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.     

The role of RuvA octamerization for RuvAB function in vitro and in vivo

RuvA plays an essential role in branch migration of the Holliday junction by RuvAB as part of the RuvABC pathway for processing Holliday junctions in Escherichia coli. Two types of RuvA-Holliday junction complexes have been characterized: 1) complex I containing a single RuvA tetramer and 2) complex II in which the junction is sandwiched between two RuvA tetramers. The functional differences between the two forms are still not clear. To investigate the role of RuvA octamerization, we introduced three amino acid substitutions designed to disrupt the E. coli RuvA tetramer-tetramer interface as identified by structural studies. The mutant RuvA was tetrameric and interacted with both RuvB and junction DNA but, as predicted, formed complex I only at protein concentrations up to 500 nm. We present biochemical and surface plasmon resonance evidence for functional and physical interactions of the mutant RuvA with RuvB and RuvC on synthetic junctions. The mutant RuvA with RuvB showed DNA helicase activity and could support branch migration of synthetic four-way and three-way junctions. However, junction binding and the efficiency of branch migration of four-way junctions were affected. The activity of the RuvA mutant was consistent with a RuvAB complex driven by one RuvB hexamer only and lead us to propose that one RuvA tetramer can only support the activity of one RuvB hexamer. Significantly, the mutant failed to complement the UV sensitivity of E. coli DeltaruvA cells. These results indicate strongly that RuvA octamerization is essential for the full biological activity of RuvABC.     

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.     

RuvA is a sliding collar that protects Holliday junctions from unwinding while promoting branch migration

The RuvAB proteins catalyze branch migration of Holliday junctions during DNA recombination in Escherichia coli. RuvA binds tightly to the Holliday junction, and then recruits two RuvB pumps to power branch migration. Previous investigations have studied RuvA in conjunction with its cellular partner RuvB. The replication fork helicase DnaB catalyzes branch migration like RuvB but, unlike RuvB, is not dependent on RuvA for activity. In this study, we specifically analyze the function of RuvA by studying RuvA in conjunction with DnaB, a DNA pump that does not work with RuvA in the cell. Thus, we use DnaB as a tool to dissect RuvA function from RuvB. We find that RuvA does not inhibit DnaB-catalyzed branch migration of a homologous junction, even at high concentrations of RuvA. Hence, specific protein-protein interaction is not required for RuvA mobilization during branch migration, in contrast to previous proposals. However, low concentrations of RuvA block DnaB unwinding at a Holliday junction. RuvA even blocks DnaB-catalyzed unwinding when two DnaB rings are acting in concert on opposite sides of the junction. These findings indicate that RuvA is intrinsically mobile at a Holliday junction when the DNA is undergoing branch migration, but RuvA is immobile at the same junction during DNA unwinding. We present evidence that suggests that RuvA can slide along a Holliday junction structure during DnaB-catalyzed branch migration, but not during unwinding. Thus, RuvA may act as a sliding collar at Holliday junctions, promoting DNA branch migration activity while blocking other DNA remodeling activities. Finally, we show that RuvA is less mobile at a heterologous junction compared to a homologous junction, as two opposing DnaB pumps are required to mobilize RuvA over heterologous DNA.     

RuvAB is essential for replication forks reversal in certain replication mutants

Inactivated replication forks may be reversed by the annealing of leading- and lagging-strand ends, resulting in the formation of a Holliday junction (HJ) adjacent to a DNA double-strand end. In Escherichia coli mutants deficient for double-strand end processing, resolution of the HJ by RuvABC leads to fork breakage, a reaction that we can directly quantify. Here we used the HJ-specific resolvase RusA to test a putative role of the RuvAB helicase in replication fork reversal (RFR). We show that the RuvAB complex is required for the formation of a RusA substrate in the polymerase III mutants dnaEts and holD, affected for the Pol III catalytic subunit and clamp loader, and in the helicase mutant rep. This finding reveals that the recombination enzyme RuvAB targets forks in vivo and we propose that it directly converts forks into HJs. In contrast, RFR occurs in the absence of RuvAB in the dnaNts mutant, affected for the processivity clamp of Pol III, and in the priA mutant, defective for replication restart. This suggests alternative pathways of RFR.     

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.     

Interaction of Branch Migration Translocases with the Holliday Junction-resolving Enzyme and Their Implications in Holliday Junction Resolution

Double-strand break repair involves the formation of Holliday junction (HJ) structures that need to be resolved to promote correct replication and chromosomal segregation. The molecular mechanisms of HJ branch migration and/or resolution are poorly characterized in Firmicutes. Genetic evidence suggested that the absence of the RuvAB branch migration translocase and the RecU HJ resolvase is synthetically lethal in Bacillus subtilis, whereas a recU recG mutant was viable. In vitro RecU, which is restricted to bacteria of the Firmicutes phylum, binds HJs with high affinity. In this work we found that RecU does not bind simultaneously with RecG to a HJ. RuvB by interacting with RecU bound to the central region of HJ DNA, loses its nonspecific association with DNA, and re-localizes with RecU to form a ternary complex. RecU cannot stimulate the ATPase or branch migration activity of RuvB. The presence of RuvB·ATPγS greatly stimulates RecU-mediated HJ resolution, but the addition of ATP or RuvA abolishes this stimulatory effect. A RecU·HJ·RuvAB complex might be formed. RecU does not increase the RuvAB activities but slightly inhibits them.     

Functional significance of octameric RuvA for a branch migration complex from Thermus thermophilus

The RuvAB complex promotes migration of Holliday junction at the late stage of homologous recombination. The RuvA tetramer specifically recognizes Holliday junction to form two types of complexes. A single tetramer is bound to the open configuration of the junction DNA in complex I, while the octameric RuvA core structure sandwiches the same junction in complex II. The hexameric RuvB rings, symmetrically bound to both sides of RuvA on Holliday junction, pump out DNA duplexes, depending upon ATP hydrolysis. We investigated functional differences between the wild-type RuvA from Thermus thermophilus and mutants impaired the ability of complex II formation. These mutant RuvA, exclusively forming complex I, reduced activities of branch migration and ATP hydrolysis, suggesting that the octameric RuvA is essential for efficient branch migration. Together with our recent electron microscopic analysis, this finding provides important insights into functional roles of complex II in the coordinated branch migration mechanism.     

Processing of recombination intermediates by the RecG and RuvAB proteins of Escherichia coli.

The RuvAB, RuvC and RecG proteins of Escherichia coli process intermediates in recombination and DNA repair into mature products. RuvAB and RecG catalyse branch migration of Holliday junctions, while RuvC resolves these structures by nuclease cleavage around the point of strand exchange. The overlap between RuvAB and RecG was investigated using synthetic X- and Y-junctions. RuvAB is a complex of RuvA and RuvB, with RuvA providing the DNA binding subunit and RuvB the ATPase activity that drives branch migration. Both RuvA and RecG form defined complexes with each of the junctions. The gel mobilities of these complexes suggests that the X-junction attracts two tetramers of RuvA, but mainly monomers of RecG. Dissociation of the junction in the presence of ATP requires high levels of RuvAB. RecG is shown to have a much higher specific activity to the extent that very little of this protein would be required to match RuvAB in vivo. Both proteins also dissociate a Y-junction, which is consistent with helicase activity. However, RecG shows no ability to unwind more conventional substrates and the suggestion is made that its helicase activity is directed towards specific DNA structures such as junctions.     

The RuvAB branch migration complex can displace topoisomerase IV.quinolone.DNA ternary complexes

Quinolone antimicrobial drugs target both DNA gyrase and topoisomerase IV (Topo IV) and convert these essential enzymes into cellular poisons. Topoisomerase poisoning results in the inhibition of DNA replication and the generation of double-strand breaks. Double-strand breaks are repaired by homologous recombination. Here, we have investigated the interaction between the RuvAB branch migration complex and the Topo IV.quinolone.DNA ternary complex. A strand-displacement assay is employed to assess the helicase activity of the RuvAB complex in vitro. RuvAB-catalyzed strand displacement requires both RuvA and RuvB proteins, and it is stimulated by a 3'-non-hybridized tail. Interestingly, Topo IV.quinolone.DNA ternary complexes do not inhibit the translocation of the RuvAB complex. In fact, Topo IV.quinolone.DNA ternary complexes are reversed and displaced from the DNA upon their collisions with the RuvAB complex. These results suggest that the RuvAB branch migration complex can actively remove quinolone-induced covalent topoisomerase.DNA complexes from DNA and complete the homologous recombination process in vivo.     

Electron microscopic single particle analysis of a tetrameric RuvA/RuvB/Holliday junction DNA complex

During the late stage of homologous recombination in prokaryotes, RuvA binds to the Holliday junction intermediate and executes branch migration in association with RuvB. The RuvA subunits form two distinct complexes with the Holliday junction: complex I with the single RuvA tetramer on one side of the four way junction DNA, and complex II with two tetramers on both sides. To investigate the functional roles of complexes I and II, we mutated two residues of RuvA (L125D and E126K) to prevent octamer formation. An electron microscopic analysis indicated that the mutant RuvA/RuvB/Holliday junction DNA complex formed the characteristic tripartite structure, with only one RuvA tetramer bound to one side of the Holliday junction, demonstrating the unexpected stability of this complex. The novel bent images of the complex revealed an intriguing morphological similarity to the structure of SV40 large T antigen, which belongs to the same AAA+ family as RuvB.     

RuvAB-mediated branch migration does not involve extensive DNA opening within the RuvB hexamer

The Escherichia coli RuvA and RuvB proteins promote the branch migration of Holliday junctions during the late stages of homologous recombination and DNA repair (reviewed in [1]). Biochemical and structural studies of the RuvAB-Holliday junction complex have shown that RuvA binds directly to the Holliday junction [2] [3] [4] [5] [6] and acts as a specificity factor that promotes the targeting of RuvB [7] [8], a hexameric ring protein that drives branch migration [9] [10] [11]. Electron microscopic visualisation of the RuvAB complex revealed that RuvA is flanked by two RuvB hexamers, which bind DNA arms that lie diametrically opposed across the junction [8]. ATP-dependent branch migration occurs as duplex DNA is pumped out through the centre of each ring. Because RuvB possesses well-conserved helicase motifs and RuvAB exhibits a 5'-3' DNA helicase activity in vitro [12], the mechanism of branch migration is thought to involve DNA opening within the RuvB ring, which provides a single strand for the unidirectional translocation of the protein along DNA. We have investigated whether the RuvB ring can translocate along duplex DNA containing a site-directed interstrand psoralen crosslink. Surprisingly, we found that the crosslink failed to inhibit branch migration. We interpret these data as evidence against a base-by-base tracking model and suggest that extensive DNA opening within the RuvB ring is not required for DNA translocation by RuvB.     

The acidic pin of RuvA modulates Holliday junction binding and processing by the RuvABC resolvasome

Holliday junctions are four-way branched DNA structures formed during recombination, replication and repair. They are processed in Escherichia coli by the RuvA, RuvB and RuvC proteins. RuvA targets the junction and facilitates loading of RuvB helicase and RuvC endonuclease to form complexes that catalyse junction branch migration (RuvAB) and resolution (RuvABC). We investigated the role of RuvA in these reactions and in particular the part played by the acidic pin located on its DNA-binding surface. By making appropriate substitutions of two key amino acids (Glu55 and Asp56), we altered the charge on the pin and investigated how this affected junction binding and processing. We show that two negative charges on each subunit of the pin are crucial. They facilitate junction targeting by preventing binding to duplex DNA and also constrain branch migration by RuvAB in a manner critical for junction processing. These findings provide the first direct evidence that RuvA has a mechanistic role in branch migration. They also provide insight into the coupling of branch migration and resolution by the RuvABC resolvasome.     

Escherichia coli RuvBL268S: a mutant RuvB protein that exhibits wild-type activities in vitro but confers a UV-sensitive ruv phenotype in vivo.

The RuvABC proteins of Escherichia coli process recombination intermediates during genetic recombination and DNA repair. RuvA and RuvB promote branch migration of Holliday junctions, a process that extends heteroduplex DNA. Together with RuvC, they form a RuvABC complex capable of Holliday junction resolution. Branch migration by RuvAB is mediated by RuvB, a hexameric ring protein that acts as an ATP-driven molecular pump. To gain insight into the mechanism of branch migration, random mutations were introduced into the ruvB gene by PCR and a collection of mutant alleles were obtained. Mutation of leucine 268 to serine resulted in a severe UV-sensitive phenotype, characteristic of a ruv defect. Here, we report a biochemical analysis of the mutant protein RuvBL268S. Unexpectedly, the purified protein is fully active in vitro with regard to its ATPase, DNA binding and DNA unwinding activities. It also promotes efficient branch migration in combination with RuvA, and forms functional RuvABC-Holliday junction resolvase complexes. These results indicate that RuvB may perform some additional, and as yet undefined, function that is necessary for cell survival after UV-irradiation.     

Helicase-defective RuvB(D113E) promotes RuvAB-mediated branch migration in vitro.

In Escherichia coli, the RuvA and RuvB proteins interact at Holliday junctions to promote branch migration leading to the formation of heteroduplex DNA. RuvA provides junction-binding specificity and RuvB drives ATP-dependent branch migration. Since RuvB contains sequence motifs characteristic of a DNA helicase and RuvAB exhibit helicase activity in vitro, we have analysed the role of DNA unwinding in relation to branch migration. A mutant RuvB protein, RuvB(D113E), mutated in helicase motif II (the DExx box), has been purified to homogeneity. The mutant protein forms hexameric rings on DNA similar to those formed by wild-type protein and promotes branch migration in the presence of RuvA. However, RuvB(D113E) exhibits reduced ATPase activity and is severely compromised in its DNA helicase activity. Models for RuvAB-mediated branch migration that invoke only limited DNA unwinding activity are proposed.     

A unique beta-hairpin protruding from AAA+ ATPase domain of RuvB motor protein is involved in the interaction with RuvA DNA recognition protein for branch migration of Holliday junctions.

The Escherichia coli RuvB protein is a motor protein that forms a complex with RuvA and promotes branch migration of Holliday junctions during homologous recombination. This study describes the characteristics of two RuvB mutants, I148T and I150T, that do not promote branch migration in the presence of RuvA. These RuvB mutants hydrolyzed ATP and bound duplex DNA with the same efficiency as wild-type RuvB, but the mutants did not form a complex with RuvA and were defective in loading onto junction DNA in a RuvA-assisted manner. A recent crystallographic study revealed that Ile(148) and Ile(150) are in a unique beta-hairpin that protrudes from the AAA(+) ATPase domain of RuvB. We propose that this beta-hairpin interacts with hydrophobic residues in the mobile third domain of RuvA and that this interaction is vital for the RuvA-assisted loading of RuvB onto Holliday junction DNA.     

Escherichia coli MutS,L modulate RuvAB-dependent branch migration between diverged DNA

This study examines the interaction between Escherichia coli MutS,L and E. coli RuvAB during E. coli RecA-promoted strand exchange. RuvAB is a branch migration complex that stimulates heterologous strand exchange. Previous studies indicate that RuvAB increases the rate at which heteroduplex products are formed by RecA, that RuvA and RuvB are required for this stimulation, and that RuvAB does not stimulate homologous strand exchange. This study indicates that MutS,L inhibit the formation of full-length heteroduplex DNA between M13-fd DNA in the presence of RuvAB, such that less than 2% of the linear substrate is converted to product. Inhibition depends on the time at which MutS,L are added to the reaction and is strongest when MutS,L are added during initiation. The kinetics of the strand exchange reaction suggest that MutS,L directly inhibit RuvAB-dependent branch migration in the absence of RecA. The inhibition requires the formation of base-base mismatches and ATP utilization; no effect on RuvAB-promoted strand exchange is seen if an ATP-deficient mutant of MutS (MutS501) is included in the reaction instead of wild-type MutS. These results are consistent with a role for MutS,L in maintaining genomic stability and replication fidelity.     

Identification of RecQL1 as a Holliday junction processing enzyme in human cell lines

Homologous recombination provides an effective way to repair DNA double-strand breaks (DSBs) and is required for genetic recombination. During the process of homologous recombination, a heteroduplex DNA structure, or a ‘Holliday junction’ (HJ), is formed. The movement, or branch migration, of this junction is necessary for recombination to proceed correctly. In prokaryotes, the RecQ protein or the RuvA/RuvB protein complex can promote ATP-dependent branch migration of Holliday junctions. Much less is known about the processing of Holliday junctions in eukaryotes. Here, we identify RecQL1 as a predominant ATP-dependent, HJ branch migrator present in human nuclear extracts. A reduction in the level of RecQL1 induced by RNA interference in HeLa cells leads to an increase in sister chromatid exchange. We propose that RecQL1 is involved in the processing of Holliday junctions in human cells.     

The RuvA homologues from Mycoplasma genitalium and Mycoplasma pneumoniae exhibit unique functional characteristics

The DNA recombination and repair machineries of Mycoplasma genitalium and Mycoplasma pneumoniae differ considerably from those of gram-positive and gram-negative bacteria. Most notably, M. pneumoniae is unable to express a functional RecU Holliday junction (HJ) resolvase. In addition, the RuvB homologues from both M. pneumoniae and M. genitalium only exhibit DNA helicase activity but not HJ branch migration activity in vitro. To identify a putative role of the RuvA homologues of these mycoplasmas in DNA recombination, both proteins (RuvA(Mpn) and RuvA(Mge), respectively) were studied for their ability to bind DNA and to interact with RuvB and RecU. In spite of a high level of sequence conservation between RuvA(Mpn) and RuvA(Mge) (68.8% identity), substantial differences were found between these proteins in their activities. First, RuvA(Mge) was found to preferentially bind to HJs, whereas RuvA(Mpn) displayed similar affinities for both HJs and single-stranded DNA. Second, while RuvA(Mpn) is able to form two distinct complexes with HJs, RuvA(Mge) only produced a single HJ complex. Third, RuvA(Mge) stimulated the DNA helicase and ATPase activities of RuvB(Mge), whereas RuvA(Mpn) did not augment RuvB activity. Finally, while both RuvA(Mge) and RecU(Mge) efficiently bind to HJs, they did not compete with each other for HJ binding, but formed stable complexes with HJs over a wide protein concentration range. This interaction, however, resulted in inhibition of the HJ resolution activity of RecU(Mge).     

Characterisation of RuvAB-Holliday junction complexes by glycerol gradient sedimentation.

The Escherichia coli RuvA and RuvB proteins interact specifically with Holliday junctions to promote ATP-dependent branch migration during genetic recombination and DNA repair. In the work described here, glycerol gradient centrifugation was used to investigate the requirements for the formation of pre-branch migration complexes. Since gradient centrifugation provides a simple and gentle method to analyse relatively unstable protein-DNA complexes, we were able to detect RuvA- and RuvAB-Holliday junction complexes without the need for chemical fixation. Using 35S-labelled RuvA protein and 3H-labelled Holliday junctions, we show that RuvA acts as a helicase accessory factor that loads the RuvB helicase onto the Holliday junction by structure-specific interactions. The resulting complex contained both RuvA and RuvB, as detected by Western blotting using serum raised against RuvA and RuvB. The stoichiometry of binding was estimated to be approximately four RuvA tetramers per junction. Formation of the RuvAB-Holliday junction complex required the presence of divalent metal ions and occurred without the need for ATP. However, the stability of the complex was enhanced by the presence of ATP gamma S, a non-hydrolysable ATP analogue. The data support a model for branch migration in which structure-specific binding of Holliday junctions by RuvA targets the assembly of hexameric RuvB rings on DNA. Specific loading of the RuvB ring helicase by RuvA is likely to be the initial step towards ATP-dependent branch migration.