PriA Mediates DNA Replication Pathway Choice at Recombination Intermediates

Molecular Cell, Vol. 11, 817–826, March, 2003, Copyright 2003 by Cell Press

PriA Mediates DNA Replication Pathway Choice at Recombination Intermediates Liewei Xu and Kenneth J. Marians* Molecular Biology Program Memorial Sloan-Kettering Cancer Center New York, New York 10021

Summary We report the reconstitution of the initial steps of the double-strand break-repair pathway where joint molecule formation between a duplex DNA fragment and a circular template by the combined action of RecA, RecBCD, and the single-stranded DNA binding protein provides the substrate for replication fork formation by the restart primosome and the DNA polymerase III holoenzyme. We show that PriA dictates the pathway of replication from the recombination intermediate by inhibiting a nonspecific, strand displacement DNA synthesis reaction and favoring the formation of a bona fide replication fork. Furthermore, we find that RecO and RecR significantly stimulate this recombination-directed DNA replication reaction, and that this stimulation is modulated by the presence of RecF, suggesting that the latter protein may also act as a regulator of the pathway of resolution of the recombination intermediate. Introduction Replication fork progression can be blocked easily by such occurrences as damage to either the template bases or phosphodiester backbone, the presence of frozen proteins on the template, or modulation of the activity of the proteins of the replisome (Haber, 1999; Rothstein et al., 2000). Thus, recombinational repair of both stalled or collapsed replication forks is a major pathway for preservation of genomic integrity. Recombination between the damaged and undamaged sister chromosome arms at the replication fork serves to provide either an accurate template copy for repair of the lesion encountered, a substrate for replication restart, or both. In bacteria, where the number of replication forks is nominally restricted to only two per chromosome per generation, replication restart of collapsed replication forks becomes essential for survival of the organism (Cox et al., 2000; Kuzminov, 1995; Liu et al., 1999). Genetic studies first suggested that the phage φXtype primosomal proteins were required for replication restart. These proteins were required for the assembly of a primosome—a multienzyme priming/helicase machine—on φX DNA (Marians, 1992). Three of these proteins, DnaB, DnaC, and DnaG, were identified as the replication fork DNA helicase, a helicase loading protein, and the replication fork primase, respectively (Marians, 1992). The other four primosomal proteins, PriA, PriB, PriC, and DnaT, had no known role in chromosomal replication, and their biochemical activities were not de* Correspondence: [email protected]

fined, with the exception of PriA, which was demonstrated to be a structure-specific DNA helicase (Lasken and Kornberg, 1988; Lee and Marians, 1987) and the specificity protein for primosome assembly (Shlomai and Kornberg, 1980). Creation of strains null for PriA function demonstrated this protein to be involved in homologous recombination, survival after UV irradiation, double-strand break repair (DSBR), and both constitutive and inducible stable DNA replication (Sandler and Marians, 2000). To account for all these phenotypes, Kogoma proposed that PriA functioned to load replication forks at recombination intermediates, most likely D loops (Kogoma, 1996). This proposal was consistent with previous suggestions from Seufert and Messer (1986) and this laboratory (Zavitz and Marians, 1991, 1992) that PriA-directed replication fork assembly acted to rescue arrested replication forks. Biochemical data have supported such a role for PriA, which was shown to bind D loops with high specificity (McGlynn et al., 1997; Nurse et al., 1999) and to be able to direct the assembly of a replication fork on a nicked, double-stranded, circular DNA template that carried a D loop (Liu et al., 1999). Additional genetic analysis has indicated that there are probably at least two replication restart pathways involving the primosomal proteins, one that utilizes PriA and PriB, and another that utilizes PriB, PriC, and Rep (Sandler, 2000), which is a DNA helicase whose inactivation leads to an increase in the generation of double-strand ends at stalled replication forks (Michel et al., 1997). Replication restart can proceed in a number of ways (Cox et al., 2000; Kowalczykowski, 2000; Kuzminov, 1999; Lusetti and Cox, 2002; Rothstein et al., 2000). Forks that have stalled completely undergo nascent strand regression, where the nascent leading and lagging strands pair and the parental duplex rewinds, generating a Holliday junction. This regression reaction, by switching the template strand, can provide an accurate template for repair of the lesion. Regression can then presumably be reversed and restart effected at the restored fork. This reaction could occur, for example, when a replication fork encounters a thymidine dimer in the leading-strand template. Interestingly, RecF, RecO, and RecR are required for restart under these circumstances (Courcelle et al., 1997). Alternatively, it has been suggested that stalled forks might break, leading to replication fork collapse (Horiuchi and Fujimura, 1995). Fork breakage has, under certain conditions, been shown to require the Holliday junction resolvase, RuvC (Seigneur et al., 1998), or it may occur spontaneously. Replication forks will also collapse if they encounter a nick in one of the template strands. In either example of replication fork collapse, the only route to restoration of chromosomal replication is via the establishment of a joint molecule between the intact and broken sister arms by the combined actions of the RecBCD helicase/nuclease and the RecA homologous strand-pairing protein. The D loop formed by this event can serve as a nucleus for restoration of replication with restart primosome assembly introducing the lagging-strand machinery and the 3⬘ end

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of the invading strand acting as the primer for leadingstrand synthesis. The Holliday junction formed is then presumably resolved to disjoin the sister chromosomes. Here we report the reconstitution of this concerted recombination-directed replication reaction using RecA, RecBCD, the single-stranded DNA binding protein (SSB), the restart primosomal proteins, the DNA polymerase III holoenzyme (Pol III HE), and DNA gyrase. We show that this reaction is stimulated by the addition of RecO and RecR and modulated by RecF. Furthermore, we show that PriA directs the pathway of resolution of the joint molecule by inhibiting a nonspecific DNA synthesis reaction and favoring the establishment of a bona fide replication fork. Results Reconstitution of the Initial Steps of Double-Strand Break Repair To model recombinational repair of a collapsed replication fork, we used a supercoiled plasmid DNA (␹-1) (Xu and Marians, 2002) to represent the intact arm and an 800 nt long duplex DNA fragment to represent the broken arm of the fork (Figure 1A). The 800-mer was generated by PCR from ␹-1 DNA. A recombinational enhancer ␹ site was positioned roughly in the middle of the 800mer. The reactions discussed in this report are illustrated in Figure 1A. The duplex end of the 800-mer is a substrate for the RecBCD enzyme, which digests the DNA primarily from the 3⬘-ended strands until it encounters the ␹ site. At this point, digestion of the 5⬘ ended strand becomes the favored reaction while RecBCD promotes loading of RecA on the 3⬘ single-stranded tail. Strand invasion now takes place, resulting in the formation of a joint molecule with a D loop on the ␹-1 template DNA. This joint molecule becomes a substrate for replication fork formation mediated by the restart primosome. The recombination-directed replication reaction described in Figure 1A was reconstituted in the presence of the ␹-1 and 800-mer DNAs, RecA, RecBCD, RecO, RecR, SSB, PriA, PriB, PriC, DnaT, DnaB, DnaC, DnaG, the Pol III HE, and DNA gyrase. [␣-32P]dATP was included in the reaction to label the DNA products, which were analyzed by neutral agarose gel electrophoresis (Figure 1B, lane 1). The major DNA product (labeled “ds product” in this and all other figures) had the mobility of a late replication intermediate (item v, Figure 1A) where there are 200–300 base pairs of unreplicated parental DNA remaining. Possible explanations as to why the replication product would take this form are discussed below. Additional products visible were a small amount of form II DNA (item vi, Figure 1A) and form X DNA. The latter species, which was observed originally by Shibata and colleagues (Iwabuchi et al., 1983), is an extremely underwound form of the plasmid DNA generated because of the combined action of gyrase and the unwinding of DNA effected within the RecA filament. We have shown previously that form X is not a precursor of replication products (Xu and Marians, 2002). Additionally, a faint ladder of bands could be observed extending from the major ds product to about the position of the form II product band. These are multiply linked DNA dimers that arise if replication is completed.

The requirements for the recombination-directed replication reaction were determined by omitting individual proteins from the reaction mixture (Figures 1B and 1C). The strong dependence of the reaction on RecA, RecBCD, and SSB reflects the fact that without the presence of a joint molecule, there is no substrate for replication fork assembly. Although ␹-1 DNA carries both a pBR322-type origin of replication and oriC, RNA polymerase (Itoh and Tomizawa, 1979) and DnaA (Kaguni and Kornberg, 1984) are required for initiation at each of these origins, respectively, and neither of these proteins were included in the reaction mixture. Thus, in the absence of these proteins, independent initiation on the plasmid template itself will not occur. The weak RecBCD-independent reaction that can be observed in lane 3 of Figure 1B will be described elsewhere. In the example shown, the concerted recombination-directed DNA replication reaction was stimulated about 3-fold by the presence of RecO and RecR (Figure 1B). This effect will be discussed in detail in the next section. The reaction was nearly completely dependent on the presence of PriA, PriB, DnaT, DnaB, and DnaC (Figure 1C), because in their absence, primosome formation will not occur. Interestingly, product formation was only reduced by about two-thirds in the absence of PriC (Figure 1C, lane 6). This observation is consistent with our previous observations on PriA-directed replication fork assembly (Liu et al., 1999; Ng and Marians, 1996a) and presumably reflects the existence of the two pathways of replication restart discussed above. The reaction was also nearly completely dependent on the presence of DnaG. This observation was surprising because replication fork formation in other systems is not dependent on this protein (Hiasa and Marians, 1994; Mok and Marians, 1987). Although a small amount of label that migrates in the position expected for the single-stranded product (corresponding to item iv in Figure 1A) could be observed (Figure 1C, lane 9), this strong dependence on the primase suggests either that the mechanics of replication fork assembly on these joint molecules is somewhat different than observed previously, perhaps because of the presence of the RecA filament, or that DnaG may also be involved in priming some leading-strand synthesis. The essentially absolute requirements for the Pol III HE and gyrase reflect their requirement for DNA synthesis and replication fork progression, respectively. The nature of the DNA products was also investigated by two-dimensional gel electrophoresis (Figure 2) where the first dimension was electrophoresis through neutral agarose, and the second dimension was electrophoresis through denaturing alkaline agarose. This technique reveals the form of the nascent DNA present under each of the product bands. The major product consisted of two distinct populations of nascent DNA that differed in size (Figure 2A). The larger population contained DNA that was 5 kb or greater in length, with the majority of the material being near unit length (the plasmid is 6.04 kb). This population represents the nascent leadingstrand DNA; it is unaffected by the absence of primase (Figure 2B) (Xu and Marians, 2002). Recall also that the majority of the leading strands will not be precisely unit length because replication is not complete on the majority of the molecules. The second population included

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Figure 1. Reconstitution of RecombinationDirected DNA Replication (A) Description of the reactions considered. Joint molecules (iii) formed between the form I template DNA (ii, ␹-1 DNA in the text) and the duplex 800-mer (i) are the substrate for assembly of a replication fork in the presence of the restart primosomal proteins and the Pol III HE. When DNA gyrase is the only topoisomerase present, the major product is a late replication intermediate (LRI; v, “ds product” in all other figures), where there is still some nonreplicated parental DNA present. If topoisomerase IV is added to the reaction mixture, replication can be completed and the daughter chromosomes decatenated to give monomer form II DNA products (vi). The joint molecules are also the substrate for a generalized strand-displacement DNA synthesis reaction, termed DnaB-independent elongation in the text, where a DNA polymerase can extend the invading strand all the way around the circular template in the presence of DNA gyrase and SSB yielding a form II DNA that is linked to the displaced template strand (iv, “ss product” in all other figures). (B and C) Requirements for concerted recombination-directed replication. Reaction mixtures were incubated for 12 min at 37⬚C and analyzed as described under Experimental Procedures. The gels were dried, exposed to a phosphorimaging screen, and autoradiographed. The double-stranded product was quantitated with a phosphorimager. Incoporation (DNA syn.) is shown relative to the complete reaction in each case. In (B) and (C), 100% incorporation is 16,860 and 17,140 PSL (photostimulated luminescence), respectively. Complete reactions (lane 1 in [B] and [C]) contained ␹-1 DNA, the 800-mer, RecA, RecBCD, RecO, RecR, SSB, DNA gyrase, PriA, PriB, PriC, DnaT, DnaB, DnaC, DnaG, and the Pol III HE. (B) Requirements for recombination proteins. The indicated recombination protein was omitted from the reaction mixture. (C) Requirements for replication proteins. The indicated replication protein was omitted from the reaction mixture. ds prod, double-stranded product, equivalent to the late replication intermediate shown in Figure 1A (v); ss prod, single-stranded product, as shown in Figure 1A (iv); F II, form II DNA; F X, form X DNA.

DNA from about 0.8 to 2.0 kb in length. This is characteristic of the pattern formed by Okazaki fragments produced on the lagging strand. As expected, this population was absent when primase was omitted from the reaction mixture (Figure 2B) (Xu and Marians, 2002). Note that the form II DNA that is also a product of the reaction presents as DNA with a range of sizes in the alkaline dimension because it derives from both of the nascent strands (see Figure 1A). The signature presence of nascent leading- and lagging-strand DNA indicates that a bona fide replication fork had been formed on the recombination intermediates. In this concerted recombination-directed replication reaction, the RecA filament that accomplishes strand exchange is capable of transiting from the heteroduplex formed between the invading strand and the template DNA to the duplex template. This event provides the basis for the generation of form X DNA (Iwabuchi et al., 1983). The presence of the RecA filament on the DNA

presents a potential problem in that it might inhibit DNA synthesis or prevent completion of DNA replication. The other possible reason for the incomplete nature of DNA replication lies in the substrate binding preferences of DNA gyrase. Gyrase supports replication fork progression on the supercoiled plasmid DNA template primarily by binding ahead of the fork. However, because gyrase wraps about 150 base pairs of DNA about itself (Klevan and Wang, 1980), a point is reached in the replication reaction where gyrase action becomes limited severely. Gyrase is very inefficient at removing precatenanes, the form the excess positive linkages take when they distribute behind the fork (Peter et al., 1998). Thus, in ␪-type replication reactions that contain gyrase as the only topoisomerase, replication slows considerably at this late stage and the late replication intermediate accumulates. We reasoned that if the latter explanation were the case, inclusion of topoisomerase IV (Topo IV), which

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Figure 2. Two-Dimensional Gel Electrophoresis of the Reaction Products The DNA products of standard reactions either in the presence (A) or absence (B) of DnaG were analyzed by two-dimensional gel electrophoresis as described under Experimental Procedures. Autoradiograms of the dried gels are shown. At the top of each panel for reference is an autoradiogram of the products after electrophoresis through the neutral agarose gel. DNA size markers were 5⬘[32P]HindIII fragments of bacteriophage ␭.

prefers to remove precatenanes rather than positive supercoils (Hiasa and Marians, 1996; Ullsperger and Cozzarelli, 1996), would allow the replication reaction to go to completion, whereas if the problem was the RecA filament, the Topo IV would have little effect. The inclusion of Topo IV in the reaction mixture in the presence of gyrase did allow the reaction to go to completion (Figure 3). Increasing the concentration of Topo IV in the reaction mixture shifted the position of the major product from that of a late replication intermediate to that of monomer form II DNA (see Figure 1A). Thus, RecA filament formation did not seem to pose any problem for complete replication of the template. RecF Modulates RecO and RecR Stimulation of the Recombination-Directed Replication Reaction The genes encoding RecF, RecO, and RecR form a common epistasis group (Lloyd et al., 1987, 1988; Mahdi and

Figure 3. Complete Replication Products Are Produced in the Presence of Topo IV Standard reactions containing the indicated concentrations of Topo IV were incubated and analyzed as described under Experimental Procedures.

Lloyd, 1989). These genes were uncovered in screens for recombination deficiency in recBC sbcB sbcC mutants. Although strains mutated in these genes do not exhibit a recombination deficiency in strains otherwise wildtype for recombination functions, they are sensitive to UV irradiation. The phenotypes of mutations in these genes suggested that the gene products were required for postreplication, recombinational gap repair (Clark and Sandler, 1994); it has also been suggested that they were required for replication fork restart after the encounter of a replication fork with UV-induced thymidine dimers (Courcelle et al., 1997). Initial biochemical studies showed that RecO and RecR acted together to overcome the inhibitory effect of SSB on RecA-catalyzed strand exchange reactions and that RecF inhibited the strand-pairing reaction by ablating the stimulatory effect of RecO and RecR (Umezu et al., 1993; Umezu and Kolodner, 1994). Thus, although there is no genetic evidence suggesting an involvement of these proteins in double-strand break repair, their possible involvement with replication restart and effect on joint molecule formation prompted us to examine the effect of these proteins on the concerted recombination-directed replication reaction. RecR binds to either RecO or RecF to form RecFR and RecOR complexes (Shan et al., 1997; Umezu and Kolodner, 1994; Webb et al., 1997). Thus, we examined the effect of all combinations of these proteins, as well as that of their single addition to the reaction mixture (Figure 4). Interestingly, the only protein to have a significant effect on the reaction when added individually was RecF, the addition of which resulted in a stimulation of incorporation of about 2-fold. However, the addition of both RecO and RecR resulted in a 10-fold stimulation. (The extent of stimulation by RecOR is somewhat variable. This variability probably results from order of addition preferences that we have yet to establish firmly. In the experiment shown in Figure 1, all the proteins in the reaction were added separately, whereas in the experiments shown in Figures 4 and 5, the RecFOR proteins were added to reaction mixtures that already contained all the other proteins. The more typical stimulation by

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Figure 4. Effect of RecF, RecO, and RecR on the RecombinationDirected Replication Reaction Standard reaction mixtures containing either no RecFOR or the indicated combinations of RecF, RecO, and RecR (each at 160 nM) were incubated and analyzed as described under Experimental Procedures. The amount of double-stranded product was quantitated as described in the legend to Figure 1. 100% incorporation represents 3150 PSL.

RecOR is as observed in Figure 5.) The addition of RecF to RecO and RecR resulted in a loss of the strong stimulation effected by the addition of RecO and RecR alone. The negative influence of RecF could be observed clearly when a titration of RecFOR was compared to one of RecOR (Figure 5). The titration of RecOR produced a classic rectangular hyperbola with a maximal stimulation of the reaction of about 6-fold. On the other hand, the titration of RecFOR was bell shaped, with stimulation observed at low concentrations. As the concentration of RecFOR increased, this stimulation was lost, and at higher concentrations the reaction was inhibited completely. These observations suggest that RecOR and RecF exert their influence on different steps in the reaction pathway and that RecF action could possibly be the result of a competition for another component of the reaction (see Discussion). PriA Directs Traffic on the Joint Molecules We have described previously a DnaB-independent DNA replication reaction that utilizes joint molecules formed by the action of RecA and RecBCD (Xu and Marians, 2002). DNA synthesis occurs as a result of a generalized strand-displacement reaction where the 3⬘-OH of the invading strand in the D loop is extended by the action of a DNA polymerase and is driven by negative superhelicity. The product of such a reaction is a form II DNA where one strand represents the newly synthesized DNA and the other strand is the parental template. The displaced template strand remains linked to the form II DNA (item iv, Figure 1A). Thus, the product of this reaction has a mobility that is retarded compared to that of form II DNA. An example of the product of the DnaB-independent reaction, labeled “ss prod,” is shown in lane 1 of Figure 6A. The DnaB-independent pathway represents a potentially promiscuous reaction to the cell. There is no specificity, as elongation of the invading

Figure 5. RecF Modulates the Stimulatory Activity of RecO and RecR Standard reaction mixtures containing either no RecFOR, the indicated concentrations of RecOR, or the indicated concentrations of RecFOR were incubated and analyzed as described under Experimental Procedures. Both an autoradiogram of the dried gel and a plot of the amount of double-stranded product, quantitated as described under the legend to Figure 1, versus the concentration of either RecOR or RecFOR are shown.

strand can be catalyzed by any DNA polymerase (Xu and Marians, 2002). Thus, we suspected that a means to regulate this reaction would exist. In the course of developing the concerted recombination-directed replication reaction described here, it became obvious that PriA was the agent that regulated DNA replication pathway choice on the recombinant joint molecules. The DnaB-independent elongation reaction requires only RecA, RecBCD, SSB, DNA gyrase, and the Pol III HE (Xu and Marians, 2002). The introduction of PriA into such reaction mixtures results in the inhibition of DnaBindependent elongation (Figure 6A, lanes 2–6). PriA inhibition was very efficient, with about 1.1 nM PriA required for 50% inhibition. This concentration of PriA is essentially identical to the Kd for PriA binding to a threestranded junction with a 5⬘ single-stranded tail (1.3 nM) and in the same range as the Kd for PriA binding to D loops (6 nM) (Nurse et al., 1999). Two possible modes of action could explain the observed inhibition by PriA: either the protein could be unwinding the D loop formed, and thus simply remove the substrate for the DNA synthesis reaction, or the binding of PriA itself to the D loop caused inhibition. To distinguish between these two possibilities, we asked if a mutant PriA protein, PriAK230R,

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Figure 6. PriA Directs Traffic on Recombination Intermediates (A) PriA inhibits the DnaB-independent elongation reaction. Reaction mixtures containing ␹-1 DNA, the 800-mer, RecA, RecBCD, SSB, DNA gyrase, the Pol III HE, and the indicated concentrations of either the wild-type or K230R PriA were incubated and analyzed as described under Experimental Procedures. Both an autoradiogram of the dried gel and a plot of the amount of double-stranded product, quantitated as described under the legend to Figure 1, versus the concentration of either wild-type or K230R PriA are shown. (B) PriA mediates DNA replication pathway choice on joint molecules. Standard reaction mixtures containing either the indicated concentrations of either wild-type or K230R PriA were incubated and analyzed as described under Experimental Procedures. Both an autoradiogram of the dried gel and a plot of the amount of double-stranded product, quantitated as described under the legend to Figure 1, versus the concentration of either wild-type or K230R PriA are shown.

that does not possess helicase activity (Zavitz and Marians, 1992), could also inhibit the DnaB-independent elongation reaction. Remarkably, not only did PriAK230R inhibit the DnaBindependent elongation reaction, it did so about 20-fold more efficiently than the wild-type protein, with 50% inhibition being achieved at a concentration of 60 pM (Figure 6A). The most reasonable interpretation of these results is that inhibition of the DnaB-independent reaction is brought about by PriA binding directly to the D loop formed, preventing polymerase access to the 3⬘-OH end of the invading strand. This action is also consistent with the location of PriA when bound to a D loop, as determined by DNase I footprinting (Liu and Marians, 1999), which showed that PriA bound to the intersection of the two template strands and the 3⬘ end of the invading strand. It is likely that the difference in amount of helicase-deficient PriA required for inhibition compared to wild-type reflects the fact that the latter can leave the D loop, whereas the former remains bound stably. The extremely low concentration of the PriA K230R required for inhibition of the DnaB-independent

elongation reaction suggests that the protein is actually measuring the level of active templates in the reaction and that a 1:1 complex of PriA bound to the D loop is sufficient for inhibition. Previous studies have demonstrated that the helicase activity of PriA was not required for primosome assembly on either φX174 viral DNA (Zavitz and Marians, 1992) or form II DNA templates that carried a D loop (Liu et al., 1999). We therefore asked whether this was the case with the concerted recombination-directed replication reaction. Interestingly, whereas the PriAK230R variant could support the reaction, the wild-type protein was considerably more effective (Figure 6B). This experiment also shows clearly that PriA directs the choice of DNA replication pathway from the joint molecules. In the reaction shown in lane 1, all the primosomal proteins are present except PriA, and the product formed is that of the DnaB-independent reaction. As either wild-type (lanes 2–6) or K230R (lanes 8–12) PriA is added to the reaction mixture, the DnaB-independent reaction is inhibited and bona fide replication fork formation, as indicated by the shift to the ds product, predominates.

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Discussion Replication errors are one of the major sources of genomic instability in both prokaryotes and eukaryotes. These errors can take many forms but can be divided into two major categories: mutations that arise either because of misincorporation of nucleotides or chromosomal rearrangements. Many chromosomal rearrangements are thought to arise as a result of either replication fork stalling or collapse. Recombinational repair provides the major housekeeping correction process for collapsed replication forks. Replication fork restart is often an obligate step that follows upon repair of the DNA damage. Replication restart is, by necessity, origin independent, and initiation occurs in many cases in a recombination-directed manner, effectively coupling these two major pathways of DNA metabolism. We report here the reconstitution of a concerted recombination-directed DNA replication reaction that models many of the steps associated with the repair of a collapsed replication fork: recombinational repair of double-strand breaks and subsequent replication restart. The recombination-directed DNA replication reaction utilizes the core of the homologous recombination machinery in E. coli, RecA, and RecBCD to form joint molecules between a duplex DNA and a supercoiled DNA template. Replication fork formation is then nucleated by PriA-directed assembly of the restart primosome at the D loop on the template DNA. The Pol III HE is presumably attracted by the available 3⬘-OH of the invading strand that serves to prime the nascent leading strand. Establishment of the ␶-DnaB interaction (Kim et al., 1996; Yuzhakov et al., 1996) couples the HE to the primosome, forming a bona fide replisome capable of replicating the entire template. Joint molecules formed by the action of RecA and RecBCD can be the substrate for a number of reactions that all have somewhat different outcomes. The heteroduplex formed can be branch migrated either by the growing RecA filament itself or by the branch migration enzymes RuvAB and RecG. Or, replication fork formation can be initiated. Little is known currently about how a recombinant joint formed between the two sister arms of a replicating chromosome is apportioned between these competing reactions. It could be that recombination between the sister arms that occurs behind the replication fork during, for example, postreplication gap repair, which does not involve a double-strand break, is processed solely by recombination enzymes and that it is only recombination joints initiated by a doublestrand break that are substrates for the restart primosome. This scenario, however, appears to require a mechanism that would allow PriA to distinguish the D loops formed in the two different instances. Such a mechanism is not immediately apparent, and this important aspect of DNA metabolism remains to be addressed. It is clear, however, that the recombination proteins involved in joint molecule formation do not present an obstacle for either recognition of the D loop by PriA or subsequent replication fork progression. We have shown in a previous report (Xu and Marians, 2002) that the dynamic nature of the RecA filament enables its clearance from the recombination joint and is an essen-

tial feature of the ability of polymerases to access the 3⬘-OH of the invading strand in the joint molecule. Our current studies indicate that the presence of a RecA filament on the double-stranded template is also not an obstacle to replication fork progression. In the reaction described here, the generation of form X DNA as a reaction product indicates that the RecA filament that forms the heteroduplex between the invading strand and the template is capable of moving into the double-stranded template DNA. Nevertheless, complete molecules could be generated if topoisomerase IV was included in the reaction mixture. Thus, if the RecA filament does present an impediment to replication fork progression, its rapid disassembly clearly seems to have been programmed to permit DNA replication subsequent to joint molecule formation. Part of the role of traffic cop on the recombination joint is clearly played by PriA. Replication pathway choice was dictated by the presence of this protein. In its absence, even when all the other restart primosomal proteins were present, the nonspecific, DnaB-independent replication reaction predominated, whereas when PriA was present, only bona fide replication fork assembly was observed. The affinity of PriA for the recombination intermediate was very high, with the helicase-deficient protein exhibiting a Kd in the range of 60 pM, indicating that PriA is likely to be a major factor in determining usage of the D loop as a substrate. In contrast to both previous biochemical and genetic observations, the helicase activity of PriA was required for maximal stimulation of the recombination-directed replication reaction. The role of the PriA helicase in the cell has been unclear. The PriAK230R protein supports replication of the complementary strand of φX174 viral DNA more efficiently than the wild-type protein (Zavitz and Marians, 1992). A similar pattern was observed when a form II DNA carrying a preformed D loop was used as a template (Liu et al., 1999). These observations were consistent with the fact that strains null for PriA activity could be restored to essentially wild-type levels of homologous recombination, UV resistance, viability, and SOS induction by the provision of the priAK230R allele (priA300) in trans (Sandler et al., 1996). Subsequent studies, however, where the phenotypes of single point mutations of the chromosomal priA were examined (Sandler et al., 2001) demonstrated that whereas priA300 behaved essentially as wild-type either alone or in combination with inactivation of priC, the priA300 priB302 combination phenocopied the effect of a complete disruption of priA. These findings were interpreted as meaning that there was a strong requirement for the PriA helicase activity in the PriA- and PriC-dependent pathway and less of a requirement for the helicase activity in the PriA- and PriB-dependent pathway. While our observations are consonant with this genetic requirement for the PriA helicase activity, our reconstituted system is, however, dependent on PriB, not PriC. There are other indications as well that the PriA helicase activity can play a role in replication fork repair. PriA helicase activity is required for replication of the cointegrate during transposition of phage Mu (Jones and Nakai, 1999). It is thought in this instance that the helicase activity acts to remove nascent lagging-strand DNA allowing deposition of DnaB onto the thus exposed

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lagging-strand template (Jones and Nakai, 2001). Similarly, extragenic suppressors (srgA) of the UV sensitivity of recG mutations were found to occur in priA (Al-Deib et al., 1996). These priA mutations were shown to affect the helicase activity of the protein, altering its specificity and activity (Gregg et al., 2002), suggesting that the PriA helicase could not be tolerated in its wild-type form in the absence of RecG. These authors suggested that the toxicity of the PriA helicase in the absence of RecG reflected events at stalled replication forks where the lagging strand had extended past the leading strand. Under normal circumstances, RecG would catalyze a regression reaction at a stalled fork that would allow either the nascent lagging strand to serve as a template to extend the nascent leading strand, or the nascent lagging strand could be trimmed back to the active 3⬘ end of the nascent leading strand. Regression would also act to provide a template for repair of whatever damage had caused the fork to stall in the first place. Once the damage had been repaired and the nascent strands processed, reversal of regression would regenerate a complete fork structure at which PriA could direct replication restart. In the absence of RecG, PriA would act to load DnaB to the structure lacking a leading strand, an action presumably deleterious to the cell. This model is supported by the activity of the SrgA PriA proteins, which retained their capacity to unwind complete fork structures, but had lost the ability to unwind the lagging strand at a fork that did not possess a leading strand (Gregg et al., 2002). Why is the wild-type protein significantly more effective in supporting the recombination-directed replication reaction than the PriAK230R mutant protein? Inactivation of the PriA helicase activity does not affect either primosome assembly, the helicase activity of DnaB in the primosome, or the spectrum of primers synthesized by the primosome (Zavitz and Marians, 1992). Thus, it is unlikely that the observed stimulation of the recombination-directed replication reaction by the PriA helicase activity reflects a requirement for this activity during either replication fork assembly or progression. Helicase activity of PriA on the displaced strand of the D loop might act to unwind the template upstream of the recombination joint, possibly aiding branch migration and tending to stabilize the D loop by acting to incorporate all of the invading strand. However, PriA action in this direction would be opposite the direction of replication fork progression, and although there is no formal evidence that PriA remains with and proceeds with the restart primosome in a replication fork, all data on primosome formation indicates that it is a stable complex of proteins (Arai et al., 1981; Low et al., 1981; Ng and Marians, 1996a, 1996b). PriA helicase activity on the template strand in the heteroduplex would likely act to unwind the invading strand, clearly a nonproductive event. Alternatively, helicase activity in this direction that initiated at the intersection between the 3⬘ end of the invading strand and the two template strands could act to generate an unwinding bubble that might facilitate loading of DnaB. Again, this seems unlikely because the preformed D loop on the aforementioned form II template that we have used previously was only 42 nt long, and in that reaction, the K230R protein was 3- to 5-fold more effective than the wild-type. Rather, we sug-

gest that the requirement for the helicase activity actually reflects a requirement for the ATPase activity of the protein. ATP hydrolysis by DNA helicases generally cause their affinity for single-stranded DNA to decrease significantly, ultimately allowing the proteins to translocate along the DNA (Velankar et al., 1999). Thus, it could be that ATP hydrolysis by PriA is required to permit the protein to dissociate from some DNA structure and encourage replication fork progression. This explanation is consistent with the nearly 20-fold higher affinity of the K230R mutant protein for the substrate than the wild-type protein. Clearly, however, we are far from a position where we understand all aspects of these reactions and the incongruence between the current biochemical findings and the genetic studies will only be resolved by additional investigation. We have also demonstrated in this report that recombination-directed replication is stimulated by the RecO and RecR proteins and inhibited by the presence of RecF. This observation is consistent with previous biochemical studies that indicated that RecF, RecO, and RecR, which are in the same epistasis group, do not appear to act as a unit. Kolodner and colleagues’ (Umezu et al., 1993; Umezu and Kolodner, 1994) original observations demonstrated that RecOR stimulated strand exchange by allowing RecA to form a filament on single-stranded DNA that was coated with SSB. The presence of RecF inhibited this stimulation. Cox and colleagues (Bork et al., 2001; Shan et al., 1997; Webb et al., 1997) have observed that whereas RecOR stimulates RecA-catalyzed strand pairing, RecFR acts to prevent the extension of the RecA filament on dsDNA. They suggested that these properties would be advantageous for proteins that acted to promote postreplication gap repair by focusing the RecA filament to, and containing it in, the gap. These authors also pointed out that RecF could, in this capacity, be considered a switch, acting to disrupt the action of RecOR when it was no longer needed and promoting some other step in the reaction pathway. The behavior of RecF in the system described here is consistent with this possible mode of action. Experimental Procedures DNAs and Proteins RecA (Griffith and Shores, 1985) and RecBCD (Roman and Kowalczykowski, 1989) were prepared as described. RecF was prepared from K38/pGP1-2(pBLW20) by sequential chromatography on phosphocellulose and Q-Sepharose, followed by gel filtration through Superose 12, a combination of the previously published procedures (Griffin and Kolodner, 1990; Webb et al., 1995). RecO was prepared from BL21(DE3)(pBLW21) by sequential chromatography on Q-Sepharose, Bio-Rex 70, hydroxylapatite, and butyl-sepharose, a combination and modification of previously published procedures (Luisi-DeLuca and Kolodner, 1994; Shan et al., 1997). RecR was prepared from Bl21(DE3)(pBLW22) by sequential chromatography on phenyl sepharose, CL-4B blue-sepharose, and Q-Sepharose followed by gel filtration through Superose 12, a modification of the procedure of Umezu et al. (1993). The RecF, RecO, and RecR expression plasmids were the kind gift of Michael Cox (University of Wisconsin). SSB, DNA gyrase, topoisomerase IV, and the Pol III HE (as Pol III* and ␤) were prepared as described previously (Hiasa and Marians, 1996). Preparations of the restart primosomal proteins are described in Marians (1995). pBR␹-1 DNA and the 800-mer were as described in Xu and Marians (2002).

Reconstitution of Double-Strand Break Repair 825

Concerted Recombination-Directed DNA Replication Standard reaction mixtures (20 ␮l) containing 50 mM HEPES-KOH (pH 8.0), 16 mM MgOAc2, 10 mM DTT, 100 ␮g/ml bovine serum albumin, 2 mM ATP, 40 ␮M [␣-32P]dATP (1000–2000 cpm/pmol), 40 ␮M dCTP, dGTP, and TTP, 7.5 mM creatine phosphate, 62 ng/ml creatine kinase, 12.5 nM 800-mer, 1.1 nM pBR␹-1 DNA, 3.5 ␮M RecA, 1 nM RecBCD, 160 nM RecO, 160 nM RecR, 0.8 ␮M SSB, 16 nM DNA gyrase, 5 nM PriA, 5 nM PriB, 5 nM PriC, 60 nM DnaB, 0.96 ␮M DnaC, 300 nM DnaG, 160 nM DnaT, 20 nM Pol III*, and 20 nM ␤ subunit of the HE were incubated at 37⬚C for 12 min. Reactions were terminated by the addition of EDTA, SDS, and proteinase K to 25 mM, 1%, and 100 ␮g/ml, respectively, followed by incubation at 37⬚C for 30 min. The samples were analyzed by electrophoresis through vertical 1% agarose gels at 1.5V/cm for 15 hr using 50 mM Tris-HCl (pH 8.3 at 23⬚C), 40 mM NaOAc, 1 mM EDTA as the electrophoresis buffer. Gels were dried, exposed to phosphorimager screens, and autoradiographed. Two-Dimensional Gel Electrophoresis Two reaction mixtures were mixed together and then applied equally to two adjacent lanes of a neutral agarose gel as described above. After electrophoresis, the two lanes were excised from the gel. One lane was dried to serve as a reference. The other lane was soaked in 50 mM NaOH-1 mM EDTA and then inserted into a 0.5% horizontal alkaline agarose gel. Electrophoresis followed at 1.5V/cm for 15 hr using 30 mM NaOH, 1 mM EDTA as the electrophoresis buffer. The gel was neutralized by soaking in 5% trichloroacetic acid, dried, and autoradiographed. Acknowledgments These studies were supported by NIH grant GM34557. Received: November 16, 2002 Revised: January 15, 2003 References Al-Deib, A.A., Mahdi, A.A., and Lloyd, R.G. (1996). Modulation of recombination and DNA repair by the RecG and PriA helicases of Escherichia coli K-12. J. Bacteriol. 178, 6782–6789. Arai, K., Low, R., Kobori, J., Shlomai, J., and Kornberg, A. (1981). Mechanism of dnaB protein action. V. Association of dnaB protein, protein n⬘, and other repriming proteins in the primosome of DNA replication. J. Biol. Chem. 256, 5273–5280. Bork, J.M., Cox, M.M., and Inman, R.B. (2001). The RecOR proteins modulate RecA protein function at 5⬘ ends of single-stranded DNA. EMBO J. 20, 7313–7322. Clark, A.J., and Sandler, S.J. (1994). Homologous genetic recombination: the pieces begin to fall into place. Crit. Rev. Microbiol. 20, 125–142. Courcelle, J., Carswell-Crumpton, C., and Hanawalt, P.C. (1997). recF and recR are required for the resumption of replication at DNA replication forks in Escherichia coli. Proc. Natl. Acad. Sci. USA 94, 3714–3719. Cox, M.M., Goodman, M.F., Kreuzer, K.N., Sherratt, D.J., Sandler, S.J., and Marians, K.J. (2000). The importance of repairing stalled replication forks. Nature 404, 37–41. Gregg, A.V., McGlynn, P., Jaktaji, R.P., and Lloyd, R.G. (2002). Direct rescue of stalled DNA replication forks via the combined action of PriA and RecG helicase activities. Mol. Cell 9, 241–251. Griffin, T.J., 4th, and Kolodner, R.D. (1990). Purification and preliminary characterization of the Escherichia coli K-12 recF protein. J. Bacteriol. 172, 6291–6299. Griffith, J., and Shores, C.G. (1985). RecA protein rapidly crystallizes in the presence of spermidine: a valuable step in its purification and physical characterization. Biochemistry 24, 158–162.

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