Identification of a Region of Escherichia coli DnaB Required for

Functional Interaction with DnaG at the Replication Fork*

Pearl Chang‡ and Kenneth J. Marians‡§

‡Molecular Biology Graduate Program, Weill Graduate School of Medical

Sciences of Cornell University, New York, NY 10021

§Molecular Biology Program, Memorial Sloan-Kettering Cancer Center,

New York, NY 10021.

∗Supported by NIH grant GM34557

Running Title: DnaG Binding Pocket on DnaB

Copyright 2000 by The American Society for Biochemistry and Molecular Biology, Inc.

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SUMMARY

The fundamental activities of the replicative primosomes

of Escherichia coli are provided by DnaB, the replication fork

DNA helicase, and DnaG, the Okazaki fragment primase. As we

have demonstrated previously, DnaG is recruited to the

replication fork via a transient protein-protein interaction with

DnaB. Here, using site-directed amino acid mutagenesis, we

have defined the region on DnaB required for this protein-

protein interaction. Mutations in this region of DnaB affect the

DnaB-DnaG interaction during both general priming-directed

and X174 complementary strand DNA synthesis, as well as at

replication forks reconstituted in rolling circle DNA replication

reactions. The behavior of the purified mutant DnaB proteins

in the various replication systems suggests that access to the

DnaG binding pocket on DnaB may be restricted at the

replication fork.

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In bacteria, the DNA unwinding and Okazaki fragment-priming

functions at the replication fork are provided by a primosome, a

multienzyme conglomerate that moves processively along the lagging-

strand template (1). There are two primosomes in Escherichia coli, one

that forms in a DnaA-directed fashion at the chromosomal origin, oriC,

and one that forms at recombination intermediates to restart stalled or

aborted replication forks (2). The replicative primosome formed at oriC

requires DnaB, DnaC, and DnaG for assembly, whereas the replication

restart primosome [formerly the φX174-type primosome (3)], which can

form at D loops (4), requires PriA, PriB, DnaT, and possibly PriC, in

addition to the former three proteins, for assembly (3).

Primosomes provide both the DNA unwinding and Okazaki

fragment-priming functions of the replisome. In the case of each of the

bacterial primosomes, these activities are provided by DnaB and DnaG,

respectively. To form a replication fork, DnaB must be placed onto single-

stranded (ss)1 DNA that is coated with the single-stranded DNA-binding

protein (SSB). Whereas DnaB itself can bind to naked ssDNA, it is

prevented from doing so in vivo because it is found in a stoichiometric

complex with DnaC (5). DnaC, which has a cryptic ssDNA binding activity

that is activated when it is complexed with DnaB (6), can transfer DnaB to

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naked ssDNA, but not to SSB-coated DNA. This mechanism presumably

prevents promiscuous loading of DnaB to any region of the chromosome

that happens to become single-stranded. Thus, DnaB must be directed to

specific regions of the DNA by the action of other proteins that somehow

manage to create an SSB-free region of ssDNA. At oriC this is

accomplished by a protein-protein interaction between DnaA and DnaB

(7). During replication fork reactivation, PriA identifies the site for

restart primosome loading (2, 3, 8) and it is probably a protein-protein

interaction between DnaT and DnaB that mediates transfer of DnaB to

SSB-coated DNA (9).

Initial studies demonstrated that DnaG, which had been identified

as a primase (10, 11), was not present in restart primosomes formed in

the absence of DNA synthesis and isolated by gel filtration bound to

φX174 ss (circular) [ss(c)] DNA. In order for primer synthesis to occur,

DnaG had to be added back to those protein-DNA complexes (12, 13). We

showed that this was also the case at active replication forks, i. e., DnaG

did not remain permanently associated with the replication fork, rather, a

new molecule of DnaG was recruited from solution to synthesize the

primer for each new Okazaki fragment (14). This distributive action, with

respect to the cycle of Okazaki fragment synthesis, of DnaG at the

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replication fork acts to regulate the size of the nascent lagging-strand

fragments. Thus, Okazaki fragment size is inversely related to the

concentration of DnaG in the reaction mixture (14, 15).

Using partial proteolysis to resolve DnaG into independent domains,

we demonstrated that the C-terminal 16 kDa of the protein were not

required for primer synthesis, but were required for DnaG activity in any

replication assay that also required DnaB (16). Because the isolated C-

terminal fragment of DnaG could compete with the intact protein at the

replication fork and cause Okazaki fragment size to be altered, we

concluded that this domain mediated a protein-protein interaction

between DnaB and DnaG that acted to recruit DnaG to the replication

fork. Subsequent studies indicated that the C-terminal 16 amino acids of

DnaG were crucial to the interaction with DnaB (17). For example, at

identical concentrations, DnaG Q576A directs the synthesis of Okazaki

fragments that are at least 15-fold longer in size than those directed by

the wild-type protein (18).

Here we report the isolation of reciprocal mutations in DnaB that

specifically affect the DnaB-DnaG interaction at the replication fork. As

was the case with the mutant DnaG proteins, the mutant DnaB proteins

direct the synthesis of larger Okazaki fragments at the replication fork

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than the wild-type protein. These mutations lie in the N-terminal region

of DnaB, mapping very close together in the crystal structure (19), and do

not affect the ability of the mutant proteins to act as replication fork DNA

helicases. Interestingly, the mutant proteins display a different spectrum

of activities in a number of DNA replication systems that utilize DnaB,

suggesting that the DnaB-DnaG interaction at the replication fork is

further modulated by another factor.

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MATERIALS AND METHODS

Reagents, DNAs, Enzymes, and Replication Proteins—Restriction

enzymes were from Amersham. pET15b plasmid DNA was from Novogen.

Oligonucleotides were from Integrated DNA Technologies. Bacteriophage

f1AY-7/M and f1R229-A/33 ss(c) DNAs (20), as well as φX174 viral DNA,

were prepared as described previously (21). PriA, PriB, PriC, DnaT, DnaC,

and DnaG were purified as described (22). Subunits of the DNA

polymerase III holoenzyme (Pol III HE) were purified as indicated: core

(23), β (24), τ and γ (25), δ and δ′ by an unpublished procedure2, and χψ

(26) and were the kind gift of Dr. Charles McHenry (University of

Colorado, Denver). SSB was purified according to Minden and Marians

(27).

Construction of Mutated dnaB Alleles and Isolation of the Mutant

Proteins—The precise dnaB open reading frame was removed from pET3c-

dnaB (22) by digestion with NdeI and BamHI and inserted into NdeI- and

BamHI-digested pET15b to give pET15b-dnaB. This results in the addition

of 20 amino acids onto the N-terminus of DnaB. This tag includes a

hexahistidine sequence and a thrombin cleavage site. Mutant alleles

encoding the E32A, E32K, and Y105A amino acid substitutions in DnaB

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were engineered according to the Stratagene Quick Change technique as

per the manufacturer’s instructions. Mutated alleles were completely

sequenced before use.

For purification, BL21(DE3)pLysS (Novagen) carrying either a wild-

type or mutant pET21a-dnaB plasmid was grown in 12 liters of L broth

supplemented with 0.4% glucose and 0.5 mg/ml ampicillin to A600 = 0.4.

IPTG was then added to 0.4 mM and the synthesis of the target protein

was induced for 2 h. The cells were harvested and resuspended in 50 mM

Tris-HCl (pH 8.0 at 4 °C) and 10% sucrose to 50% w/v, frozen in liquid N2,

and stored at –80 °C.

Because of the extreme overproduction, DnaB was followed during

purification by SDS-PAGE. The cell suspension was thawed quickly and

brought to 1 mM PMSF, 0.1% Brij-58, and 0.2 mg/ml lysozyme. The

suspension was incubated briefly on ice as required for cell lysis and then

sedimented in the Sorvall A820 rotor at 37,000 rpm for 1 h at 4 °C. The

lysate was applied immediately to a 10 ml column of Ni-NTA-Agarose

(Qiagen) equilibrated in 50 mM Tris-HCl (pH 7.5 at 4 °C), 50 mM NaCl, 1

mM PMSF, and 10% sucrose. The column was then washed with two

column volumes of 50 mM Tris-HCl (pH 7.5 at 4 °C), 50 mM NaCl, 1 mM

PMSF, 10 mM imidazole-HCl (pH 8.0), and 10% glycerol. DnaB was eluted

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from the column with a 10-column volume gradient of 10-300 mM

imidazole-HCl (pH 8) in the same buffer. Fractions (0.5 ml) containing

DnaB were pooled and dialyzed overnight against ATP agarose buffer [50

mM Tris-HCl (pH 7.5 at 4 °C), 1 mM EDTA, 10 mM MgCl2,1 mM DTT, 0.1

mM PMSF, 50 mM NaCl, and 20% glycerol). The dialyzed fraction was

applied to and eluted from an ATP-agarose column, and dialyzed into

storage buffer as described previously (22). An SDS-PAGE gel of the

purified wild-type, E32A, E32K, and Y105A mutant DnaBs is shown in Fig.

1. Multiple experiments demonstrated that the N-His tag had essentially

no effect on DnaB activity. As an example, titrations comparing the

activity of wild-type DnaB and N-His DnaBE32A (a mutant DnaB with

activities indistinguishable from wild type, see Results) in the rolling

circle DNA replication assay is shown in Fig. 2.

X174 ss(c) Replicative Form (RF) DNA Replication and General

Priming—The standard reaction buffer was 50 mM HEPES-KOH (pH 8.0 at

30 °C), 10 mM MgOAc, 10 mM dithiothreitol, 0.01 mg/ml rifampicin, and

0.2 mg/ml bovine serum albumin. Both assays were stopped by the

addition of 100 µl of 0.2 M NaPPi. After addition of 100 µl of 1 mg/ml

heat-denatured salmon sperm DNA as carrier, trichloroacetic acid-

insoluble radioactivity was then determined. For the φX174 ss(c) → RF

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assay, reaction mixtures (25 µl) contained the standard buffer, φX174

ss(c) DNA (220 pmol as nt), 750 ng SSB, 1 mM ATP, 100 µM CTP, GTP, and

UTP, 40 µM dNTPs including [3H]dTTP (150 cpm/pmol),12 nM either wild-

type or mutant DnaB, 72 nM DnaC, 10 nM DnaT, 15 nM PriA, 15 nM PriB,

15 nM PriC, 10 nM DNA polymerase III HE (Pol III HE), and the indicated

concentrations of DnaG. Reactions were incubated at 30 °C for 10 min.

For the general priming assay, reaction mixtures (25 µl) contained the

standard buffer, φX174 ss(c) DNA (330 pmol as nt), 1 mM ATP, 200 µM

CTP, GTP, and UTP, 40 µM dNTPs including [3H]dTTP (150 cpm/pmol), 12

nM DnaB, 10 nM DNA Pol III HE, and the indicated concentrations of

DnaG. The reaction mixture was incubated at 30 °C for 15 min.

Rolling Circle DNA Replication—Tailed form II (TFII) DNA was

prepared as described by Mok and Marians (20). For rolling-circle DNA

replication with the complete restart primosome, reaction mixtures (12 µl)

containing 50 mM HEPES-KOH (pH 7.9), 12 mM MgOAc, 10 mM DTT, 5 µM

ATP, 80 mM KCl, 0.1 mg/ml BSA, 1.1 µM SSB, 0.42 nM TFII DNA, 3.2 nM

DnaB, 56 nM DnaC, 680 nM DnaG, 28 nM DnaT, 2.5 nM PriA, 2.5 nM PriB,

2.5 nM PriC, and 28 nM Pol III HE were preincubated at 30 °C for 2 min.

NTPs were added to final concentrations of 1 mM ATP, 200 µM GTP, 200

µM CTP, and 200 µM UTP, and dNTPs to 40 µM and the reaction was

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incubated for 2 min at 30 °C (stage 1). [α-32P]dATP (2000-4000

cpm/pmol) was added to the reaction mixture and the incubation was

continued at 30 °C for an additional 10 min (stage 2). For rolling circle

DNA replication with only DnaB and DnaG, reaction mixtures were

identical except that PriA, PriB, PriC, and DnaT were omitted and the

concentrations of DnaB and DnaC were increased to 80 nM and 1 µΜ,

respectively. In addition, the SSB was added along with the nucleotides

during the stage 1 incubation rather than at the start of the incubation.

DNA synthesis was quenched by addition of EDTA to 40 mM. Total DNA

synthesis was determined by assaying an aliquot of the reaction mixture

for acid insoluble radioactivity. DNA products were analyzed by alkaline

gel electrophoresis as described (20).

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RESULTS

Identification of Mutant DnaB Proteins Altered in their Interaction

with DnaG at the Replication Fork—DnaB activity is crucial to the proper

function of the replisome. Not only does the protein provide the DNA

unwinding necessary for replication fork propagation, it also serves to

attract DnaG to the replication fork via a protein-protein interaction. In

addition, another protein-protein interaction between DnaB and the τ

subunit of the DNA Pol III HE literally cements the replisome together,

stimulating the helicase activity of DnaB (28) and defining which of the

two polymerase cores in the holoenzyme becomes the leading-strand

polymerase (29, 30). Thus, understanding replication fork function

requires observation of the effects of disrupting these interactions. In

order to define the regions on DnaB that are involved in these important

protein-protein interactions, we have subjected dnaB to alanine-scanning

and charge-reversal mutagenesis. The mutated proteins are expressed,

purified by a combination of Ni-NTA agarose and ATP-agarose affinity

chromatography, and the initial screening of their biochemical phenotype

performed using rolling circle DNA replication.

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Rolling circle DNA replication is established on a tailed form II DNA

template by the addition of the replication restart primosomal proteins

(including the mutant DnaB under consideration), SSB, and the DNA Pol

III HE. We use this system as the initial screen because, as we have

documented previously (14, 20), it accurately mimics the behavior of the

cellular DNA replication fork. Moreover, the products of rolling circle

DNA replication are cleanly resolved by alkaline agarose gel

electrophoresis into a large leading-strand population that barely enters

the gel and a population of Okazaki fragments that is typically centered

about 1.5-2.5 kb in length. Thus, mutant DnaBs affected in the functions

described above can therefore easily be identified as a result of the

predicted effect on the products of the reaction.

When incorporated into the replisome, DnaB proteins that have

become modified in their ability to interact with DnaG should exhibit, at

identical concentrations of primase, a population of Okazaki fragments of

altered size compared to those made at replication forks reconstituted

with the wild-type protein. This is because, as described above, Okazaki

fragment size is controlled by the cycle of DnaG binding to and

dissociating from DnaB at the replication fork. Thus, any change in the

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affinity of this interaction will result in a change in the average size of the

population of Okazaki fragments synthesized.

We have identified two single amino acid substitutions in DnaB that

fulfill these predictions. These mutant DnaB proteins were culled from a

set of mutant proteins engineered by substituting charged amino acid

residues that were conserved among DnaB proteins on the assumption

that these residues were more likely to reside on the surface of the

protein and thus altering them might affect protein-protein interactions.

When wild-type DnaB was used to reconstitute rolling circle

replication, Okazaki fragment size reached its minimum as a function of

DnaG concentration between 100 and 200 nM (Fig. 3). In fact, the

average size of Okazaki fragments synthesized at these two concentrations

was nearly identical. Reduction of the DnaG concentration below 100 nM

resulted in a large increase in Okazaki fragment size, such that at 25 nM,

it was not possible to determine the average size of the fragments because

the population of lagging-strand products had merged with the

population of leading-strand products.

Replication forks reconstituted with DnaB E32A produced Okazaki

fragment populations that were identical to those made in the presence of

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the wild-type protein (Fig. 3), however, those containing DnaB E32K

consistently produced Okazaki fragments that were about 3-fold longer

than those synthesized by replication forks containing either the wild-

type or E32A DnaB (Fig. 3). Okazaki fragments produced by replication

forks containing DnaB Y105A were even longer (Fig. 3). Note that in Fig.

3, the lowest concentration of DnaG in the titration of DnaB Y105A is

nearly 90% greater than the highest value in the titration for either the

wild-type, E32A, or E32K DnaBs. And even at 3 µM DnaG, Okazaki

fragments synthesized by replication forks containing DnaB Y105A are

still larger than those synthesized by replication forks containing the

wild-type protein with DnaG at 100 nM. A conservative estimate is that at

equivalent concentrations of DnaG, the Okazaki fragments synthesized by

replication forks containing DnaB Y105A are at least 15-fold larger than

those synthesized by replication forks containing the wild-type DnaB.

Given that DnaB is also the replication fork DNA helicase, the

observed variation in Okazaki fragment size as a function of the DnaB

present at the fork could arise for one of two reasons. It could be, as

described above, that the mutations actually affected the affinity of the

protein-protein interaction between DnaB and DnaG. On the other hand,

it could also be that the mutations affected the rate of replication fork

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progression. The size of an Okazaki fragment is essentially the distance

on the lagging-strand template between two successful DnaG-primed

initiation events by the lagging-strand polymerase. Because in the rolling

circle system the nascent leading strand is the lagging-strand template,

Okazaki fragment size can also be made to vary at a fixed concentration of

DnaG by altering the rate at which the lagging-strand template is

generated, i. e., by altering the rate of DnaB-catalyzed unwinding at the

replication fork. Although we considered this explanation unlikely—in

this scenario DnaB Y105A would have to have at least a 15-fold greater

rate of DNA unwinding at the replication fork than the wild-type

protein—we compared the rate of replication fork progression for the

wild-type and mutant proteins directly.

The rate of replication fork progression sustained by replisomes

containing either the wild-type or mutant DnaBs was assessed by

sampling rolling circle replication reactions in 10 sec intervals from the

start of the incubation and analyzing the products by alkaline agarose gel

electrophoresis (Fig. 4). The change in the length of the longest leading

strand present is a direct measure of the rate of replication fork

movement. As evident in Fig. 4, the size of the nascent leading strand was

identical at each time point for the wild-type and three mutant DnaBs.

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We thus conclude that neither the E32A, E32K, nor Y105A amino acid

substitutions has affected, in any gross manner, the ability of that

particular DnaB to act as the replication fork DNA helicase. Thus, the

variation in Okazaki fragment size observed with replication forks

containing the mutant DnaB proteins is very likely the result of an

alteration of the affinity of the interaction between the mutated DnaB and

DnaG.

The Mutant DnaB Proteins Behave Differently in Single-stranded

DNA Priming Systems than they do at the Replication Fork—The results

described above suggested that the interaction between DnaG and DnaB

Y105A was more severely altered than the interaction between DnaG and

DnaB E32K. If this were the case, it should also hold true in the general

priming reaction where only DnaB and DnaG are present with the HE. In

this reaction, DnaB binds to the protein-free ss(c) DNA and then serves to

attract DnaG to synthesize a primer that is then elongated by the HE.

Alterations in the affinity of the interaction between DnaB and DnaG can

therefore be directly read out from the dose response curve of DnaG

concentration.

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Surprisingly, both the E32K and Y105A DnaBs behaved identically

in the general priming reaction (Fig. 5). At subsaturating levels of

primase, about 2- to 2.5-fold higher concentrations of DnaG were

required to support an equivalent amount of nucleotide incorporation as

wild-type DnaB when these two mutant proteins were present in the assay.

As expected, DnaB E32A did not exhibit any defect in this assay, if

anything, it might been somewhat more active than the wild type.

The general priming data would have predicted that both DnaB

E32K and DnaB Y105A would show similar defects at replication forks.

However, although at the same concentration of DnaG the lagging-strand

products formed in the presence of either mutant protein are clearly

larger than those formed in the presence of the wild-type, the Okazaki

fragments formed by the DnaB Y105 forks are much larger than those

formed by the DnaB E32K forks. We considered that this apparent

difference might be because there are probably more proteins present on

the DNA at replication forks formed in the rolling circle system, which

utilizes all the restart primosomal proteins, than in the general priming

system, which utilizes only DnaB and DnaG. We therefore compared the

activity of the mutant protein during synthesis of the complementary

strand of φX174 ss(c) DNA.

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In this assay, SSB-coated φX viral DNA is converted to the RF by the

formation of a restart primosome at the primosome assembly site. The

primosome catalyzes primer synthesis and the primer is elongated by the

HE to form the complementary strand. Once again, both the E32K and

Y105A DnaBs required higher concentrations of primase to sustain the

same level of nucleotide incorporation as the wild-type protein (Fig. 6).

In this case, the defect exhibited by DnaB Y105A was somewhat greater

than that exhibited by DnaB E32K. Thus, it was possible that the presence

of other primosomal proteins at the replication fork might alter the

interaction between DnaB and DnaG and exacerbate the effect of the

Y105A amino acid substitution. This predicts that the E32K and Y105A

DnaBs should behave identically at replication forks reconstituted in the

presence of only DnaB, DnaC, and DnaG.

The E32K and Y105A DnaBs Maintain their Differential Defects in

Replication Forks Formed only with DnaB and DnaG—Typically, we use all

the restart primosomal proteins to form replication forks in the rolling

circle system. This is because loading of DnaB to DNA by DnaC is

relatively inefficient. Auxiliary proteins are required to maximize the

process. At oriC, this is accomplished by DnaA, which has been shown to

interact with DnaB (31). Effectively, the combination of PriA, PriB, DnaT,

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and possibly PriC (32, 33) act as the equivalent of DnaA at the

primosome assembly site on φX viral DNA and at recombination

intermediates (8). However, replication forks can be formed in the rolling

circle system in the absence of PriA, PriB, PriC, and DnaT if the

concentration of DnaB and DnaC is increased 15- to 20-fold (20). In

addition, the reaction has to be staged somewhat differently because

DnaC cannot load DnaB to SSB-coated DNA . Thus, DnaB and DnaC are

exposed to the TFII template first for a short period of time and then SSB

is added.

Interestingly, the dramatic difference between the Y105A and E32K

DnaBs was maintained at replication forks formed in the absence of PriA,

PriB, PriC, and DnaT (Fig. 7). Replication forks formed in the presence of

the E32K protein consistently gave Okazaki fragments that were, at

equivalent concentrations of DnaG, about 2- to 3-fold longer than those

synthesized at replication forks formed in the presence of the DnaB E32A

(which is essentially identical to wild type). On the other hand, at the

DnaG concentrations shown, Okazaki fragments produced by replication

forks formed in the presence of DnaB Y105A were very long and could

barely be distinguished from the leading-strand DNA.

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In order to prove that Okazaki fragments were, in fact, being made

at replication forks formed in the presence of DnaB Y105A, the ability of a

restriction enzyme to digest the DNA products formed was examined.

BamHI will only digest the rolling circle DNA product if both leading- and

lagging-strand DNA had been synthesized, producing a duplex DNA tail.

This was the case when DNA made by replication forks containing wild-

type DnaB was treated with BamHI (Fig. 8, lanes 1 and 2). In the absence

of primase, so no Okazaki fragments could be synthesized, DNA made by

replication forks formed with DnaB Y105A was resistant to BamHI

treatment (Fig. 8, lanes 3 and 4). As the concentration of primase was

increased, DNA made by replication forks containing DnaB Y105A became

progressively more sensitive to BamHI digestion, being essentially

completely digested at 200 nM primase (Fig. 8, lanes 5-10). Thus, it is

clear that even though they could not be distinguished from leading-

strand DNA under these conditions, Okazaki fragments were being made.

These data therefore suggest that either the architecture of the

replication fork itself or some interaction between DnaB and a polymerase

subunit restricts or modifies access to the DnaG binding pocket on DnaB

that is defined by the E32A and Y105A amino acid substitutions.

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DISCUSSION

The interaction between DnaB and DnaG is of crucial importance to

the replisome. These two proteins form the core of the replicative

primosome, providing both the DNA unwinding function, via the 5′ → 3′

DNA helicase activity of DnaB, and the Okazaki fragment priming

function, via the oligoribonucleotide synthetase activity of DnaG,

necessary for proper replication fork propagation.

In E. coli, the DnaB-DnaG interaction is transient (1). At first glance,

this seems to create an inefficiency at the replication fork. Because

Okazaki fragments are an average of about 2 kb in length and the speed

of replication fork propagation is nearly 1000 nt/sec, a new primer for

lagging-strand DNA synthesis must be manufactured at least once every

two sec. Thus, it would seem reasonable to expect that the primase would

remain permanently associated with the replisome, waiting to synthesize a

new primer as soon as it was needed. However, this is not the case. At

the E. coli replication fork, DnaG acts distributively with respect to a cycle

of Okazaki fragment synthesis (14). That is, a molecule of DnaG

associates with the replication fork via a protein-protein interaction with

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DnaB (17), synthesizes a primer, and then leaves the fork to be replaced

by a different molecule of DnaG that will synthesize the next primer.

The cyclical association of DnaG with the replication fork proved to

be a regulatory feature governing the size of Okazaki fragments (15, 18).

This is because the size of an Okazaki fragment is determined by the

distance between two successful initiation events by the lagging-strand

polymerase on the lagging-strand template and the frequency of primer

synthesis is governed by the cycle of association/dissociation of DnaG

with DnaB (18). Thus, a complete understanding of replisome function

requires a thorough understanding of the dynamics of this protein-

protein interaction.

We have previously reported our determination that the C-terminal

16 amino acids of DnaG were crucial for the functional interaction

between DnaG and DnaB at the replication fork (17). We demonstrated

that single amino acid substitutions in this region affected the period of

the Okazaki fragment clock, leading to the synthesis of Okazaki fragments

of altered size compared to those directed by the wild-type enzyme. Here

we have use directed single amino acid substitutions to localize the

reciprocal region of DnaB.

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Two mutant DnaB proteins were described that exhibited, when

incorporated into replication forks, an alteration in the size of Okazaki

fragments synthesized when compared to those synthesized at identical

concentrations of DnaG by replication forks that contained the wild-type

protein. The two amino acid residues mutated, Glu32 and Tyr105, lie very

close together in the crystal structure of the N-terminal domain of DnaB

(Fig. 8). This structure includes amino acid residues 15-128, although the

first residue for which electron density can be observed is Pro26 (19). The

structured is dimeric, with each monomer composed of six α helices, five

of which are wrapped around a central helix. The overall dimensions of

the structure are 25 Å x 25 Å x 35 Å, consistent with the size of the

globular vertices observed by electron microscopy (37, 38). Both Glu32

and Tyr 105, aspects of which are as close together as 7 Å, are surface

exposed residues. Thus, it would appear that these amino acid residues

contribute to a binding pocket for DnaG on DnaB.

The interaction between DnaB and DnaG is difficult to observe

physically. DnaG does not remain associated with the restart primosome

when it is isolated on φX174 DNA (12, 13, 33), nor can the interaction be

detected by either gel filtration chromatography or glycerol gradient

sedimentation. Interestingly, the interaction between DnaB and DnaG

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from Bacillus stearothermophilus is very stable at room temperature and

can be detected by gel filtration (34). Presumably, this interaction is less

stable at the normal growth temperature of this thermophile. The

interaction between the E. coli proteins has been observed by affinity

matrix chromatography and ELISA (35). However, these techniques give

only relative descriptions of the binding and are very difficult to

quantitate. Our preliminary data using surface plasmon resonance

suggests that the E32K and Y105A amino acid substitutions do alter the

binding affinity between the mutant DnaB and DnaG (data not shown).

Previous studies have addressed assignment of the various activities

of DnaB to particular regions of the protein. Nakayama et al. (36)

demonstrated, using partial proteolysis, that DnaB was composed of a N-

terminal domain of about 12 kDa, named fragment 3, corresponding

roughly to amino acid residues 14-136, a C-terminal domain of about 33

kDa, named fragment 2, corresponding roughly to amino acid residues

172-470, and a linker region between these two domains. Electron

microscopic examination of the structure of DnaB confirm that a single

protomer of the DnaB hexamer appears with two globular domains, one

large and one small, connected by a hinge (37, 38).

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Fragment 2 appears to provide the primary hexamerization contacts

with fragment 3 providing additional stabilization via dimer contacts.

And, indeed, as described above, the crystal structure of what is

essentially fragment 3 is that of a dimer. Fragment 2 is responsible for

DNA binding and ATPase activity, whereas both fragments 2 and 3 are

required for helicase activity (39, 40). Based on the relative activity of

the DnaB fragments in general priming, φX174 complementary strand

synthesis, and protection of DnaC from inactivation by NEM, the initial

structure-function studies of Nakayama et al. (36) suggested that

fragment 3 was also the site of binding to both DnaG and DnaC.

Previous studies have yielded some information on the region of

DnaB involved in the interaction with DnaG. In an investigation of the

role of the linker region, Stordal and Maurer (41) found that purified

Salmonella typhimurium mutant DnaB proteins carrying the I135N,

I141T, and L156P amino acid substitutions were all defective in the

general priming reaction. Unfortunately, none of these amino acid

residues are present in the fragment 3 crystal structure of Fass et al. (19),

so their proximity to the region defined in this report cannot be assessed.

However, given that hinge regions are, by definition, very flexible, it is

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certainly possible that these amino acid residues are involved in

determining the DnaG binding pocket as well.

Lu et al. (35) assessed the ability of in vitro translated, truncated

derivatives of DnaB to be retained by an N-terminal glutathione S-

transferase-DnaG chimera bound to a glutathione affinity resin, and

concluded that the region between amino acid residues 211 and 256,

which falls in fragment 2, was important for binding of DnaG. Based on

these data, these authors constructed three double mutants: D212A,

D213A; K216A, K217A; and D253A, K254A and assayed their ability to

interact with DnaB by ELISA. Only the former two mutant DnaB proteins

exhibited a defect in binding DnaG. These mutant proteins also exhibited

a decreased ability to sustain primer synthesis on M13 ss(c) DNA in the

presence of DnaG.

Given the lack of a crystal structure of the entire DnaB molecule, it

is, of course, difficult to determine whether the region encompassing

amino acid residues 212-217 is anywhere near the region defined by Glu32

and Y105. Existing evidence would argue that these two regions of the

protein were actually quite distant from each other. Studies by Egelman

et al. (42) have shown that the bacteriophage T7 gene 4 helicase/primase,

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which is a member of the DnaB family of helicases (43), is a bilobed

molecule oriented such that the N-terminal primase domain is a small

toroid abutting the C-terminal helicase region, which is a large toroid. In

other words, the primase doughnut sits on top of the helicase doughnut

and at the replication fork, the lagging-strand template is likely to run

through the center of the toroidal structure.

Sawaya et al. (44) have solved the crystal structure of a portion of

the helicase domain of the bacteriophage gene 4 protein. Although this

structure is corkscrew-like and not hexameric, a hexameric projection can

be made. When mapped to this projection, the region including amino

acid residues 212-217 is likely to be near the C-terminal face of the

helicase domain of DnaB and not near the N-terminal face, which would

presumably abut the region defined by Glu32 and Y105. If these

speculations prove accurate, it is difficult to see how the regions on DnaB

defined by Glu32 and Tyr105 and the region including amino acid residues

212-217 could come together to participate in the same DnaG binding

pocket. On the other hand, it should be noted that in the case of the B.

stearothermophilus DnaB, Bird et al. (34) concluded that the DnaG

interaction surface was composed of regions from both the N- and C-

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terminal domains of the protein. Complete resolution of this issue awaits

more crystal structures of DnaB.

Although at equivalent concentrations of DnaG, replication forks

reconstituted with DnaB Y105A manufactured significantly larger Okazaki

fragments than forks reconstituted with DnaB E32K, both mutant proteins

exhibited the same quantitative defect in the general priming reaction,

which is presumably a better direct test of the affinity of the interaction

between DnaB and DnaG than is the intact fork. Even though there was

some suggestion that when all the components of the restart primosome

were present, the defect exhibited by DnaB Y105A was more severe than

that exhibited by DnaB E32K, the possible presence of PriA, PriB, PriC, and

DnaT at the replication fork could not be used to explain the difference

between the two mutant DnaB proteins. This is because the differential

defect was maintained in replication forks reconstituted in the presence

of only the mutant DnaB, DnaC, and DnaG. These observations suggest

that, at the replication fork, access to the DnaG binding pocket on DnaB

that includes Glu32 and Tyr105 is either restricted by interaction with either

SSB or one of the subunits of the polymerase or that a protein-protein

interaction between either SSB or a polymerase subunit and DnaB alters

the affinity of the interaction between DnaB and DnaG.

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FOOTNOTES

1 The abbreviations used are: ss, single stranded; SSB, the E. coli single-

stranded DNA-binding protein; ss(c), single-stranded circular; RF,

replicative form; Pol III HE, the E. coli DNA polymerase III holoenzyme;

TFII, tailed form II.

2 Olson, M., Carter, J., Dallmann, H. G., and McHenry, C. S., personal

communication.

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ACKNOWLEDGMENTS

We thank James Berger for providing the coordinates of the structure of

DnaB fragment 3.

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Figure Legends

Fig. 1. SDS-PAGE analysis of the wild-type and mutant DnaB

proteins. One microgram of the wild-type (lane 1), E32A (lane 2), E32K

(lane 3), and Y105A (lane 4) DnaB proteins was analyzed by SDS-PAGE

through a 10% gel. The gel was stained with Coomassie Brilliant Blue and

the image recorded using a BioRad Gel Doc imaging system. The faint

bands present in all lanes represent proteolytic products corresponding to

fragments 1 and 2 (36).

Fig. 2. Comparison of the activity of N-His-tagged and wild-

type DnaB. Standard rolling circle replication reactions containing

either wild-type or N-His-DnaB E32A at the indicated concentrations

(DnaB concentration increases 3-fold in lanes 1-5 and 6-10 from left to

right) were performed and analyzed as indicated under “Materials and

Methods.” A, alkaline agarose gel electrophoresis of the reaction

products. B, graphic analysis of the incorporation of [−32P]dAMP into acid-

insoluble product for each reaction shown in panel A. r—r, wild type;

m—m, N-His-DnaB E32A.

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Fig. 3. The E32K and Y105A mutant DnaB proteins display

an altered response to variation of the concentration of

primase. A, standard rolling circle replication reactions containing the

indicated DnaB protein and varying concentrations of DnaG (increasing 2-

fold from left to right) were incubated, processed, and analyzed as

described under “Materials and Methods.” B, phosphorimager traces of

the DNA products made at 100 nM DnaG for the wild-type, E32A, and

E32K DnaB proteins and at 750 nM DnaG for DnaB Y105A.

Fig. 4. The E32K and Y105A amino acid substitutions in

DnaB do not affect the rate of replication fork progression.

Standard rolling circle replication reactions containing the indicated DnaB

proteins were incubated at 30 °C. Aliquots (2 µl) were withdrawn at the

indicated times from the start of the incubation and the reactions

quenched by rapid mixing with 50 mM EDTA (10 µl). DNA products were

analyzed by alkaline agarose gel electrophoresis as described under

“Materials and Methods.”

Fig. 5. Activity of the wild-type and mutant DnaB proteins

in general priming. Standard general priming reactions containing

either the wild-type or mutant DnaB proteins and the indicated

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concentrations of DnaG were incubated and analyzed as described under

“Materials and Methods”. The left-hand panel is an exploded view of the

right-hand panel. Wild-type DnaB, r—r; DnaB E32A, ◊—◊; DnaB E32K,

¦—¦; DnaB Y105A, ∆—∆.

Fig. 6. Activity of the wild-type and mutant DnaB proteins

in X174 complementary strand synthesis. Standard φX174

complementary strand synthesis reactions containing either the wild-type

or mutant DnaB proteins and the indicated concentrations of DnaG were

incubated and analyzed as described under “Materials and Methods”.

The right-hand panel is an exploded view of the left-hand panel. Wild-

type DnaB, r—r ; DnaB E32A, ◊—◊; DnaB E32K, ¦—¦ ; DnaB Y105A, ∆—∆

.

Fig. 7. The E32K and Y105A mutant DnaB proteins maintain

their differential defects at replication forks formed only with

DnaB and DnaG. Rolling circle replication reactions containing the TFII,

SSB, the Pol III HE, DnaC, the indicated DnaB, and varying concentrations

of DnaG (increasing 2-fold from left to right) were incubated, processed,

and analyzed as described under “Materials and Methods.”

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Fig. 8. Okazaki fragments are made at replication forks

containing DnaB Y105A. Rolling circle replication reactions

containing the TFII DNA template, SSB, the Pol III HE, DnaC, the indicated

concentration of DnaG, and either wild-type or Y105A DnaB were

incubated for 10 min at 30 °C. The reactions were terminated by heating

at 65 °C for 10 min. Each reaction was then divided in two and one half

was treated with the BamHI restriction endonuclease. The DNA products

were then analyzed by alkaline agarose gel electrophoresis.

Fig. 9. Location of Glu32 and Tyr 105 on the crystal structure

of DnaB fragment 3. A space-filling representation of the structure of

DnaB fragment 3. Amino acid residues involved in the dimer interface are

colored magenta. Tyr105 is yellow and Glu32 is green. The figure was made

using RasMac 2.6. Note that Fass et al. (19) assign Ala2 as the N-terminal

amino acid of DnaB. Thus, in their structure, the amino acids residues

referred to here as Tyr105 and Glu32 are listed as Tyr104 and Glu31.

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Pearl Chang and Kenneth J. Marianswith DnaG at the replication fork

Identification of a region of Escherichia coli DnaB required for functional interaction

published online May 31, 2000J. Biol. Chem. 

  10.1074/jbc.M001800200Access the most updated version of this article at doi:

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