Available online at www.sciencedirect.com

Bacterial replicases and related polymerases

Charles S McHenry

Bacterial replicases are complex, tripartite replicative replication that are conserved among all life forms are

machines. They contain a polymerase, Pol III, a b2 processivity illustrated in Figure 1.

factor and a DnaX complex ATPase that loads b2 onto DNA

and chaperones Pol III onto the newly loaded b2. Many Structure and function of a, the catalytic

bacteria encode both a full length t and a shorter g form of subunit

DnaX by a variety of mechanisms. The polymerase catalytic Like all polymerases, Pol III a contains palm, thumb and

subunit of Pol III, a, contains a PHP domain that not only binds fingers domains, in the shape of a cupped right hand

2+

to prototypical e Mg -dependent exonuclease, but also Figure 2. However, apo-enzyme structures of the full

2+

contains a second Zn -dependent proofreading length Thermus aquaticus (Taq) and a truncated version of

exonuclease, at least in some bacteria. Replication of the E. coli (Eco) a subunit revealed a big surprise: the palm

chromosomes of low GC Gram-positive bacteria require two domain has the basic fold of the X family of DNA

Pol IIIs, one of which, DnaE, appears to extend RNA primers a polymerases, which includes the slow, non-processive

only short distance before handing the product off to the major Pol bs [1 ,2 ].

replicase, PolC. Other bacteria encode a second Pol III (ImuC)

that apparently replaces Pol V, required for induced A ternary complex of a dideoxy-terminated primer-tem-

mutagenesis in E. coli. Approaches that permit simultaneous plate, incoming dNTP and full length Taq a provided

biochemical screening of all components of complex bacterial significant insight into the function of Pol III a [3 ].

replicases promise inhibitors of specific protein targets and Among the template-primer induced conformational

reaction stages. changes are movement of the thumb domain toward the

DNA bound by the palm to make contacts with the sugar-

Address phosphate backbone in the minor groove. The polymerase

Department of Chemistry and Biochemistry, University of Colorado,

contacts the template from its terminus to a position 12

Boulder 80309, United States

nucleotides behind the primer terminus. The fingers

domain creates a wall at the end of the primer terminus

Corresponding author: McHenry, Charles S

([email protected]) that forces a sharp kink in the emerging template strand.

In the ternary complex structure of a Gram-positive Pol

Current Opinion in Chemical Biology 2011, 15:587–594

III, two novel elements, not present in Eco or Taq a, were

2+

This review comes from a themed issue on identified [4]. The four-Cys Zn binding motif, shown

Molecular Machines earlier by Neal Brown and colleagues to be required for

Edited by Stephen Benkovic and Kevin D. Raney

activity, serves an apparently structural function and is

not part of the catalytic site. DNA binding through the

Available online 19th August 2011

thumb domain comes primarily from two b strands that

1367-5931/$ – see front matter interact with the minor groove [4].

# 2011 Elsevier Ltd. All rights reserved.

PHP domain

DOI 10.1016/j.cbpa.2011.07.018

A PHP domain (initially identified as having homology to

phosphoesterases) is found in a wide variety of bacterial

polymerases, including bacterial Pol bs (Figure 2). Initially,

Introduction it was proposed that this region might be involved in

Cellular chromosomal replicases from all branches of life pyrophosphate hydrolysis, but such an activity has not

are tripartite. They contain a polymerase (Pol III in been found [1]. This domain, at least in Taq Pol III,

2+

bacteria and Pol d and e in eukaryotes), a sliding clamp contains a second proofreading activity that is Zn -de-

2+

processivity factor (b2 in bacteria and PCNA in eukar- pendent [5 ] and also binds the classical Mg -based proof-

yotes), and a clamp loader (DnaX complex (DnaXcx) in reading subunit, e [6]. The structure of Taq a revealed a

bacteria and RFC in eukaryotes). By themselves, repli- cluster of nine residues in the PHP domain that chelate

cative polymerases do not exhibit special properties that three metal ions [2]. A structure of a Gram-positive PolC

distinguish them from other polymerases, but together PHP domain also binds three metals, using the same nine

with the sliding clamp and clamp loader they become ligands expected from the homologous Taq PHP structure.

highly processive. b2 forms a ring that surrounds the Kuriyan and colleagues, from the structure of Eco a,

DNA template and tethers the polymerase to it, enabling pointed out a channel between the polymerase active site

processive replication. Other features of bacterial and the proposed PHP active site [1].

www.sciencedirect.com Current Opinion in Chemical Biology 2011, 15:587–594

588 Molecular Machines

Figure 1

(a) (b) (c) 5´ 3´ β (E) 2 RNA (D) δ DnaG SSB ´ (A) DnaX Pol III Primer χψ γ δ Primase Complex

Dna Xcx τ τ Primase (C) (B) DnaB SSB β β χ 3´ 2 2 Helicase ψ αεθ 5´ δ δ´ β Helicase α α γ τ τ ε ε Pol III αεθ θ θ β 5´ 3´

Current Opinion in Chemical Biology

DNA Polymerase III Holoenzyme contacts at the replication fork. (a) A hexameric helicase translocates down the lagging strand template, splitting two

strands apart in advance of the leading strand replicase, Pol III HE. Single-stranded regions of the lagging strand template are coated by SSB. Primase

interacts with the helicase and synthesizes short RNA primers for Okazaki fragment synthesis that are extended by the Pol III HE until a signal is

received to recycle to the next primer synthesized at the replication fork. For clarity, this view is drawn with a discrete DnaXcx on each Pol III; they are

actually shared between the leading and lagging strand polymerase (dotted line). (b) Details of known subunit interactions within the Pol III HE. In

addition, there is a transient interaction between d and, perhaps, additional DnaXcx subunits with b2 during the clamp loading reaction. (c) A cartoon of

the replication fork showing relevant protein-protein interactions, including dimerization of the leading and lagging strand polymerases through contact

of domain V of t with a. A contact between domain IV of two ts and two DnaB protomers anchors the replicase to the helicase, placing all replication

fork components into one replisome.

b2 binding domain the b1–b2 loop were changed to serine [8]. The proces-

A structure of Taq a revealed a well-organized b2-binding sivity of the mutant polymerase was decreased, an effect

domain with a HhH motif that together with its flanking that was rescued by the presence of the t-complex [8].

loops binds dsDNA [3] (Figure 2). The b2 binding con- The latter observation would seem to suggest that

sensus sequence is presented in a loop that is oriented although the OB-fold contributes to ssDNA affinity

adjacent to dsDNA as it exits the polymerase, in the and processivity, it is not the processivity sensor, or at

correct position to bind b2 as it surrounds DNA. The b2 least that the residues mutated are not the key interactors.

binding domain rotates 208 and swings down into position

as the enzyme binds DNA [3], a reorientation that is In fact, the entire polymerase active site may function as

apparently driven energetically by the HhH motif bind- the processivity switch [9 ]. Steitz and colleagues [3] have

ing to DNA and probably coupled to conformational elegantly demonstrated a conformational change induced

changes of the thumb, palm, OB fold, and PHP domains. by substrate binding in which several elements move that

include placement of the b2 binding domain in a position

OB-fold domain where it can productively interact with the b2 clamp on

The structure of the ternary complex of Taq a with DNA. The geometry and spatial constraints around the

primer-template and incoming dNTP reveals a striking active site when the exiting template is double-stranded

conformational change that includes the OB-fold mov- might make insertion of the last nucleotide energetically

ing to a position near the single-strand template distal unfavorable. Upon insertion, the product might lose

to the primer [3]. The path of the emerging template, affinity for the active site, triggering a reversal of the

which can be traced from electron density of the ribose- conformational changes that occurred upon primer-tem-

phosphate backbone, appears to come close to the OB- plate and dNTP binding, switching the polymerase to a

fold. The element of the OB-fold that comes closest to low-processivity mode.

the ssDNA template, the b1–b2 loop, often contributes

to ssDNA binding [7]. However, the b1–b2–b3 face t-binding domain

that commonly interacts with ssDNA [7] appears to face The second half of the C-terminal domain in the Taq a

away from the emerging template, toward the t-binding structure revealed a domain containing an incompletely

domain. conserved sequence that binds weakly to b2, but is not

required for processive replication in vitro or function in

A test of the importance of the OB-fold motif was made vivo. This domain is loosely packed against the OB fold,

using a mutant in which three basic residues located in with many polar residues in the interface [2] (Figure 2).

Current Opinion in Chemical Biology 2011, 15:587–594 www.sciencedirect.com

Bacterial replicases McHenry 589

Figure 2

(a) 778 511 964 272 1078 1160 430 560 D555 D401, 403 php palm thumb palm fingers β binding OB-fold τ binding β HhH binding loop

(b) PHP

Fingers Thumb Palm

OB fold

β-binding loop β binding τ binding

Current Opinion in Chemical Biology

Modular organization of Pol III a. (a) The residue numbers that define domain borders in E. coli a are shown above the bar in black. The positions of the

three catalytic aspartates are indicated. The locations of the b binding loop and the dsDNA binding HhH motif are shown below the bar. (b) A space-

filling representation of the Taq Pol III a structure [2 ], with the domains colored the same as in (a). The view is to the side where the dsDNA product

emerges from the central channel.

Mutational studies support the importance of this sub- Pre-steady state kinetic studies of the b2 loading reaction

domain in binding t [10]. Possible sites of interaction of have been performed by Bloom and colleagues. ATP

this extreme C-terminal domain with t, derived from a serves as an allosteric effector, increasing the affinity of

genetic screen, have been reviewed recently [9]. the DnaXcx for b2 and DNA, bringing all reaction partici-

pants together [11 ]. In a study investigating the rate of

Mechanism of clamp loading and initiation binding of b2 and primed DNA to gcx, it was found that b2

complex formation bound to gcx-ATP at a diffusion-controlled rate. By con-

Before DNA elongation begins, the Pol III holoenzyme trast, primed DNA bound slowly (limited by the slow

(HE) forms an initiation complex in an ATP-dependent ATP-induced conformational change within the gcx of

1

reaction. This reaction is often artificially divided into an 3.3 s ) [11 ]. At replication forks, an ordered mechan-

ATP-dependent clamp loading reaction in which a b2 ring ism may be further enforced by the availability of primers

is loaded around DNA and a subsequent association of for DNA replication that are only synthesized ca. every

Pol III with the loaded clamp. Sliding clamps require 2 s. Another investigation addressed the relative order of

ATP-powered clamp loaders for their assembly around b2 release, primer-template release and ATP hydrolysis

DNA. The E. coli clamp loader is composed of seven in the final stages of the gcx-catalyzed loading reaction

subunits (Figure 1). The ATP binding subunit that drives [12 ]. Once a gcx–b2–ATP complex bound DNA,

assembly is encoded by dnaX, which directs the synthesis hydrolysis of three ATPs was triggered, followed by

of two proteins, t and g, by programmed ribosomal DNA release, then b2. It is not yet known when b2

frameshifting. The shorter g contains three domains; t becomes closed in this process.

contains two additional domains (termed IV and V,

respectively) that bind the replicative helicase DnaB6 The x and c subunits of the DnaXcx perform several

and Pol III. functions. The Bloom laboratory demonstrated important

www.sciencedirect.com Current Opinion in Chemical Biology 2011, 15:587–594

590 Molecular Machines

functional consequences that correlate nicely with a determine if a cryptic interaction occurs at the analogous

structure determined by Kuriyan and colleagues that site in E. coli.

reveals conformational changes induced by the inter-

action of an N-terminal peptide of c with g3 in a complex Hydrolysis of an ATP by each DnaX protomer may not be

0

with dd [13,14 ]. It was concluded that c stabilizes an required for initiation complex formation

ATP-induced conformational state that binds DNA [13]. Current models propose that the E. coli DnaXcx binds and

The structure also reveals how binding is restricted to a hydrolyzes three ATPs in a synchronous wave during the

single copy of xc: the N-terminus of c snakes through a clamp loading process [18]. Recently, both the g and t

collar of three DnaX domain IIIs in an asymmetric way forms of DnaX with the crucial Lys in the Walker A motif

with different contacts with each, but in a way that changed to a Glu have been purified [19 ]. Together with

precludes further c association [14 ]. their wild-type counterparts, these mutants have been

assembled into DnaX complexes and the ten possible

Binding of the c peptide stabilizes a conformation that resulting complexes purified and characterized. Surpris-

binds the ATP analog, ADP-BeF3, in all three DnaX sites ingly, complexes containing only one active ATP binding

[14 ]. DNA binding favors a similar conformational DnaX protomer function in initiation complex formation.

change. This contrasts with structures in the absence This contradicts proposals regarding unique functions of

of c where the DnaX subunit C (Figure 1b) is in a individual DnaX protomers or the requirement for a

conformation that cannot bind ATP. The DNA–ADP– synchronous wave of ATP hydrolysis to be absolutely

0

BeF3–g3dd –c peptide structure adopts a ‘notched screw required for b2 loading.

cap’ conformation in which the ATPase subunits are

symmetrically oriented in a spiral arrangement and con- However, a very recent pre-steady-state kinetic study has

tacts are made with the phosphates of the template strand confirmed that ATP hydrolysis at multiple sites is

through basic residues and a-helical dipoles. No contacts required for the fastest rates of initiation complex for-

are made with the primer, other than a stack between d mation [20 ]. A DnaX complex containing only two active

Tyr316 and the terminal base of the primer. An interest- DnaX ATPases drives initiation complex formation 30-

ing conjecture was made that this arrangement would fold more slowly than a complex with three active

allow for accommodating both RNA and DNA primers ATPases. This same study showed that the benefit from

[14 ]. It will be interesting to see, in future studies, chaperoning by t-containing DnaX complexes is 100-fold.

whether b2 is open when bound to this assembly and Thus, a complex containing t and only two active ATPase

whether the ATP sites remain equivalent. subunits supports complex formation faster than a three-

ATPase g complex, the often used model assembly for b2

DnaX complex chaperones Pol III onto newly loaded b2 loading studies. It is likely that the presence of t and

Recently, it has been demonstrated that t-containing chaperoning changes the kinetic pathway for initiation

DnaX complexes serve another role in initiation com- complex formation [20 ].

plex formation: they chaperone the associated polymer-

ase onto the b2 just loaded by the same complex [15 ]. Possible new mechanism for recycling b2 at the

Chaperoning significantly increases the rate of initiation replication fork

complex formation and drops the Pol III concentration Until recently, it has been thought at Pol III*, when it

required for efficient assembly. Both of these features cycles from a completed Okazaki fragment, leaves the old

are probably crucial for the cell to sustain a rate of b2 behind and that a new b2 needs to be loaded on the

initiation complex formation sufficient to support the next primer [21]. That may still be the most frequent

rate of Okazaki synthesis required for chromosomal event, but recent single-molecule experiments suggest

replication. that b2 might be recycled during Okazaki fragment

synthesis [22]. Elongating complexes synthesize Okazaki

Initiation complex formation with t-containing DnaXcx fragments in the absence of added b2 in a flow system

is stimulated by SSB, suggesting a role for SSB beyond where unbound b2 was washed away. The authors

that of protecting the DNA template. The only known acknowledge that an alternative interpretation could be

SSB-interacting protein within Pol III HE is x. The x- that the lagging strand polymerase could function without

SSB interaction has been shown to be important for b2 under the conditions they used [22]. Indeed, the

allowing Pol III to replicate SSB-coated DNA and for presence of t-containing DnaX complexes and inter-

stabilizing Pol III on DNA in strand displacement reac- action of x with SSB coating the lagging strand stabilizes

tions [16]. Yet, the stimulation by SSB can occur in the the replicase on replication forks [16]. The effective high

absence of x, revealing that an additional, undiscovered local concentration of the lagging strand polymerase

site for SSB interaction exists [15 ]. Polard and col- created by its association with the leading strand poly-

leagues have detected a stable interaction between B. merase through a t–t bridge within the DnaX complex

subtilis (Bsu) SSB and Bsu DnaE [17]. It would be inter- might support a b-less reaction. Thus, definitive testing

esting to identify the SSB binding site in Bsu DnaE and for the presence of b2 with the lagging strand replicase

Current Opinion in Chemical Biology 2011, 15:587–594 www.sciencedirect.com

Bacterial replicases McHenry 591

under the conditions used should be pursued in future level of synthesis greater than that achieved by DnaE

experiments. alone [26]. Using a specific PolC inhibitor (HBEMAU)

[28] that traps PolC in a dead end complex, drastic

DnaX complex composition and assembly within cells inhibition is observed when Pol C is added to DnaE

The DnaX complex can be constructed, in vitro, to contain RNA primer extension reactions, indicating the handoff

any combination of the t and g dnaX gene products [23]. A to PolC occurs early in the reaction [26].

trimeric Pol III HE containing only t was constructed in

vitro and was shown to function in reconstituted rolling In E. coli, under appropriate conditions, Pol II, Pol IV and

circle reactions [24]. A suggestion that it might represent Pol V can invade elongating Pol III HE and gain access to

the authentic replicase was made even though this form of the primer terminus [29,30]. Initially, a toolbelt model

the enzyme has never been isolated in preparations was proposed in which multiple polymerases might bind

obtained from cells. All published preparations of Pol III to multiple sites in an oligomeric sliding clamp proces-

HE obtained from cells contain both t-subunits and g- sivity factor and switch out at the replication fork. How-

subunits [9]. A recent report using Slimfield fluorescence ever, interactions of polymerases with sliding clamps are

microscopy, a technique that can visualize replication weak when the polymerase cannot gain access to the

proteins tagged with YPet within foci of live cells, primer terminus to acquire binding energy, and the off-

suggested three Pol IIIs and three ts are present per rates are fast. Furthermore, Sutton and colleagues have

replisome [25]. The error was large enough to make this provided evidence that would limit polymerase inter-

determination ambiguous (3.1 1.1 as, for example). The action to one set of adjacent sites on one half of the b2

interpretation was that the replicase contains three ts and sliding clamp [31]. So, if b2 is the initial contact with an

three Pol IIIs, but the potential for other Pol III assemblies exogenous polymerase, other interactions and contacts

being in the vicinity performing mismatch or gap proces- must be present to drive, and perhaps regulate, the

sing was not considered. Interestingly, mutation of dnaX to exchange process. Goodman, Maki and colleagues have

eliminate g increased the t stoichiometry 30% [25]—the provided evidence that, in addition to the known E. coli

amount expected with a Pol III HE dominated by a t2g Pol IV-b2 interaction, additional contacts between Pol IV

stoichiometry. Other issues with interpretation and quanti- and a component of Eco Pol III* (Pol III + t-containing

fication are discussed elsewhere [9]. DnaXcx) triggers polymerase release [32]. Future research

in this area requires an investigation of the role of the

Replication of Gram-positive bacteria uses DnaXcx in the polymerase exchange mechanism.

two DNA polymerase IIIs

In low-GC Gram-positive bacteria, two Pol IIIs exist, Many bacteria use a special mutagenic Pol III

termed PolC and DnaE. They are homologous, but PolC in place of Pol V

has some of its domains rearranged, and it contains an In E. coli, a specialized class of PolY polymerases serves

endogenous proofreading activity. DnaE is more closely the role of induced mutagenesis and stress-induced adap-

related to E. coli Pol III. A rolling circle replication system tive modifications. With the sequencing of multiple bac-

using 13 purified B. subtilis replication proteins has been terial genomes, it has become apparent that many bacteria

reconstituted [26 ]. This system appears to accurately have two E. coli-like dnaEs in their genomes (not in-

mimic the reaction at the replication fork of a Gram- cluding the PolC/DnaE combinations), with the second

positive bacterium, in terms of both its correspondence one apparently replacing Pol V, the major polymerase

with genetic requirements and the replication fork rate in responsible for induced mutagenesis in E. coli [33,34,35 ].

vivo. Leading strand replication requires 11 proteins,

including the Pol III encoded by polC. In addition to In the most advanced studies in this area to date, it has

these 11 proteins, lagging strand replication requires been demonstrated that in Mycobacterium tuberculosis

DnaE and primase [26]. This is consistent with proposals (Mtb), ImuB interacts with the replicative DnaE and

for a lagging strand role for DnaE [27]. However, the ImuC (previously called DnaE2, see Table 1) and ImuA

elongation rate of DnaE is too slow (25 nt/s) to keep up [36 ]. ImuB, in spite of being closely homologous to

with the replication fork. By contrast, PolC supports a PolY error-prone polymerases, does not contain the triad

physiologically relevant elongation rate (500 nt/s). PolC of conserved Pol III catalytic acidic residues [36] and thus

discriminates against RNA primers; DnaE uses RNA must be inactive as a polymerase. Mutation of ImuC’s

primers efficiently [26]. These characteristics suggest a predicted catalytic Asp residues ablates function

role for Bsu DnaE analogous to eukaryotic Pol a that (induced mutagenesis). Thus, ImuC is the error-prone

extends RNA primers initially and then hands them off to polymerase in Mtb and presumably in other organisms

a processive replicase. that contain ImuA/B/C and lack Pol V homologs. Yet,

ImuB interacts with b2, but ImuC does not. Thus, it

Consistent with the eukaryotic Pol a role, model systems appears that ImuB serves an important gatekeeper role,

using RNA primed ssDNA show inefficient use by PolC, interacting with b2, the replicative DnaE, and the error

with a marked stimulation by low levels of DnaE to a prone polymerase ImuC.

www.sciencedirect.com Current Opinion in Chemical Biology 2011, 15:587–594

592 Molecular Machines

Table 1

Three classes of DNA Polymerase IIIs

Model Organism Name (or proposed name) Former Names Function Accessory Factors

0

E. coli a, DnaE PolC Extension of RNA primers b2, DnaXcx (t2 gdd xc)

Rapid, processive chromosomal replication

0

B. subtilis PolC DnaF Rapid, processive chromosomal replication b2, DnaXcx (t3dd )

DnaE Extension of RNA primers b2, DnaXcx

P. aeruginosa DnaE Extension of RNA primers b2, DnaXcx

Rapid, processive chromosomal replication

ImuC DnaE2 Error-prone replication, induced mutagenesis b2, DnaXcx?, ImuA, ImuB

There has been considerable confusion in the literature peptides that bind to and inhibit the b2 sliding clamp and

about the relationship between DnaE-like polymerases DnaI helicase loader [39,40]. And, coliphage N4 produces

that coexist in PolC containing strains and those that a peptide inhibitor of E. coli DnaXcx that functions

coexist with replicative DnaEs. To provide criteria for through interaction with the d subunit [41 ]. Crystal

distinguishing them, a comparison was made of the con- structures of complexes of these peptides and their tar-

sensus sequences resulting from a comprehensive ImuC gets should provide data that could support library design

alignment with an alignment of DnaEs that exist alone or and development of small molecule inhibitors.

with PolCs. Both of the latter are very similar and readily

distinguishable from ImuC. Key differences map to the Conclusions

active site and the end of the DNA binding channel [35]. There have been several recent advances in our under-

If these changes relax substrate binding in a way that standing of the structure and function of bacterial repli-

diminishes fidelity and permits bypass, this would appear cases. Structures of the apoenzyme and ternary complex

to be consistent with the error-prone function attributed forms of Pol III offer novel insights into the functions of a

to ImuC. replicase and interaction with its processivity factor. These

structures probably will provide a starting point for future

Chemical biology of DNA replication work to understand the structural basis of interaction with

DNA replication is an essential process for the prolifer- DnaXcx, formation of initiation complexes upon the

ation of all pathogens and offers a largely unexplored initiation of Okazaki fragment synthesis and regulated

target for development of novel antibacterials. Most of dissociation upon completion, and partitioning of the pri-

the subunits of the bacterial DNA replication apparatus mer terminus to proofreading exonucleases.

are essential, suggesting that their inhibition should lead

to blockage of cell proliferation or death. This has been New insight has emerged into the mechanism of

validated by a class of compounds, 6-anilinouracils, tar- initiation complex formation both in terms of the order

geted to the polymerase subunit of the Gram-positive of assembly and the discovery that the E. coli clamp loader

replicase, PolC. These compounds are not only potent also functions to chaperone the associated polymerase

biochemical inhibitors, but specifically block DNA repli- onto the newly loaded clamp. Future work will probably

cation in Gram-positive bacteria [37]. While screens tar- reveal if a reversal of this chaperoning reaction is respon-

geting individual replicase subunits have been described, sible for dissociation of elongating complexes upon the

complete bacterial replicases have been explored only completion of Okazaki fragment synthesis and polymer-

recently by chemical genetic approaches [38]. In a trial ase exchange at the replication fork.

screen with a small (20,000-compound) library against full

replication systems derived from model Gram-negative Reconstitution of a prototypical Gram-positive rolling

and Gram-positive organisms in parallel, it was possible to circle replication system that mimics the enzymology

distinguish compounds that inhibited the replicase of a of fork progression revealed the function of a second

single species from compounds that exhibited broad replicative polymerase: the extension of RNA primers

spectrum potential. Counterscreens against non-ortholo- before handoff to the principle replicase. Such a function

gous enzymes with related activities revealed those com- was previously thought to be reserved to Pol a in eukar-

pounds that are most likely to be target-specific. yotic systems. Future research will probably focus on the

mechanism of the handoff between two polymerases, a

Another source of useful in vitro inhibitors to support reaction that may provide a general prototype for poly-

mechanistic studies derives from the discovery of bac- merase exchange during ongoing replication. Yet other

teriophages that express peptides directed toward shut- bacteria contain two Pol IIIs for a different purpose: the

ting down cellular processes, including DNA replication. second Pol III replaces a PolY polymerase for purposes of

For example, some Staphylococcus aureus phages produce induced mutagenesis. The development of high through-

Current Opinion in Chemical Biology 2011, 15:587–594 www.sciencedirect.com

Bacterial replicases McHenry 593

12. Anderson SG, Thompson JA, Paschall CO, O’Donnell M,

put screens that include all proteins of complete replica-

Bloom LB: Temporal correlation of DNA binding, ATP

tion systems promises specific small molecules that block hydrolysis, and clamp release in the clamp loading reaction

catalyzed by the Escherichia coli gamma complex.

these reactions at specific stages, providing new tools for

Biochemistry 2009, 48:8516-8527.

biochemical and cell physiological investigations. The order of dissociation of components from the DnaXcx upon ATP

hydrolysis during the b loading reaction is addressed.

Acknowledgements 13. Anderson SG, Williams CR, O’Donnell M, Bloom LB: A function

I thank Diane Hager for preparation of figures and Melissa Stauffer, PhD, of for the c subunit in loading the Escherichia coli DNA

Scientific Editing Solutions, for editing the manuscript. Work from the polymerase sliding clamp. J Biol Chem 2007, 282:7035-7045.

author’s laboratory was supported by Grant RO1 GM060273 from the

14. Simonetta KR, Kazmirski SL, Goedken ER, Cantor AJ, Kelch BA,

National Institute of General Medical Sciences and Grant MCB-0919961

McNally R, Seyedin SN, Makino DL, O’Donnell M, Kuriyan J: The

from the National Science Foundation.

mechanism of ATP-dependent primer-template recognition

by a clamp loader complex. Cell 2009, 137:659-671.

This paper is the first report of an apparently active DnaXcx. It shows all

References and recommended reading

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Papers of particular interest, published within the period of review,

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c subunit to the DnaXcx and reveals important conformational changes

induced by this interaction.

of special interest

15. Downey CD, McHenry CS: Chaperoning of a replicative

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clamp by the clamp loader. Mol Cell 2010, 37:481-491.

This paper reveals that the DnaXcx clamp loader also chaperones the

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The structure of T. aquaticus DNA

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polC reveals unique DNA binding and fidelity determinants. Chem 2010, 285:29049-29053.

Proc Natl Acad Sci USA 2008, 105:20695-20700. This paper shows that DnaXcxs that contain fewer than three active

ATPases can function in initiation complex formation.

5. Stano NM, Chen J, McHenry CS: A coproofreading Zn(2+)-

dependent exonuclease within a bacterial replicase. Nat Struct 20. Downey CD, Crooke E, McHenry CS: Polymerase chaperoning

Mol Biol 2006, 13:458-459. and multiple ATPase sites enable the E. coli DNA polymerase

2+

This work represents the first report of a novel Zn -dependent second III holoenzyme to rapidly form initiation complexes. J Mol Biol

proofreading activity within Pol III. 2011, in press. doi:10.1016/j.jmb.2011.07.051.

This paper quantifies the kinetic advantage in initiation complex formation

6. Wieczorek A, McHenry CS: The NH(2)-terminal php domain of

through chaperoning and multiple ATPases. It shows that the slow ATPgS

the a subunit of the E. coli replicase binds the e proofreading

supported initiation complex formation is accompanied by hydrolysis.

subunit. J Biol Chem 2006, 281:12561-12567.

21. Stukenberg PT, Turner J, O’Donnell ME: An explanation for

7. Theobald DL, Mitton-Fry RM, Wuttke DS: Nucleic acid

lagging strand replication: polymerase hopping among DNA

recognition by OB-fold proteins. Annu Rev Biophys Biomol

sliding clamps. Cell 1994, 78:877-887.

Struct 2003, 32:115-133.

22. Tanner NA, Tolun G, Loparo JJ, Jergic S, Griffith JD, Dixon NE, van

8. Georgescu RE, Kurth I, Yao NY, Stewart J, Yurieva O, O’Donnell M:

Oijen AM: E. coli DNA replication in the absence of free beta

Mechanism of polymerase collision release from sliding

clamps. EMBO J 2011, 30:1830-1840.

clamps on the lagging strand. EMBO J 2009,

28:2981-2991. 23. Pritchard AE, Dallmann HG, Glover BP, McHenry CS: A novel

assembly mechanism for the DNA polymerase III holoenzyme

9. McHenry CS: DNA replicases from a bacterial perspective. 0

DnaX complex: association of dd with DnaX(4) forms

Annu Rev Biochem 2011, 80:403-436. 0

DnaX(3)dd . EMBO J 2000, 19:6536-6545.

This review includes models for a new assembly mechanism of the

DnaXcx within cells and polymerase cycling upon completion of Okazaki 24. McInerney P, Johnson A, Katz F, O’Donnell M: Characterization

fragment synthesis. of a triple DNA polymerase replisome. Mol Cell 2007,

27:527-538.

10. Dohrmann PR, McHenry CS: A bipartite polymerase-

processivity factor interaction: only the internal b binding site 25. Reyes-Lamothe R, Sherratt DJ, Leake MC: Stoichiometry and

of the a subunit is required for processive replication by the architecture of active DNA replication machinery in

DNA polymerase III holoenzyme. J Mol Biol 2005, 350:228-239. Escherichia coli. Science 2010, 328:498-501.

11. Thompson JA, Paschall CO, O’Donnell M, Bloom LB: A slow ATP- 26. Sanders GM, Dallmann HG, McHenry CS: Reconstitution of the

induced conformational change limits the rate of DNA binding B. subtilis replisome with 13 proteins including two distinct

but not the rate of b-clamp binding by the Escherichia coli g replicases. Mol Cell 2010, 37:273-281.

complex clamp loader. J Biol Chem 2009, 284:32147-32157. This work represents the first reconstitution of an active replication fork in

This expands on a series of elegant pre-steady state investigations of the a Gram-positive organism using 13 purified proteins. This work also

mechanism of b loading by the DnaXcx. In this paper, the order of binding reveals a function for the second Pol III (DnaE) in replication. It elongates

of participants to the DnaXcx is addressed. a nascent RNA primer and hands the product off to the PolC replicase.

www.sciencedirect.com Current Opinion in Chemical Biology 2011, 15:587–594

594 Molecular Machines

27. Dervyn E, Suski C, Daniel R, Bruand C, Chapuis J, Errington J, This review presents a model for distinguishing Pol IIIs involved in

Janniere L, Ehrlich SD: Two essential DNA polymerases at the mutatgenesis from Pol IIIs that serve a replicative function and suggests

bacterial replication fork. Science 2001, a nomenclature sytem for organisms containing more than one Pol III.

294:1716-1719.

36. Warner DF, Ndwandwe DE, Abrahams GL, Kana BD,

28. Tarantino PM, Zhi C, Gambino JJ, Wright GE, Brown NC: 6- Machowski EE, Venclovas C, Mizrahi V: Essential roles for

0

Anilinouracil-based inhibitors of Bacillus subtilis DNA imuA - and imuB-encoded accessory factors in DnaE2-

polymerase III: antipolymerase and antimicrobial structure- dependent mutagenesis in Mycobacterium tuberculosis. Proc

activity relationships based on substitution at uracil N3. J Med Natl Acad Sci USA 2010, 107:13093-13098.

Chem 1999, 42:2035-2040. This important work is the first to address molecular interactions that

occur in ImuC (second DnaE-like polymerases) in induced mutagenesis.

29. Friedberg EC, Lehmann AR, Fuchs RP: Trading places: how do

DNA polymerases switch during translesion DNA synthesis? 37. Daly JS, Giehl TJ, Brown NC, Zhi C, Wright GE, Ellison RT III: In

Mol Cell 2005, 18:499-505. vitro antimicrobial activities of novel anilinouracils which

selectively inhibit DNA polymerase III of Gram-positive

30. Patel M, Jiang Q, Woodgate R, Cox MM, Goodman MF: A new

bacteria. Antimicrob Agents Chemother 2000, 44:2217-2221.

model for SOS-induced mutagenesis: how RecA protein

activates DNA polymerase V. Crit Rev Biochem Mol Biol 2010,

38. Dallmann HG, Fackelmayer OJ, Tomer G, Chen J, Wiktor-

45:171-184.

Becker A, Ferrara T, Pope C, Oliveira MT, Burgers PM, Kaguni LS,

McHenry CS: Parallel multiplicative target screening against

31. Heltzel JM, Maul RW, Scouten Ponticelli SK, Sutton MD: A model

divergent bacterial replicases: identification of specific

for DNA polymerase switching involving a single cleft and the

inhibitors with broad spectrum potential. Biochemistry 2010,

rim of the sliding clamp. Proc Natl Acad Sci USA 2009, 49:2551-2562.

106:12664-12669.

39. Liu J, Dehbi M, Moeck G, Arhin F, Bauda P, Bergeron D, Callejo M,

32. Furukohri A, Goodman MF, Maki H: A dynamic polymerase

Ferretti V, Ha N, Kwan T et al.: Antimicrobial drug discovery

exchange with Escherichia coli polymerase IV replacing

through bacteriophage genomics. Nat Biotechnol 2004,

polymerase III on the sliding clamp. J Biol Chem 2008,

283:11260-11269. 22:185-191.

40. Belley A, Callejo M, Arhin F, Dehbi M, Fadhil I, Liu J, McKay G,

33. Boshoff HI, Reed MB, Barry CE, Mizrahi V: DnaE2 polymerase

Srikumar R, Bauda P, Ha N et al.: Competition of bacteriophage

contributes to in vivo survival and the emergence of drug

polypeptides with native replicase proteins for binding to the

resistance in Mycobacterium tuberculosis. Cell 2003,

113:183-193. DNA sliding clamp reveals a novel mechanism for DNA

replication arrest in Staphylococcus aureus. Mol Microbiol

34. Galhardo RS, Rocha RP, Marques MV, Menck CF: An SOS- 2006, 62:1132-1143.

regulated operon involved in damage-inducible mutagenesis

41. Yano ST, Rothman-Denes LB: A phage-encoded inhibitor of

in Caulobacter crescentus. Nucleic Acids Res 2005,

33:2603-2614. Escherichia coli DNA replication targets the DNA polymerase

clamp loader. Mol Microbiol 2011, 79:1325-1338.

35. McHenry CS: Breaking the rules: multiple replicases and DNA This work reveals a peptide encoded by a bacteriophage that shuts down

polymerase IIIs in bacteria. EMBO Rep 2011, 12:408-414. host DNA replication by binding to the DnaXcx.

Current Opinion in Chemical Biology 2011, 15:587–594 www.sciencedirect.com