Context-Dependent Remodeling of Rad51–DNA Complexes by Srs2 Is Mediated by a Specific Protein–Protein Interaction

The yeast Srs2 helicase removes Rad51 nucleoprotein filaments from single-stranded DNA (ssDNA), preventing DNA strand invasion and exchange by homologous recombination. This activity requires a physical interaction between Srs2 and Rad51, which stimulates ATP turnover in the Rad51 nucleoprotein filament and causes dissociation of Rad51 from ssDNA. Srs2 also possesses a DNA unwinding activity and here we show that assembly of more than one Srs2 molecule on the 3′ ssDNA overhang is required to initiate DNA unwinding. When Rad51 is bound on the double-stranded DNA, its interaction with Srs2 blocks the helicase (DNA unwinding) activity of Srs2. Thus, in different DNA contexts, the physical interaction of Rad51 with Srs2 can either stimulate or inhibit the remodeling functions of Srs2, providing a means for tailoring DNA strand exchange activities to enhance the fidelity of recombination.     

Srs2 promotes Mus81–Mms4-mediated resolution of recombination intermediates

A variety of DNA lesions, secondary DNA structures or topological stress within the DNA template may lead to stalling of the replication fork. Recovery of such forks is essential for the maintenance of genomic stability. The structure-specific endonuclease Mus81–Mms4 has been implicated in processing DNA intermediates that arise from collapsed forks and homologous recombination. According to previous genetic studies, the Srs2 helicase may play a role in the repair of double-strand breaks and ssDNA gaps together with Mus81–Mms4. In this study, we show that the Srs2 and Mus81–Mms4 proteins physically interact in vitro and in vivo and we map the interaction domains within the Srs2 and Mus81 proteins. Further, we show that Srs2 plays a dual role in the stimulation of the Mus81–Mms4 nuclease activity on a variety of DNA substrates. First, Srs2 directly stimulates Mus81–Mms4 nuclease activity independent of its helicase activity. Second, Srs2 removes Rad51 from DNA to allow access of Mus81–Mms4 to cleave DNA. Concomitantly, Mus81–Mms4 inhibits the helicase activity of Srs2. Taken together, our data point to a coordinated role of Mus81–Mms4 and Srs2 in processing of recombination as well as replication intermediates.     

Rad51 gain-of-function mutants that exhibit high affinity DNA binding cause DNA damage sensitivity in the absence of Srs2

We previously identified several rad51 gain-of-function alleles that partially suppress the requirement for RAD55 and RAD57 in DNA repair. To gain further insight into the mechanism of action of these alleles, we compared the activities of Rad51-V328A, Rad51-P339S and Rad51-I345T with wild-type Rad51, for DNA binding, filament stability, strand exchange and interaction with the antirecombinase helicase, Srs2. These alleles were chosen because they show the highest activity in suppression of ionizing radiation sensitivity of the rad57 mutant, and Val 328 and Ile 345 are conserved in the human Rad51 protein. All three mutant proteins exhibited higher affinity for single-stranded DNA (ssDNA) and showed more robust strand exchange activity with oligonucleotide substrates than wild-type Rad51, with the Rad51-I345T and Rad51-V328A proteins displaying higher activity than Rad51-P339S. However, the Srs2 antirecombinase was able to disrupt Rad51–ssDNA complexes formed with all the mutant proteins. In vivo, the rad51-I345T mutant strain exhibited high resistance to methyl methane sulfonate that was dependent on functional SRS2. These results suggest the Srs2 translocase is able to disrupt Rad51–ssDNA complexes at stalled replication forks, but in the absence of Srs2 the enhanced DNA binding of the Rad51-I345T protein is detrimental to cell survival.     

The Srs2 helicase activity is stimulated by Rad51 filaments on dsDNA: implications for crossover incidence during mitotic recombination

Saccharomyces cerevisiae Srs2 helicase was shown to displace Rad51 in vitro upon translocation on single-stranded DNA. This activity is sufficient to account for its antirecombination effect and for the elimination of otherwise dead-end recombination intermediates. Roles for the helicase activity are yet unknown. Because cells lacking Srs2 show increased incidence of mitotic crossovers, it was postulated that Srs2 promotes synthesis-dependent strand annealing (SDSA) by unwinding the elongating invading strand from the donor strand. We report here that synthetic DNA structures that mimic D loops are good substrates for the Srs2 helicase activity, that Srs2 translocates on RPA-coated ssDNA, and, furthermore, that the helicase activity is largely stimulated by the presence of Rad51 nucleoprotein filaments on double-stranded DNA. These properties strongly support the idea that Srs2 actively prevents crossovers by promoting SDSA.     

Srs2 removes deadly recombination intermediates independently of its interaction with SUMO-modified PCNA

Saccharomyces cerevisiae Srs2 helicase plays at least two distinct functions. One is to prevent recombinational repair through its recruitment by sumoylated Proliferating Cell Nuclear Antigen (PCNA), evidenced in postreplication-repair deficient cells, and a second one is to eliminate potentially lethal intermediates formed by recombination proteins. Both actions are believed to involve the capacity of Srs2 to displace Rad51 upon translocation on single-stranded DNA (ssDNA), though a role of its helicase activity may be important to remove some toxic recombination structures. Here, we described two new mutants, srs2R1 and srs2R3, that have lost the ability to hinder recombinational repair in postreplication-repair mutants, but are still able to remove toxic recombination structures. Although the mutants present very similar phenotypes, the mutated proteins are differently affected in their biochemical activities. Srs2R1 has lost its capacity to interact with sumoylated PCNA while the biochemical activities of Srs2R3 are attenuated (ATPase, helicase, DNA binding and ability to displace Rad51 from ssDNA). In addition, crossover (CO) frequencies are increased in both mutants. The different roles of Srs2, in relation to its eventual recruitment by sumoylated PCNA, are discussed.     

Srs2 DNA helicase is involved in checkpoint response and its regulation requires a functional Mec1-dependent pathway and Cdk1 activity

In Saccharomyces cerevisiae the rate of DNA replication is slowed down in response to DNA damage as a result of checkpoint activation, which is mediated by the Mec1 and Rad53 protein kinases. We found that the Srs2 DNA helicase, which is involved in DNA repair and recombination, is phosphorylated in response to intra-S DNA damage in a checkpoint-dependent manner. DNA damage-induced Srs2 phosphorylation also requires the activity of the cyclin-dependent kinase Cdk1, suggesting that the checkpoint pathway might modulate Cdk1 activity in response to DNA damage. Moreover, srs2 mutants fail to activate Rad53 properly and to slow down DNA replication in response to intra-S DNA damage. The residual Rad53 activity observed in srs2 cells depends upon the checkpoint proteins Rad17 and Rad24. Moreover, DNA damage-induced lethality in rad17 mutants depends partially upon Srs2, suggesting that a functional Srs2 helicase causes accumulation of lethal events in a checkpoint-defective context. Altogether, our data implicate Srs2 in the Mec1 and Rad53 pathway and connect the checkpoint response to DNA repair and recombination.     

Localization of recombination proteins and Srs2 reveals anti-recombinase function in vivo

Homologous recombination (HR), although an important DNA repair mechanism, is dangerous to the cell if improperly regulated. The Srs2 “anti-recombinase” restricts HR by disassembling the Rad51 nucleoprotein filament, an intermediate preceding the exchange of homologous DNA strands. Here, we cytologically characterize Srs2 function in vivo and describe a novel mechanism for regulating the initiation of HR. We find that Srs2 is recruited separately to replication and repair centers and identify the genetic requirements for recruitment. In the absence of Srs2 activity, Rad51 foci accumulate, and surprisingly, can form in the absence of Rad52 mediation. However, these Rad51 foci do not represent repair-proficient filaments, as determined by recombination assays. Antagonistic roles for Rad52 and Srs2 in Rad51 filament formation are also observed in vitro. Furthermore, we provide evidence that Srs2 removes Rad51 indiscriminately from DNA, while the Rad52 protein coordinates appropriate filament reformation. This constant breakdown and rebuilding of filaments may act as a stringent quality control mechanism during HR.     

Human DNA Helicase B Functions in Cellular Homologous Recombination and Stimulates Rad51-Mediated 5′-3′ Heteroduplex Extension In Vitro

Homologous recombination is involved in the repair of DNA damage and collapsed replication fork, and is critical for the maintenance of genomic stability. Its process involves a network of proteins with different enzymatic activities. Human DNA helicase B (HDHB) is a robust 5′-3′ DNA helicase which accumulates on chromatin in cells exposed to DNA damage. HDHB facilitates cellular recovery from replication stress, but its role in DNA damage response remains unclear. Here we report that HDHB silencing results in reduced sister chromatid exchange, impaired homologous recombination repair, and delayed RPA late-stage foci formation induced by ionizing radiation. Ectopically expressed HDHB colocalizes with Rad51, Rad52, RPA, and ssDNA. In vitro, HDHB stimulates Rad51-mediated heteroduplex extension in 5′-3′ direction. A helicase-defective mutant HDHB failed to promote this reaction. Our studies implicate HDHB promotes homologous recombination in vivo and stimulates 5′-3′ heteroduplex extension during Rad51-mediated strand exchange in vitro.     

Srs2 mediates PCNA‐SUMO‐dependent inhibition of DNA repair synthesis

Completion of DNA replication needs to be ensured even when challenged with fork progression problems or DNA damage. PCNA and its modifications constitute a molecular switch to control distinct repair pathways. In yeast, SUMOylated PCNA (S‐PCNA) recruits Srs2 to sites of replication where Srs2 can disrupt Rad51 filaments and prevent homologous recombination (HR). We report here an unexpected additional mechanism by which S‐PCNA and Srs2 block the synthesis‐dependent extension of a recombination intermediate, thus limiting its potentially hazardous resolution in association with a cross‐over. This new Srs2 activity requires the SUMO interaction motif at its C‐terminus, but neither its translocase activity nor its interaction with Rad51. Srs2 binding to S‐PCNA dissociates Polδ and Polη from the repair synthesis machinery, thus revealing a novel regulatory mechanism controlling spontaneous genome rearrangements. Our results suggest that cycling cells use the Siz1‐dependent SUMOylation of PCNA to limit the extension of repair synthesis during template switch or HR and attenuate reciprocal DNA strand exchanges to maintain genome stability.     

ATPase and DNA helicase activities of the Saccharomyces cerevisiae anti-recombinase Srs2

Saccharomyces cerevisiae SRS2 encodes an ATP-dependent DNA helicase that is needed for DNA damage checkpoint responses and that modulates the efficiency of homologous recombination. Interestingly, strains simultaneously mutated for SRS2 and a variety of DNA repair genes show low viability that can be overcome by inactivating homologous recombination, thus implicating inappropriate recombination as the cause of growth impairment in these mutants. Here, we report on our biochemical characterization of the ATPase and DNA helicase activities of Srs2. ATP hydrolysis by Srs2 occurs efficiently only in the presence of DNA, with ssDNA being considerably more effective than dsDNA in this regard. Using homopolymeric substrates, the minimal DNA length for activating ATP hydrolysis is found to be 5 nucleotides, but a length of 10 nucleotides is needed for maximal activation. In its helicase action, Srs2 prefers substrates with a 3' ss overhang, and approximately 10 bases of 3' overhanging DNA is needed for efficient targeting of Srs2 to the substrate. Even though a 3' overhang serves to target Srs2, under optimized conditions blunt-end DNA substrates are also dissociated by this protein. The ability of Srs2 to unwind helicase substrates with a long duplex region is enhanced by the inclusion of the single-strand DNA-binding factor replication protein A.     

Role of Saccharomyces Single-Stranded DNA-Binding Protein RPA in the Strand Invasion Step of Double-Strand Break Repair

The single-stranded DNA (ssDNA)-binding protein replication protein A (RPA) is essential for both DNA replication and recombination. Chromatin immunoprecipitation techniques were used to visualize the kinetics and extent of RPA binding following induction of a double-strand break (DSB) and during its repair by homologous recombination in yeast. RPA assembles at the HO endonuclease-cut MAT locus simultaneously with the appearance of the DSB, and binding spreads away from the DSB as 5′ to 3′ exonuclease activity creates more ssDNA. RPA binding precedes binding of the Rad51 recombination protein. The extent of RPA binding is greater when Rad51 is absent, supporting the idea that Rad51 displaces RPA from ssDNA. RPA plays an important role during RAD51-mediated strand invasion of the MAT ssDNA into the donor sequence HML. The replication-proficient but recombination-defective rfa1-t11 (K45E) mutation in the large subunit of RPA is normal in facilitating Rad51 filament formation on ssDNA, but is unable to achieve synapsis between MAT and HML. Thus, RPA appears to play a role in strand invasion as well as in facilitating Rad51 binding to ssDNA, possibly by stabilizing the displaced ssDNA.     

Regulation of the bacteriophage T4 Dda helicase by Gp32 single-stranded DNA–binding protein

Dda, one of three helicases encoded by bacteriophage T4, has been well-characterized biochemically but its biological role remains unclear. It is thought to be involved in origin dependent DNA replication, recombination-dependent replication, anti-recombination, and recombination repair. The Gp32 protein of bacteriophage T4 plays critical roles in DNA replication, recombination, and repair by coordinating protein components of the replication fork and by stabilizing ssDNA. Previous work demonstrated that stimulation of DNA synthesis by Dda helicase appears to require direct Gp32–Dda protein–protein interactions and that Gp32 and Dda form a tight complex in the absence of ssDNA. Here we characterize the effects of Gp32–Dda physical and functional interactions through changes in the duplex DNA unwinding and ATPase activities of Dda helicase in the presence of different variants of Gp32 and different DNA repair and replication intermediate structures. Results show that Gp32–Dda interactions can be enhancing or inhibitory, depending on the Gp32 domain seen by Dda. Protein–protein interactions with Gp32 stimulate the unwinding activity of Dda, an effect associated with increased turnover of ATP, suggesting a higher rate of ATPase-driven translocation. Dda–Gp32 interactions also promote the unwinding of DNA substrates at higher salt concentrations and in the presence of substrate-bound DNA polymerase. Conversely, the formation of Gp32 clusters on ssDNA can inhibit unwinding, suggesting that Gp32–ssDNA formation sterically regulates which portions of replication and recombination intermediates are accessible for processing by Dda helicase. The data suggest a mechanism of replication fork restart in which Gp32 promotes Dda activity in template switching while preventing premature fork progression.     

Rad52 Sumoylation Prevents the Toxicity of Unproductive Rad51 Filaments Independently of the Anti-Recombinase Srs2

The budding yeast Srs2 is the archetype of helicases that regulate several aspects of homologous recombination (HR) to maintain genomic stability. Srs2 inhibits HR at replication forks and prevents high frequencies of crossing-over. Additionally, sensitivity to DNA damage and synthetic lethality with replication and recombination mutants are phenotypes that can only be attributed to another role of Srs2: the elimination of lethal intermediates formed by recombination proteins. To shed light on these intermediates, we searched for mutations that bypass the requirement of Srs2 in DNA repair without affecting HR. Remarkably, we isolated rad52-L264P, a novel allele of RAD52, a gene that encodes one of the most central recombination proteins in yeast. This mutation suppresses a broad spectrum of srs2Δ phenotypes in haploid cells, such as UV and γ-ray sensitivities as well as synthetic lethality with replication and recombination mutants, while it does not significantly affect Rad52 functions in HR and DNA repair. Extensive analysis of the genetic interactions between rad52-L264P and srs2Δ shows that rad52-L264P bypasses the requirement for Srs2 specifically for the prevention of toxic Rad51 filaments. Conversely, this Rad52 mutant cannot restore viability of srs2Δ cells that accumulate intertwined recombination intermediates which are normally processed by Srs2 post-synaptic functions. The avoidance of toxic Rad51 filaments by Rad52-L264P can be explained by a modification of its Rad51 filament mediator activity, as indicated by Chromatin immunoprecipitation and biochemical analysis. Remarkably, sensitivity to DNA damage of srs2Δ cells can also be overcome by stimulating Rad52 sumoylation through overexpression of the sumo-ligase SIZ2, or by replacing Rad52 by a Rad52-SUMO fusion protein. We propose that, like the rad52-L264P mutation, sumoylation modifies Rad52 activity thereby changing the properties of Rad51 filaments. This conclusion is strengthened by the finding that Rad52 is often associated with complete Rad51 filaments in vitro.     

Functional significance of the Rad51-Srs2 complex in Rad51 presynaptic filament disruption

The SRS2 (Suppressor of RAD Six screen mutant 2) gene encodes an ATP-dependent DNA helicase that regulates homologous recombination in Saccharomyces cerevisiae. Mutations in SRS2 result in a hyper-recombination phenotype, sensitivity to DNA damaging agents and synthetic lethality with mutations that affect DNA metabolism. Several of these phenotypes can be suppressed by inactivating genes of the RAD52 epistasis group that promote homologous recombination, implicating inappropriate recombination as the underlying cause of the mutant phenotype. Consistent with the genetic data, purified Srs2 strongly inhibits Rad51-mediated recombination reactions by disrupting the Rad51-ssDNA presynaptic filament. Srs2 interacts with Rad51 in the yeast two-hybrid assay and also in vitro. To investigate the functional relevance of the Srs2-Rad51 complex, we have generated srs2 truncation mutants that retain full ATPase and helicase activities, but differ in their ability to interact with Rad51. Importantly, the srs2 mutant proteins attenuated for Rad51 interaction are much less capable of Rad51 presynaptic filament disruption. An internal deletion in Srs2 likewise diminishes Rad51 interaction and anti-recombinase activity. We also present evidence that deleting the Srs2 C-terminus engenders a hyper-recombination phenotype. These results highlight the importance of Rad51 interaction in the anti-recombinase function of Srs2, and provide evidence that this Srs2 function can be uncoupled from its helicase activity.     

Fbh1 Limits Rad51-Dependent Recombination at Blocked Replication Forks

Controlling the loading of Rad51 onto DNA is important for governing when and how homologous recombination is used. Here we use a combination of genetic assays and indirect immunofluorescence to show that the F-box DNA helicase (Fbh1) functions in direct opposition to the Rad52 orthologue Rad22 to curb Rad51 loading onto DNA in fission yeast. Surprisingly, this activity is unnecessary for limiting spontaneous direct-repeat recombination. Instead it appears to play an important role in preventing recombination when replication forks are blocked and/or broken. When overexpressed, Fbh1 specifically reduces replication fork block-induced recombination, as well as the number of Rad51 nuclear foci that are induced by replicative stress. These abilities are dependent on its DNA helicase/translocase activity, suggesting that Fbh1 exerts its control on recombination by acting as a Rad51 disruptase. In accord with this, overexpression of Fbh1 also suppresses the high levels of recombinant formation and Rad51 accumulation at a site-specific replication fork barrier in a strain lacking the Rad51 disruptase Srs2. Similarly overexpression of Srs2 suppresses replication fork block-induced gene conversion events in an fbh1Δ mutant, although an inability to suppress deletion events suggests that Fbh1 has a distinct functionality, which is not readily substituted by Srs2.Homologous recombination (HR) is often described as a double-edged sword: it can maintain genome stability by promoting DNA repair, while its injudicious action can disturb genome stability by causing gross chromosome rearrangement (GCR) or loss of heterozygosity (LOH). Both GCR and LOH are potential precursors of diseases such as cancer, and consequently there is need to control when and how HR is used.A key step in most HR is the loading of the Rad51 recombinase onto single-stranded DNA (ssDNA), which forms a nucleoprotein filament (nucleofilament) that catalyzes the pairing of homologous DNAs and subsequent strand invasion (32). This is a critical point at which recombination can be regulated through the removal of the Rad51 filament (60). Early removal can prevent strand invasion altogether, freeing the DNA for alternative processing. Later removal may limit unnecessary filament growth, free the 3′-OH of the invading strand to prime DNA synthesis, and ultimately enable ejection of the invading strand, which is important for the repair of double-strand breaks (DSBs) by synthesis-dependent strand annealing (SDSA). SDSA avoids the formation of Holliday junctions that can be resolved into reciprocal exchange products (crossovers), which may result in GCR or LOH if the recombination is ectopic or allelic, respectively.One enzyme that appears to be able to control Rad51 in the aforementioned manner is the yeast superfamily 1 DNA helicase Srs2 (42). In Saccharomyces cerevisiae, Srs2 is recruited to stalled replication forks by the SUMOylation of PCNA, and there it appears to block Rad51-dependent HR in favor of Rad6- and Rad18-dependent postreplication repair (1, 2, 35, 50, 53, 58). In vitro Srs2 can strip Rad51 from ssDNA via its DNA translocase activity (31, 62) and therefore probably controls HR at stalled replication forks by acting as a Rad51 disruptase. In accord with this, chromatin immunoprecipitation analysis has shown that Rad51 is enriched at or near replication forks in an srs2 mutant (50). Srs2 also plays an important role in crossover avoidance during DSB repair, where it is thought to promote SDSA by both disrupting Rad51 nucleofilaments and dissociating displacement (D) loops (20, 27).Srs2 is conserved in the fission yeast Schizosaccharomyces pombe (19, 43, 63) and has a close relative in bacteria called UvrD, which can similarly control HR by disrupting RecA nucleofilaments (61). However, an obvious homologue in mammals has not been detected. Recently, two mammalian members of the RecQ DNA helicase family, BLM and RECQL5, were shown to disrupt Rad51 nucleofilaments in vitro (11, 25), although in the case of BLM, this activity appears to be relatively weak (5, 55). Nevertheless these data have led to speculation that both BLM and RECQL5 might perform a function similar to that of Srs2 in vivo (6). Certainly mutational inactivation of either helicase results in elevated levels of HR and genome instability, with an associated increased rate of cancer (23, 25). However, BLM and RECQL5 are not the only potential Rad51 disruptases in mammals; a relative of Srs2 and UvrD called FBH1 was recently implicated in this role by genetic studies of its orthologue in S. pombe and by its ability to partially compensate for the loss of Srs2 in S. cerevisiae, which, unlike S. pombe, lacks an FBH1 orthologue (15). FBH1 is so named because of an F box near its N terminus—a feature that makes it unique among DNA helicases (28). The F box is important for its interaction with SKP1 and therefore the formation of an E3 ubiquitin ligase SCF (SKP1-Cul1-F-box protein) complex (29). The targets of this complex are currently unknown. In S. pombe, mutations within Fbh1''s F-box block interaction with Skp1 and prevent Fbh1 from localizing to the nucleus and forming damage-induced foci therein (57). Fbh1''s role in constraining Rad51 activity in S. pombe is evidenced by the increase in spontaneous Rad51 foci and accumulation of UV irradiation-induced Rad51-dependent recombination intermediates in an fbh1Δ mutant (47). Moreover, loss of both Fbh1 and Srs2 in S. pombe results in a synergistic reduction in cell viability, and like Srs2, Fbh1 is essential for viability in the absence of the S. pombe RecQ family DNA helicase Rqh1, which processes recombination intermediates (47, 48). In both cases the synthetic interaction is suppressed by deleting rad51, suggesting that Fbh1 works in parallel with Srs2 and Rqh1 to prevent the formation of toxic recombination intermediates. In yeast, Rad51-mediated recombination is dependent on Rad52 (Rad22 in S. pombe), which is believed to promote the nucleation of Rad51 onto DNA that is coated with the ssDNA binding protein replication protein A (RPA) (18, 32). Intriguingly, the genotoxin sensitivity and recombination deficiency of a rad22 mutant are suppressed in a Rad51-dependent manner by deleting fbh1 (48). This suggests that Fbh1 and Rad22 act in opposing ways to modulate the assembly of the Rad51 nucleofilament. Although current data indicate a role for Fbh1 in controlling HR, the only evidence so far that Fbh1 limits recombinant formation is in chicken DT40 cells, for which a modest increase in sister chromatid exchange has been noted when FBH1 is deleted (30).Here we present in vivo evidence suggesting that Fbh1 does indeed act as a Rad51 disruptase, which is dependent on its DNA helicase/translocase activity. We confirm predictions that this activity works in opposition to Rad22 for the loading of Rad51 onto DNA and show that Fbh1''s modulation of Rad51 activity, while not essential for limiting spontaneous direct-repeat recombination, is critical for preventing recombination at blocked replication forks. Finally, we highlight similarities and differences between Fbh1 and Srs2, based on their mutant phenotypes and relative abilities to suppress recombination when overexpressed. Overall our data affirm that Fbh1 is one of the principal modulators of Rad51 activity in fission yeast and therefore may play a similar role in vertebrates.     

Srs2 enables checkpoint recovery by promoting disassembly of DNA damage foci from chromatin

Following DNA repair, checkpoint signalling must be abated to resume cell cycling in a phenomenon known as checkpoint recovery. Although a number of genes have been implicated in the recovery process, it is still unknown whether checkpoint recovery is caused by a signalling network activated by DNA repair or whether it is the result of the loss of DNA structures that elicit the checkpoint. Here we show that checkpoint recovery can be uncoupled from bulk chromosome DNA repair if single-stranded (ss) DNA persists. This situation occurs in cells that are deficient in the Srs2 helicase, a protein that antagonizes Rad51. We report that srs2Δ cells fail to eliminate Ddc2 and RPA subnuclear foci following bulk chromosome repair due to the persistence of ssDNA. In contrast to cells with DNA double-strand breaks that remain unrepaired, srs2Δ cells remove the 9-1-1 checkpoint clamp from chromatin after repair. However, despite the loss of the 9-1-1 clamp, Dpb11 remains associated with chromatin to promote checkpoint activity. Our work indicates that Srs2 promotes checkpoint recovery by removing Rad51 after DNA repair. A failure to remove Rad51 causes persistence of ssDNA and the checkpoint signal. Therefore, we conclude that cells initiate recovery when the DNA structures that elicit the checkpoint are eliminated.     

Role of ATP hydrolysis in the antirecombinase function of Saccharomyces cerevisiae Srs2 protein

Mutants of the Saccharomyces cerevisiae SRS2 gene are hyperrecombinogenic and sensitive to genotoxic agents, and they exhibit a synthetic lethality with mutations that compromise DNA repair or other chromosomal processes. In addition, srs2 mutants fail to adapt or recover from DNA damage checkpoint-imposed G2/M arrest. These phenotypic consequences of ablating SRS2 function are effectively overcome by deleting genes of the RAD52 epistasis group that promote homologous recombination, implicating an untimely recombination as the underlying cause of the srs2 mutant phenotypes. TheSRS2-encodedproteinhasasingle-stranded (ss) DNA-dependent ATPase activity, a DNA helicase activity, and an ability to disassemble the Rad51-ssDNA nucleoprotein filament, which is the key catalytic intermediate in Rad51-mediated recombination reactions. To address the role of ATP hydrolysis in Srs2 protein function, we have constructed two mutant variants that are altered in the Walker type A sequence involved in the binding and hydrolysis of ATP. The srs2 K41A and srs2 K41R mutant proteins are both devoid of ATPase and helicase activities and the ability to displace Rad51 from ssDNA. Accordingly, yeast strains harboring these srs2 mutations are hyperrecombinogenic and sensitive to methylmethane sulfonate, and they become inviable upon introducing either the sgs1Delta or rad54Delta mutation. These results highlight the importance of the ATP hydrolysisfueled DNA motor activity in SRS2 functions.     

Functions of the Snf2/Swi2 family Rad54 motor protein in homologous recombination

Homologous recombination is a central pathway to maintain genomic stability and is involved in the repair of DNA damage and replication fork support, as well as accurate chromosome segregation during meiosis. Rad54 is a dsDNA-dependent ATPase of the Snf2/Swi2 family of SF2 helicases, although Rad54 lacks classical helicase activity and cannot carry out the strand displacement reactions typical for DNA helicases. Rad54 is a potent and processive motor protein that translocates on dsDNA, potentially executing several functions in recombinational DNA repair. Rad54 acts in concert with Rad51, the central protein of recombination that performs the key reactions of homology search and DNA strand invasion. Here, we will review the role of the Rad54 protein in homologous recombination with an emphasis on mechanistic studies with the yeast and human enzymes. We will discuss how these results relate to in vivo functions of Rad54 during homologous recombination in somatic cells and during meiosis. This article is part of a Special Issue entitled: Snf2/Swi2 ATPase structure and function.     

The involvement of Srs2 in post-replication repair and homologous recombination in fission yeast

Homologous recombination is important for the repair of double-strand breaks and daughter strand gaps, and also helps restart stalled and collapsed replication forks. However, sometimes recombination is inappropriate and can have deleterious consequences. To temper recombination, cells have employed DNA helicases that unwind joint DNA molecules and/or dissociate recombinases from DNA. Budding yeast Srs2 is one such helicase. It can act by dissociating Rad51 nucleoprotein filaments, and is required for channelling DNA lesions to the post-replication repair (PRR) pathway. Here we have investigated the role of Srs2 in controlling recombination in fission yeast. Similar to budding yeast, deletion of fission yeast srs2 results in hypersensitivity to a range of DNA damaging agents, rhp51-dependent hyper-recombination and synthetic sickness when combined with rqh1^– that is suppressed by deleting rhp51, rhp55 or rhp57. Epistasis analysis indicates that Srs2 and the structure-specific endonuclease Mus81–Eme1 function in a sub-pathway of PRR for the tolerance/repair of UV-induced damage. However, unlike in Saccharomyces cerevisiae, Srs2 is not required for channelling lesions to the PRR pathway in Schizosaccharomyces pombe. In addition to acting as an antirecombinase, we also show that Srs2 can aid the recombinational repair of camptothecin-induced collapsed replication forks, independently of PRR.     

Srs2 helicase prevents the formation of toxic DNA damage during late prophase I of yeast meiosis

Proper repair of double-strand breaks (DSBs) is key to ensure proper chromosome segregation. In this study, we found that the deletion of the SRS2 gene, which encodes a DNA helicase necessary for the control of homologous recombination, induces aberrant chromosome segregation during budding yeast meiosis. This abnormal chromosome segregation in srs2 cells accompanies the formation of a novel DNA damage induced during late meiotic prophase I. The damage may contain long stretches of single-stranded DNAs (ssDNAs), which lead to aggregate formation of a ssDNA binding protein, RPA, and a RecA homolog, Rad51, as well as other recombination proteins inside of the nuclei, but not that of a meiosis-specific Dmc1. The Rad51 aggregate formation in the srs2 mutant depends on the initiation of meiotic recombination and occurs in the absence of chromosome segregation. Importantly, as an early recombination intermediate, we detected a thin bridge of Rad51 between two Rad51 foci in the srs2 mutant, which is rarely seen in wild type. These might be cytological manifestation of the connection of two DSB ends and/or multi-invasion. The DNA damage with Rad51 aggregates in the srs2 mutant is passed through anaphases I and II, suggesting the absence of DNA damage-induced cell cycle arrest after the pachytene stage. We propose that Srs2 helicase resolves early protein-DNA recombination intermediates to suppress the formation of aberrant lethal DNA damage during late prophase I.