1
promoter are face of the helix dependent. Nucleic Acids Res. 20: 719-726.

Ninnemann, O., Koch, C. and Kahmann, R. (1992) The E. coli fis promoter is
subject to stringent control and autoregulation. EMBO J. 11: 1075-1083.

 

179

Nozaki, N., Okazaki, T. and Ogawa, T. (1988) In vitro transcription of the origin
region of replication of the Escherichia coli chromosome. J. Brol. Chem. 263:
14176-14183.

Numrych, T. E., Gumport, R. I. and Gardner, J. F. (1991) A genetic analysis of Xis
and FIS interactions with their binding sites in bacteriophage lambda. J.
Bacteriol. 173: 5954-5963.

Numrych, T. E., Gumport, R. I. and Gardner, J. F. (1992) Characterization of the
bacteriophage lambda excisionase (Xis) protein: the C-terminus is requrred for
Xis-integrase cooperativity but not for DNA binding. EMBO J. 11: 3797-
3806.

Ogasawara, N., Moriya, S., von Meyenburg, K., Hansen, F. G. and Yoshikawa, H.
(1985) Conservation of genes and their organization in the chromosomal
replication origin region of Bacillus subtilis and Escherichia coli. EMBO J.
4: 3345-3350.

Ogasawara, N. and Yoshikawa, H. (1992) Genes and their organization in the
replication origin region of the bacterial chromosome. Mol. Micro. 6: 629-634.

Ogawa, T., Baker, T. A., van der Ende, A. and Kornberg, A. (1985) Initiation of
enzymatic replication at the origin of the Escherichia coli chromosome:
contributions of RNA polymerase and primase. Proc. Natl. Acad. Sci., U.S.A.
82: 3562-3566.

Ogawa, T. and Okazaki, T. (1991) Concurrent transcription from the gid and mioC
promoters activates replication of an Escherichia coli minichromosome. Mol.
Gen. Genet. 230: 193-200.

Ogawa, T. and Okazaki, T. (1994) Cell cycle-dependent transcription from the gid
and mioC promoters of Escherichia coli. J. Bacteriol. 176: 1609-1615.

Ogawa, T., Pickett, G. G., Kogoma, T. and Kornberg, A. (1984) RNase H confers
specificity in the dnaA-dependent initiation of replication at the unique origin

of the Escherichia coli chromosome in vivo and in vitro. Proc. Natl. Acad.
Sci., U.S.A. 81: 1040-1044.

Ogden, G. B., Pratt, M. J. and Schaechter, M. (1988) The replicative origin of the

E. coli chromosome binds to cell membranes only when hemimethylated. Cell
54: 127-135.

Ohmori, H., Kimura, M., Nagata, T. and Sakakibara, Y. (1984) Structural analysis of
the dnaA and dnaN genes of Escherichia coli. Gene 28: 159-170.

Oka, A., Sasaki, H., Sugirnoto, K. and Takanami, M. (1984) Sequence organization
of replication origin of the Escherichia coli K- 12 chromosome. J. Mol. Biol.
176: 443-458.

 

180

Oka, A., Sugirnoto, K., Takanami, M. and Hirota, Y. (1980) Replication origin of
the Escherichia coli K-12 chromosome: the size and structure of the minimum
DNA segment carrying the information for autonomous replciation. Mol. Gen.
Genet. 178: 9-20.

Ortega, S., Lanka, E. and Diaz, R. (1986) The involvement of host replication
proteins and of specific origin sequences in the in vitro replication of
miniplasmid R1 DNA. Nucleic Acids Res. 14: 4865-4879.

Osuna, R., Finkel, S. E. and Johnson, R. C. (1991) Identification of two functional
regions in Fis: the N-terminus is required to promote Hin-mediated DNA
inversion but not lambda excision. EMBO J. 10: 1593-1603.

Parada, C. A. and Marians, K. J. (1991) Mechanism of DnaA protein-dependent
pBR322 DNA replication. DnaA protein-mediated trans-strand loading of the
DnaB protein at the origin of pBR322 DNA. J. Biol.. Chem. 266: 18895-

1 8906

Pettijohn, D. E. (1988) Histone-like proteins and bacterial chromosome structure.
J. Biol. Chem. 263: 12793-12796.

Pierucci, O., Helrnstetter, C. E., Rickert, M., Weinberger, M. and Leonard, A. C.
(1987) Overexpression of the dnaA gene in Escherichia coli B/r: chromosome

and minichromosome replication in the presence of rifampin. J. Bacteriol. 169:
1 87 1-1 877 .

Polaczek, P. (1990) Bending of the origin of replication of E. coli by binding of
IHF at a specific site. New Biol. 2: 265-271.

Polaczek, P. and Wright, A. (1990) Regulation of expression of the dnaA gene in
gcherichia coli: role of the two promoters and the DnaA box. New Biol. 2:
4-5 82.

Pontiggia, A., Negri, A., Beltrame, M. and Bianchi, M. E. (1993) Protein HU Binds
Specrfically to Kinked DNA. Mol. Micro. 7: 343-350.

Pritchard,.R. H., Barth, RT. and Collins, J. (1969) Control of DNA synthesis in
bacterra. Symp. Soc. Gen. Microbiol. 19: 263-297.

Pruss, G. J ., Manes, S. H. and Drlica, K. (1982) Escherichia coli DNA
toporsomerase I mutants: increased supercoiling is corrected by mutations near
gyrase genes. Cell 31: 35-42.

Ream, L. W., Margossian, L., Clark, A. J ., Hansen, F. G. and von Meyenburg, K.

(1980) Genetic and physical mapping of recF in Escherichia coli K—12. Mol.
Gen. Genet. 180: 115-121.

 

181

Rice, R. A., Yang, S., Mizuuchi, K. and Nash, H. A. (1996) Crystal structure of an
IHF-DNA complex: a protein-induced DNA U-tum. Cell 87: 1295-1306.

Richet, E., Abcarian, P. and Nash, H. A. (1986) The interaction of recombination
proteins with supercoiled DNA: defining the role of supercoiling in lambda
integrative recombination. Cell 46: 1011-1021.

Robinson, A. C., Collins, J. F. and Donachie, W. D. (1987) Prokaryotic and
eukaryotic cell-cycle proteins [letter]. Nature 328: 766.

Rokeach, L. A. and Zyskind, J. W. (1986) RNA terminating within the E. coli
origin of replication: stringent regulation and control by DnaA protein. Cell
46: 763-771.

Ross, W., Thompson, J. F., Newlands, J. T. and Gourse, R. L. (1990) E. coli FIS
protein activates ribosomal RNA transcription in vitro and in vivo. EMBO J.
9: 3733-3742.

Roth, A. and Messer, W. (1995) The DNA binding domain of the initiator protein
DnaA. EMBO J. 14: 2106-2111.

Roth, A., Urmoneit, B. and Messer, W. (1994) Functions of histone-like proteins in
the initiation of DNA replication at oriC of Escherichia coli. Biochimie 76:
917-923.

Rouviere, Y. J. and Gros, F. (1975) Characterization of a novel, low-molecular-
weight DNA-binding protein from Escherichia coli. Proc. Natl. Acad. Sci.
U.S.A. 72: 3428-3432.

Rouviere, Y. J ., Yaniv, M. and Gerrnond, J. E. (1979) E. coli DNA binding protein
HU forms nucleosomelike structure with circular double-stranded DNA. Cell
17: 265-274.

Russell, D. W. and Zinder, N. D. (1987) Hemirnethylation prevents DNA
replication in E. coli. Cell 50: 1071-1079.

Sakakibara, Y., Tsukano, H. and Sako, T. (1981) Organization and transcription of
the dnaA and dnaN genes of Escherichia coli. Gene 13: 47-55.

Sakakibara, Y. and Yuasa, S. (1982) Continuous synthesis of the dnaA gene
product of Escherichia coli in the cell cycle. Mol. Gen. Genet. 186: 87—94.

Samitt, C. E., Hansen, F. G., Miller, J. F. and Schaechter, M. (1989) In vivo studies

of DnaA binding to the origin of replication of Escherichia coli. EMBO J. 8:
989-993.

 

182

Schaefer, C. and Messer, W. (1988) Termination of the Escherichia coli aan
transcript. The DnaA protein/dnaA box complex blocks transcribing RNA
polymerase. Gene 73: 347-354.

Schaefer, C. and Messer, W. (1989) Directionality of DnaA protein/DNA .
interaction. Active orientation of the DnaA protein/dnaA box complex 1n
transcription termination. EMBO J. 8: 1609-1613.

Schaefer, C. and Messer, W. (1991) DnaA protein/DNA interaction. Modulation of
the recognition sequence. Mol. Gen. Genet. 226: 34-40.

Schaper, S. and Messer, W. (1995) Interaction of the initiator protein DnaA of
Escherichia coli with its DNA target. J. Biol. Chem. 270: 17622-17626.

Schaus, N., O'Day, K., Peters, W. and Wright, A. (1981) Isolation and
characterization of amber mutations in gene dnaA of Escherichia coli K-12. J.
Bacteriol. 145: 904-913.

Schauzu, M. A., Kucherer, C., Kolling, R., Messer, W. and Lother, H. (1987)
Transcripts within the replication origin, oriC, of Escherichia coli. Nucleic
Acids Res. 15: 2479-2497.

Segall, A. M., Goodman, SD, and Nash, HA. (1994) Architectural elements in
nucleoprotein complexes: interchangeability of specific and non-secific DNA
binding proteins. EMBO J. 13: 4536-4548.

Sekimizu, K., Bramhill, D. and Kornberg, A. (1987) ATP activates DnaA protein in
initiating replication of plasmids bearing the origin of the E. coli chromosome.
Cell 50: 259-265.

Sekimizu, K., Bramhill, D. and Kornberg, A. (1988a) Sequential early stages in the
in vitro initiation of replication at the origin of the Escherichia coli
chromosome. J. Biol. Chem. 263: 7124-7130.

Sekimizu, K. and Kornberg, A. (1988) Cardiolipin activation of DnaA protein, the
initiation protein of replication in Escherichia coli. J. Biol. Chem. 263: 7131-
7 1 35.

Sekimizu, K., Yung, B. Y. and Kornberg, A. (1988b) The DnaA protein of
Escherichia coli. Abundance, improved purification, and membrane binding.
J. Biol. Chem. 263: 7136-7140.

Shimizu, M., Miyake, M., Kanke, F., Matsumoto, U. and Shindo, H. (1995)
Characterization of the binding of HU and IHF, homologous histone-like

proteins of Escherichia coli, to curved and uncurved DNA. Biochim.
Biophys. Acta. 1264: 330-336.

 

183

Sigal, N., Delius, H., Kornberg, T., Gefter, M. L. and Alberts, B. (1972) A DNA-
unwinding protein isolated from Escherichia coli: its interaction wrth DNA
and with DNA polymerases. Proc. Natl. Acad. Sci., U.S.A. 69: 3537-3541.

Skarstad, K., Baker, T. A. and Kornberg, A. (1990) Strand separation required for
initiation of replication at the chromosomal origin of E. coli is facilitated by a
distant RNA-~DNA hybrid. EMBO J. 9: 2341-2348.

Skarstad, K., Boye, E. and Steen, H. B. (1986) Timing of initiation of chromosome

replication in individual Escherichia coli cells [published erratum appears in
EMBO J. 1986 Nov;5(1 1):3074]. EMBO J. 5: 1711-1717.

Skarstad, K., Lobner-Olesen, A., Atlung, T., von Meyenburg, K. and Boye, E.
(1989) Initiation of DNA replication in Escherichia coli after overproduction
of the DnaA protein. Mol. Gen. Genet. 218: 50—56.

Skarstad, K., Steen, H. B. and Boye, E. (1983) Cell cycle parameters of slowly

growing Escherichia coli B/r studied by flow cytometry. J. Bacteriol. 154:
656-662.

Skarstad, K., Steen, H. B. and Boye, E. (1985) Escherichia coli DNA distributions
measured by flow cytometry and compared with theoretical computer
simulations. J. Bacteriol. 163: 661-668.

Skarstad, K., Thony, B., Hwang, D. S. and Kornberg, A. (1993) A novel binding

protein of the origin of the Escherichia coli chromosome. J. Biol. Chem. 268:
5365-5370.

Skarstad, K., von Meyenburg, K., Hansen, F. G. and Boye, E. (1988) Coordination
of chromosome replication initiation in Escherichia coli: effects of different
dnaA alleles. J. Bacteriol. 170: 852-858.

Skovgaard, O. and Hansen, F. G. (1987) Comparison of dnaA nucleotide
sequences of Escherichia coli, Salmonella typhimurium, and Serratia
marcescens. J. Bacteriol. 169: 3976-3981.

Slater, S., Wold, S., Lu, M., Boye, E., Skarstad, K. and Kleckner, N. (1995) E. coli
Squ protein binds oriC in two different methyl-modulated reactions

appropriate to its roles in DNA replication initiation and origin sequestration.
Cell 82: 927-936.

Smith, D. W., Garland, A. M., Herman, G., Enns, R. E., Baker, T. A. and Zyskind, J.
W. (1985) Importance of state of methylation of oriC GATC sites in initiation
of DNA replication in Escherichia coli. EMBO J. 4: 1319-1326.

Stenzel, T. T., MacAllister, T. and Bastia, D. (1991) Cooperativity at a distance
promoted by the combined action of two replication initiator proteins and a

 

184

DNA bending protein at the replication origin of pSClOl [published erratum
appears in Genes Dev 1991 Dec;5(12A):2362]. Genes & Dev. 5: 1453-1463.

Stuitje, A. R., de Wind, N., van der Spek, J. C., Pors, T. H. and Meijer,.M. (1986)
Dissection of promoter sequences involved in transcriptronal activation of the
Escherichia coli replication origin. Nucleic Acids Res. 14: 2333-2344.

Stuitje, A. R. and Meijer, M. (1983) Maintenance and incompatibility of plasmids
carrying the replication origin of the Escherichia coli chromosome: evidence
for a control region of replication between oriC and asnA. Nucleic Acids Res.
11: 5775-5791.

Sun Lee, Y. and Su Hwang, D. (1997) Occlusion of RNA polymerase by
oligomerization of DnaA protein over the dnaA promoter of Escherichia coli.
J. Biol. Chem. 272: 83-88.

Surette, M. G., Lavoie, B. D. and Chaconas, G. (1989) Action at a distance in Mu
DNA transposition: an enhancer-like element is the site of action of
supercoiling relief activity by integration host factor (IHF). EMBO J. 8: 3483-
3489.

Sutton (1996) Genetic and biochemical dissection of functional domains of the
Escherichia coli replication initiation protein DnaA. PhD. dissertation

Svitil, A. L., Cashel, M. and Zyskind, J. W. (1993) Guanosine tetraphosphate
inhibits protein synthesis in vivo. A possible protective mechanism for
starvation stress in Escherichia coli. J. Biol. Chem. 268: 2307-2311.

Tanaka, H., Yasuzawa, K., Kohno, K., Goshima, N., Kano, Y., Saiki, T. and Irnamoto,
F. (1995) Role of HU proteins in forming and constraining supercoils of
chromosomal DNA in Escherichia coli. Mol. Gen. Genet. 248: 518-526.

Tanaka, I., Appelt, K., Dijk, J ., White, S.W., and Wilson, KS. (1984) 3-A resoultion
structure of a protein with histone-like roperties in prokaryotes. Nature 310:
376-3 8 1 .

Tanaka, M. and Hiraga, S. (1985) Negative control of oriC plasmid replication by
transcription of the oriC region. Mol. Gen. Genet. 200: 21-26.

Theisen, P. W., Grimwade, J. E., Leonard, A. C., Bogan, J. A. and Helrnstetter, C. E.
(1993) Correlation of gene transcription with the time of initiation of
chromosome replication in Escherichia coli. Mol. Micro. 10: 575-584.

Thompson, J. F. and Landy, A. (1988) Empirical estimation of protein-induced
DNA bending angles: applications to lambda site-specific recombination
complexes. Nucleic Acids Res. 16: 9687-9705.

 

185

Thompson, J. F., Moitoso, de Vargas, L., Koch, C., Kahmann, R. and Landy, A.
(1987) Cellular factors couple recombination with growth phase:
characterization of a new component in the lambda site-specific
recombination pathway. Cell 50: 901-908.

Thony, B., Hwang, D. S., Fradkin, L. and Kornberg, A. (1991) iciA, an Escherichia
coli gene encoding a specific inhibitor of chromosomal initiation of replication
in vitro. Proc. Natl. Acad. Sci., U.S.A. 88: 4066-4070.

Trueba, F. J ., Neijssel, O. M. and Woldringh, C. L. (1982) Generality of the growth
kinetics of the average individual cell in different bacterial populatrons. J.
Bacteriol. 150: 1048-1055.

van der Ende, A., Baker, T. A., Ogawa, T. and Kornberg, A. (1985) Initiation of
enzymatic replication at the origin of the Escherichia coli chromosome:

primase as the sole priming enzyme. Proc. Natl. Acad. Sci., U.S.A. 82: 3954-
3958.

von Freiesleben, U. and Rasmussen, K. V. (1992) The level of supercoiling affects
the regulation of DNA replication in Escherichia coli. Res. Microbiol. 143:
655-663.

von Freiesleben, U., Rasmussen, K. V. and Schaechter, M. (1994) Squ limits

DnaA activity in replication from oriC in Escherichia coli. Mol. Micro. 14:
763-772.

von Meyenburg, K., Hansen, F. G. (1980) The origin of replication, oriC, of
Escherichia coli chromsome: Genes near oriC and construction of oriC
deletion mutations. UCLA symp. Mol. Cell. Biol. 19: 137-159.

von Meyenburg, K., Boye, E., Skarstad, K., Koppes, L. and Kogoma, T. (1987)
Mode of initiation of constitutive stable DNA replication in RNase H-
defective mutants of Escherichia coli K-12. J. Bacteriol. 169: 2650-2658.

von Meyenburg, K. and Hansen, F. G. (1987) Regulation of chromosome
replication. p. 1555-1577 in Neidhardt, F. C., Ingraham, J. L., Low, K. B.,
Magasanik, B., Schaechter, M. and Umbarger, H. E. (eds.),Escherichia coli
and Salmonella typhimurium. Cellular and Molecular Biology. American
Society for Microbiology, Washington, D. C.

Wagg, J. C. (1996) DNA Topoisomerases. Annual Review of Biochemistry 65:
25-692.

Wang, Q. P. and Kaguni, J. M. (1987) Transcriptional repression of the dnaA gene
of Escherichia coli by DnaA protein. Mol. Gen. Genet. 209: 518-525.

Wang, Q. P. and Kaguni, J. M. (1989) DnaA protein regulates transcriptions of the
rpoH gene of Escherichia coli. J. Biol. Chem. 264: 7338-7344.

186

Wang, S., Cosstick, R., Gardner, J. F. and Gumport, R. I. (1995) The specific
binding of Escherichia coli integration host factor involves both major and
minor grooves of DNA. Biochem 34: 13082-13090.

Wechsler, J. A. (1975) Genetic and phenotypic characterization of dnaC
mutations. J. Bacteriol. 121: 594-599.

Wechsler, J. A. and Gross, J. D. (1971) Escherichia coli mutants temperature-
sensitive for DNA synthesis. Mol. Gen. Genet. 113: 273-284.

Wickner, S. and Hurwitz, J. (1975) Interaction of Escherichia coli dnaB and
dnaC(D) gene products in vitro. Proc. Natl. Acad. Sci., U.S.A. 72: 921-925.

Wold, S., Crooke, E. and Skarstad, K. (1996) The Escherichia coli FIS protein
prevents initiation of DNA replication from oriC in vitro. Nucleic Acids Res.
24: 3527-3532.

Wu, F., Goldberg, I. and Filutowicz, M. (1992) Roles of a 106-bp origin enhancer
and Escherichia coli DnaA protein in replication of plasmid R6K. Nucleic
Acids Res. 20: 811-817.

Xia, W. and Dowhan, W. (1995) Phosphatidylinositol cannot substitute for
phosphatidylglycerol in supporting cell growth of Escherichia coli. J.
Bacteriol. 177: 2926-2928.

Xu, J. and Johnson, R. C. (1995) Identification of genes negatively regulated by
Fis: Fis and RpoS comodulate growth-phase-dependent gene expression in
Escherichia coli. J. Bacteriol. 177: 938-947.

Xu, Y. C. and Bremer, H. (1988) Chromosome replication in Escherichia coli
induced by oversupply of DnaA. Mol. Gen. Genet. 211: 138-142.

Yang, C. C. and Nash, H. A. (1989) The interaction of E. coli IHF protein with its
specific binding sites. Cell 57: 869-880.

Yoshikawa, H. and Ogasawara, N. (1991) Structure and function of DnaA and the

DnaA-box in eubacteria: evolutionary relationships of bacterial replication
origins. Mol. Micro. 5: 2589-2597.

Yung, B. Y., Crooke, E. and Kornberg, A. (1990) Fate of the DnaA initiator
protein in replication at the origin of the Escherichia coli chromosome in
vitro. J. Biol. Chem. 265: 1282-1285.

Yung, B. Y. and Kornberg, A. (1988) Membrane attachment activates DnaA
protein, the initiation protein of chromosome replication in Escherichia coli.
Proc. Natl. Acad. Sci., U.S.A. 85: 7202-7205.

 

187

Yung, B. Y. and Kornberg, A. (1989) The DnaA initiator protein binds separate
domains in the replication origin of Escherichia coli. J. Biol. Chem. 264:
6146-6150.

Zulianello, L., de la Gorgue, de Rosny, E., P., v. U., van de Putte, P. and Goosen, N.
(1994) The HimA and HimD subunits of integration host factor can specifically
bind to DNA as homodimers. EMBO J. 13: 1534-15340.

Zyskind, J. W., Cleary, J. M., Brusilow, W. S., Harding, N. E. and Srrrith, D. W.
(1983) Chromosomal replication origin from the marine bacterium Vibrio

harveyi functions in Escherichia coli: oriC consensus sequence. Proc. Natl.
Acad. Sci., U.S.A. 80: 1164—1168.

Zyskind, J. W., Deen, L. T. and Smith, D. W. (1977) Temporal sequence of events
during the initiation process in Escherichia coli deoxyribonucleic acid

replication: roles of the dnaA and dnaC gene products and ribonucleic acid
polymerase. J. Bacteriol. 129: 1466-1475.

Zyskind, J. W., Smith, D. W. (1977) Novel Escherichia coli dnaB mutant: Direct
involvement of the dnaB252 gene product in the synthesis of an origin-
ribonulceic acid replication. J. Bacteriol. 129: 1476-1486.

HICHIGRN ST TE UNIV. LIBRRRIES
llllllllll1lllllllllllllllllllllllllllllllllllllllllll
31293015795929