Formation of a stable RuvA protein double tetramer is required for efficient branch migration in vitro and for replication fork reversal in vivo

In bacteria, RuvABC is required for the resolution of Holliday junctions (HJ) made during homologous recombination. The RuvAB complex catalyzes HJ branch migration and replication fork reversal (RFR). During RFR, a stalled fork is reversed to form a HJ adjacent to a DNA double strand end, a reaction that requires RuvAB in certain Escherichia coli replication mutants. The exact structure of active RuvAB complexes remains elusive as it is still unknown whether one or two tetramers of RuvA support RuvB during branch migration and during RFR. We designed an E. coli RuvA mutant, RuvA2(KaP), specifically impaired for RuvA tetramer-tetramer interactions. As expected, the mutant protein is impaired for complex II (two tetramers) formation on HJs, although the binding efficiency of complex I (a single tetramer) is as wild type. We show that although RuvA complex II formation is required for efficient HJ branch migration in vitro, RuvA2(KaP) is fully active for homologous recombination in vivo. RuvA2(KaP) is also deficient at forming complex II on synthetic replication forks, and the binding affinity of RuvA2(KaP) for forks is decreased compared with wild type. Accordingly, RuvA2(KaP) is inefficient at processing forks in vitro and in vivo. These data indicate that RuvA2(KaP) is a separation-of-function mutant, capable of homologous recombination but impaired for RFR. RuvA2(KaP) is defective for stimulation of RuvB activity and stability of HJ·RuvA·RuvB tripartite complexes. This work demonstrates that the need for RuvA tetramer-tetramer interactions for full RuvAB activity in vitro causes specifically an RFR defect in vivo.     

ruvA and ruvB mutants specifically impaired for replication fork reversal

Replication fork reversal (RFR) is a reaction that takes place in Escherichia coli at replication forks arrested by the inactivation of a replication protein. Fork reversal involves the annealing of the leading and lagging strand ends; it results in the formation of a Holliday junction adjacent to DNA double-strand end, both of which are processed by recombination enzymes. In several replication mutants, replication fork reversal is catalysed by the RuvAB complex, originally characterized for its role in the last steps of homologous recombination, branch migration and resolution of Holliday junctions. We present here the isolation and characterization of ruvA and ruvB single mutants that are impaired for RFR at forks arrested by the inactivation of polymerase III, while they remain capable of homologous recombination. The positions of the mutations in the proteins and the genetic properties of the mutants suggest that the mutations affect DNA binding, RuvA-RuvB interaction and/or RuvB-helicase activity. These results show that a partial RuvA or RuvB defect affects primarily RFR, implying that RFR is a more demanding reaction than Holliday junction resolution.     

Recombination proteins and rescue of arrested replication forks

Recombination proteins play crucial roles in the rescue of inactivated replication forks in Escherichia coli. The enzymes that catalyze the repair of DNA double-strand breaks by a classical strand-exchange reaction (RecBCD, RecA) act in two well-characterized fork repair pathways. They repair the DNA double-strand end made when a replication fork runs into a single-strand interruption. They reset the DNA double-strand end generated by replication fork reversal when a component of the replication machinery is inactivated. In addition, recombination proteins also act at replication forks in ways other than the classical strand-exchange reaction. For example, the RuvAB enzyme that catalyzes Holliday junction branch-migration during homologous recombination is also able to catalyze replication fork reversal in certain replication mutants, i.e. to convert certain blocked replication forks into Holliday junctions. Finally, some of the actions of recombination proteins after replication impairment are still unclear, as for example in UV-irradiated cells, where RecFOR and RecA catalyze gap repair but also participate, in a yet undefined way, in "replisome reactivation".     

Mus81-Eme1 and Rqh1 involvement in processing stalled and collapsed replication forks

The processing of stalled replication forks and the repair of collapsed replication forks are essential functions in all organisms. In fission yeast DNA junctions at stalled replication forks appear to be processed by either the Rqh1 DNA helicase or Mus81-Eme1 endonuclease. Accordingly, we show that the hypersensitivity to agents that cause replication fork stalling of mus81, eme1, and rqh1 mutants is suppressed by a Holliday junction resolvase (RusA), as is the synthetic lethality of a mus81(-) rqh1(-) double mutant. Recombinant Mus81-Eme1, purified from Escherichia coli, readily cleaves replication fork structures but cleaves synthetic Holliday junctions relatively poorly in vitro. From these data we propose that Mus81-Eme1 can process stalled replication forks before they have regressed to form a Holliday junction. We also implicate Mus81-Eme1 and Rqh1 in the repair of collapsed replication forks. Here Mus81-Eme1 and Rqh1 seem to function on different substrates because RusA can substitute for Mus81-Eme1 but not Rqh1.     

ruvA Mutants that resolve Holliday junctions but do not reverse replication forks

RuvAB and RuvABC complexes catalyze branch migration and resolution of Holliday junctions (HJs) respectively. In addition to their action in the last steps of homologous recombination, they process HJs made by replication fork reversal, a reaction which occurs at inactivated replication forks by the annealing of blocked leading and lagging strand ends. RuvAB was recently proposed to bind replication forks and directly catalyze their conversion into HJs. We report here the isolation and characterization of two separation-of-function ruvA mutants that resolve HJs, based on their capacity to promote conjugational recombination and recombinational repair of UV and mitomycin C lesions, but have lost the capacity to reverse forks. In vivo and in vitro evidence indicate that the ruvA mutations affect DNA binding and the stimulation of RuvB helicase activity. This work shows that RuvA's actions at forks and at HJs can be genetically separated, and that RuvA mutants compromised for fork reversal remain fully capable of homologous recombination.     

The DNA repair helicase UvrD is essential for replication fork reversal in replication mutants

Replication forks arrested by inactivation of the main Escherichia coli DNA polymerase (polymerase III) are reversed by the annealing of newly synthesized leading- and lagging-strand ends. Reversed forks are reset by the action of RecBC on the DNA double-strand end, and in the absence of RecBC chromosomes are linearized by the Holliday junction resolvase RuvABC. We report here that the UvrD helicase is essential for RuvABC-dependent chromosome linearization in E. coli polymerase III mutants, whereas its partners in DNA repair (UvrA/B and MutL/S) are not. We conclude that UvrD participates in replication fork reversal in E. coli.     

UvrD controls the access of recombination proteins to blocked replication forks

Blocked replication forks often need to be processed by recombination proteins prior to replication restart. In Escherichia coli, the UvrD repair helicase was recently shown to act at inactivated replication forks, where it counteracts a deleterious action of RecA. Using two mutants affected for different subunits of the polymerase III holoenzyme (Pol IIIh), we show here that the anti-RecA action of UvrD at blocked forks reflects two different activities of this enzyme. A defective UvrD mutant is able to antagonize RecA in cells affected for the Pol IIIh catalytic subunit DnaE. In this mutant, RecA action at blocked forks specifically requires the protein RarA (MgsA). We propose that UvrD prevents RecA binding, possibly by counteracting RarA. In contrast, at forks affected for the Pol IIIh clamp (DnaN), RarA is not required for RecA binding and the ATPase function of UvrD is essential to counteract RecA, supporting the idea that UvrD removes RecA from DNA. UvrD action on RecA is conserved in evolution as it can be performed in E. coli by the UvrD homologue from Bacillus subtilis, PcrA.     

sbcB sbcC null mutations allow RecF-mediated repair of arrested replication forks in rep recBC mutants

We have proposed previously that, in Escherichia coli, blockage of replication forks can lead to the reversal of the fork. Annealing of the newly synthesized strands creates a double-stranded end adjacent to a Holliday junction. The junction is migrated away from the DNA end by RuvAB and can be cleaved by RuvC, while RecBCD is required for the repair of the double-stranded tail. Consequently, the rep mutant, in which replication arrests are frequent and fork reversal occurs, requires RecBCD for growth. We show here that the combination of sbcB sbcCD null mutations restores the viability to rep recBC mutants by activation of the RecF pathway of recombination. This shows that the proteins belonging to the RecF pathway are able to process the DNA ends made by the replication fork reversal into a structure that allows recombination-dependent replication restart. However, we confirm that, unlike sbcB null mutations, sbcB15, which suppresses all other recBC mutant defects, does not restore the viability of rep recBC sbcCD strains. We also show that ruvAB inactivation suppresses the lethality and the formation of double-stranded breaks (DSBs) in a rep recBC recF strain, totally deficient for homologous recombination, as well as in rep recBC mutants. This confirms that RuvAB processing of arrested replication forks is independent of the presence of recombination intermediates.     

Action of RuvAB at replication fork structures

The replicative apparatus often encounters blocks to its progression that necessitate removal of the block and reloading of the replication machinery. In Escherichia coli, a major pathway of replication restart involves unwinding of the stalled fork to generate a four-stranded Holliday junction, which can then be cleaved by the RuvABC helicase-endonuclease. This fork regression may be catalyzed by RecG but is thought to occur even in its absence. Here we test whether RuvAB helicase can also catalyze the unwinding of forked DNA to form Holliday junctions. We find that fork DNA is unwound in the direction required for Holliday junction formation only if the loading of RuvB is restricted to the parental duplex DNA arm. If the binding of RuvB is unrestricted, then RuvAB preferentially unwinds forks in the opposite direction. This is probably related to the greater efficiency of two opposed RuvB hexamers operating across a junction compared with a single hexamer. These data argue against RuvAB acting directly at damaged replication forks and imply that other mechanisms must operate in vivo to catalyze Holliday junction formation.     

Replication fork reversal in DNA polymerase III mutants of Escherichia coli: a role for the beta clamp

Certain replication mutations lead in Escherichia coli to a specific reaction named replication fork reversal: at blocked forks, annealing of the nascent strands and pairing of the template strands form a four-way junction. RuvABC-catalysed resolution of this Holliday junction causes chromosome double-strand breaks (DSBs) in a recBC context and therefore creates a requirement for the recombination proteins RecBC for viability. In the present work, two mutants were tested for replication fork reversal: a dnaEts mutant and a dnaNts mutant, affected in the alpha (polymerase) and beta (processivity clamp) subunits of DNA polymerase III holoenzyme respectively. In the dnaEts recB strain, RuvABC-dependent DSBs caused by the dnaEts mutation occurred at 37 degrees C or 42 degrees C, indicating the occurrence of replication fork reversal upon partial or complete inactivation of the DNA polymerase alpha subunit. DSB formation was independent of RecA, RecQ and the helicase function of PriA. In the dnaNts recB mutant, RuvABC-dependent DSB caused by the dnaNts mutation occurred only at semi-permissive temperature, 37 degrees C, indicating the occurrence of replication fork reversal in conditions in which the remaining activity of the beta clamp is sufficient for viability. In contrast, the dnaNts mutation did not cause chromosome breakage at 42 degrees C, a temperature at which DnaN is totally inactive and the dnaNts mutant is inviable. We propose that a residual activity of the DNA polymerase III beta clamp is required for replication fork reversal in the dnaNts mutant.     

Functional analysis of DNA replication fork reversal catalyzed by Mycobacterium tuberculosis RuvAB proteins

Initially discovered in Escherichia coli, RuvAB proteins are ubiquitous in bacteria and play a dual role as molecular motor proteins responsible for branch migration of the Holliday junction(s) and reversal of stalled replication forks. Despite mounting genetic evidence for a crucial role of RuvA and RuvB proteins in reversal of stalled replication forks, the mechanistic aspects of this process are still not fully understood. Here, we elucidate the ability of Mycobacterium tuberculosis RuvAB (MtRuvAB) complex to catalyze the reversal of replication forks using a range of DNA replication fork substrates. Our studies show that MtRuvAB, unlike E. coli RuvAB, is able to drive replication fork reversal via the formation of Holliday junction intermediates, suggesting that RuvAB-catalyzed fork reversal involves concerted unwinding and annealing of nascent leading and lagging strands. We also demonstrate the reversal of replication forks carrying hemi-replicated DNA, indicating that MtRuvAB complex-catalyzed fork reversal is independent of symmetry at the fork junction. The fork reversal reaction catalyzed by MtRuvAB is coupled to ATP hydrolysis, is processive, and culminates in the formation of an extended reverse DNA arm. Notably, we found that sequence heterology failed to impede the fork reversal activity of MtRuvAB. We discuss the implications of these results in the context of recognition and processing of varied types of replication fork structures by RuvAB proteins.     

Cells defective for replication restart undergo replication fork reversal

We have studied the fate of blocked replication forks with the use of the Escherichia coli priA mutant, in which spontaneously arrested replication forks persist owing to the lack of the major replication restart pathway. Such blocked forks undergo a specific reaction named replication fork reversal, in which newly synthesized strands anneal to form a DNA double-strand end adjacent to a four-way junction. Indeed, (i) priA recB mutant chromosomes are linearized by a reaction that requires the presence of the Holliday junction resolvase RuvABC, and (ii) RuvABC-dependent linearization is prevented by the presence of RecBC. Replication fork reversal in a priA mutant occurs independently of the recombination proteins RecA and RecR. recBC inactivation does not affect priA mutant viability but prevents priA chronic SOS induction. We propose that, in the absence of PriA, RecBC action at reversed forks does not allow replication restart, which leads to the accumulation of SOS-inducing RecA filaments. Our results suggest that types of replication blockage that cause replication fork reversal occur spontaneously.     

UV stalled replication forks restart by re-priming in human fibroblasts

Restarting stalled replication forks is vital to avoid fatal replication errors. Previously, it was demonstrated that hydroxyurea-stalled replication forks rescue replication either by an active restart mechanism or by new origin firing. To our surprise, using the DNA fibre assay, we only detect a slightly reduced fork speed on a UV-damaged template during the first hour after UV exposure, and no evidence for persistent replication fork arrest. Interestingly, no evidence for persistent UV-induced fork stalling was observed even in translesion synthesis defective, Polη(mut) cells. In contrast, using an assay to measure DNA molecule elongation at the fork, we observe that continuous DNA elongation is severely blocked by UV irradiation, particularly in UV-damaged Polη(mut) cells. In conclusion, our data suggest that UV-blocked replication forks restart effectively through re-priming past the lesion, leaving only a small gap opposite the lesion. This allows continuation of replication on damaged DNA. If left unfilled, the gaps may collapse into DNA double-strand breaks that are repaired by a recombination pathway, similar to the fate of replication forks collapsed after hydroxyurea treatment.     

A fork-clearing role for UvrD

The inactivation of a replication protein causes the disassembly of the replication machinery and creates a need for replication reactivation. In several replication mutants, restart occurs after the fork has been isomerized into a four-armed junction, a reaction called replication fork reversal. The repair helicase UvrD is essential for replication fork reversal upon inactivation of the polymerase (DnaE) or the beta-clamp (DnaN) subunits of the Escherichia coli polymerase III, and for the viability of dnaEts and dnaNts mutants at semi-permissive temperature. We show here that the inactivation of recA, recFOR, recJ or recQ recombination genes suppresses the requirement for UvrD for replication fork reversal and suppresses the lethality conferred by uvrD inactivation to Pol IIIts mutants at semi-permissive temperature. We propose that RecA binds inappropriately to blocked replication forks in the dnaEts and dnaNts mutants in a RecQ- RecJ- RecFOR-dependent way and that UvrD acts by removing RecA or a RecA-made structure, allowing replication fork reversal. This work thus reveals the existence of a futile reaction of RecA binding to blocked replication forks, that requires the action of UvrD for fork-clearing and proper replication restart.     

Substrate specificity of RusA resolvase reveals the DNA structures targeted by RuvAB and RecG in vivo

RusA endonuclease cleaves Holliday junctions by introducing paired strand incisions 5' to CC dinucleotides. Coordinated catalysis is achieved when both subunits of the homodimer interact simultaneously with cleavage sites located symmetrically. This requirement confers Holliday junction specificity. Uncoupled catalysis occurs when binding interactions are disturbed. Genetic studies indicate that uncoupling occurs rarely in vivo, and DNA cleavage is therefore restricted to Holliday junctions. We exploited the specificity of RusA to identify the DNA substrates targeted by the RuvAB and RecG branch-migration proteins in vivo. We present evidence that replication restart in UV-irradiated cells relies on the processing of stalled replication forks by RecG helicase and of Holliday junctions by the RuvABC resolvasome, and that RuvAB alone may not promote repair.     

Swi1 and Swi3 are components of a replication fork protection complex in fission yeast

Swi1 is required for programmed pausing of replication forks near the mat1 locus in the fission yeast Schizosaccharomyces pombe. This fork pausing is required to initiate a recombination event that switches mating type. Swi1 is also needed for the replication checkpoint that arrests division in response to fork arrest. How Swi1 accomplishes these tasks is unknown. Here we report that Swi1 copurifies with a 181-amino-acid protein encoded by swi3(+). The Swi1-Swi3 complex is required for survival of fork arrest and for activation of the replication checkpoint kinase Cds1. Association of Swi1 and Swi3 with chromatin during DNA replication correlated with movement of the replication fork. swi1Delta and swi3Delta mutants accumulated Rad22 (Rad52 homolog) DNA repair foci during replication. These foci correlated with the Rad22-dependent appearance of Holliday junction (HJ)-like structures in cells lacking Mus81-Eme1 HJ resolvase. Rhp51 and Rhp54 homologous recombination proteins were not required for viability in swi1Delta or swi3Delta cells, indicating that the HJ-like structures arise from single-strand DNA gaps or rearranged forks instead of broken forks. We propose that Swi1 and Swi3 define a fork protection complex that coordinates leading- and lagging-strand synthesis and stabilizes stalled replication forks.     

RecG interacts directly with SSB: implications for stalled replication fork regression

RecG and RuvAB are proposed to act at stalled DNA replication forks to facilitate replication restart. To define the roles of these proteins in fork regression, we used a combination of assays to determine whether RecG, RuvAB or both are capable of acting at a stalled fork. The results show that RecG binds to the C-terminus of single-stranded DNA binding protein (SSB) forming a stoichiometric complex of 2 RecG monomers per SSB tetramer. This binding occurs in solution and to SSB protein bound to single stranded DNA (ssDNA). The result of this binding is stabilization of the interaction of RecG with ssDNA. In contrast, RuvAB does not bind to SSB. Side-by-side analysis of the catalytic efficiency of the ATPase activity of each enzyme revealed that (−)scDNA and ssDNA are potent stimulators of the ATPase activity of RecG but not for RuvAB, whereas relaxed circular DNA is a poor cofactor for RecG but an excellent one for RuvAB. Collectively, these data suggest that the timing of repair protein access to the DNA at stalled forks is determined by the nature of the DNA available at the fork. We propose that RecG acts first, with RuvAB acting either after RecG or in a separate pathway following protein-independent fork regression.     

Dna2 helicase/nuclease causes replicative fork stalling and double-strand breaks in the ribosomal DNA of Saccharomyces cerevisiae

We have proposed that faulty processing of arrested replication forks leads to increases in recombination and chromosome instability in Saccharomyces cerevisiae and contributes to the shortened lifespan of dna2 mutants. Now we use the ribosomal DNA locus, which is a good model for all stages of DNA replication, to test this hypothesis. We show directly that DNA replication pausing at the ribosomal DNA replication fork barrier (RFB) is accompanied by the occurrence of double-strand breaks near the RFB. Both pausing and breakage are elevated in the early aging, hypomorphic dna2-2 helicase mutant. Deletion of FOB1, encoding the fork barrier protein, suppresses the elevated pausing and DSB formation, and represses initiation at rDNA ARSs. The dna2-2 mutation is synthetically lethal with deltarrm3, encoding another DNA helicase involved in rDNA replication. It does not appear to be the case that the rDNA is the only determinant of genome stability during the yeast lifespan however since strains carrying deletion of all chromosomal rDNA but with all rDNA supplied on a plasmid, have decreased rather than increased lifespan. We conclude that the replication-associated defects that we can measure in the rDNA are symbolic of similar events occurring either stochastically throughout the genome or at other regions where replication forks move slowly or stall, such as telomeres, centromeres, or replication slow zones.     

Coordinated leading- and lagging-strand synthesis at the Escherichia coli DNA replication fork. IV. Reconstitution of an asymmetric, dimeric DNA polymerase III holoenzyme.

Individually purified subunits have been used to reconstitute the action of the Escherichia coli DNA polymerase III holoenzyme (Pol III HE) at a replication fork formed in the presence of the primosome, the single-stranded DNA binding protein, and a tailed form II DNA template. Complete activity, indistinguishable from that of the intact DNA Pol III HE, could be reproduced with a combination of the DNA polymerase III core (Pol III core), the gamma.delta complex, and the beta subunit. Experiments where the Pol III core in reaction mixtures containing active replication forks was diluted suggested that the lagging-strand Pol III core remained associated continuously with the replication fork through multiple cycles of Okazaki fragment synthesis. Since the lagging-strand Pol III core must dissociate from the 3' end of the completed Okazaki fragment, this suggests that its association with the fork is via protein-protein interactions, lending credence to the idea that it forms a dimeric complex with the leading-strand Pol III core. An asymmetry in the action of the subunits was revealed under conditions (high ionic strength) that were presumably destabilizing to the integrity of the replication fork. Under these conditions, tau acted to stimulate DNA synthesis only when the primase was present (i.e. when lagging-strand DNA synthesis was ongoing). This stimulation was reflected by an inhibition of the formation of small Okazaki fragments, suggesting that, within the context of the model developed to account for the temporal order of steps during a cycle of Okazaki fragment synthesis, the presence of tau accelerated the transit of the lagging-strand Pol III core from the 3' end of the completed Okazaki fragment to the 3' end of the new primer.