Rrp1 translocase and ubiquitin ligase activities restrict the genome destabilising effects of Rad51 in fission yeast

Rad51 is the key protein in homologous recombination that plays important roles during DNA replication and repair. Auxiliary factors regulate Rad51 activity to facilitate productive recombination, and prevent inappropriate, untimely or excessive events, which could lead to genome instability. Previous genetic analyses identified a function for Rrp1 (a member of the Rad5/16-like group of SWI2/SNF2 translocases) in modulating Rad51 function, shared with the Rad51 mediator Swi5-Sfr1 and the Srs2 anti-recombinase. Here, we show that Rrp1 overproduction alleviates the toxicity associated with excessive Rad51 levels in a manner dependent on Rrp1 ATPase domain. Purified Rrp1 binds to DNA and has a DNA-dependent ATPase activity. Importantly, Rrp1 directly interacts with Rad51 and removes it from double-stranded DNA, confirming that Rrp1 is a translocase capable of modulating Rad51 function. Rrp1 affects Rad51 binding at centromeres. Additionally, we demonstrate in vivo and in vitro that Rrp1 possesses E3 ubiquitin ligase activity with Rad51 as a substrate, suggesting that Rrp1 regulates Rad51 in a multi-tiered fashion.     

The role of novel genes rrp1+ and rrp2+ in the repair of DNA damage in Schizosaccharomyces pombe

We identified two predicted proteins in Schizosaccharomyces pombe, Rrp1 (SPAC17A2.12) and Rrp2 (SPBC23E6.02) that share 34% and 36% similarity to Saccharomyces cerevisiae Ris1p, respectively. Ris1p is a DNA-dependent ATP-ase involved in gene silencing and DNA repair. Rrp1 and Rrp2 also share similarity with S. cerevisiae Rad5 and S. pombe Rad8, containing SNF2-N, RING finger and Helicase-C domains. To investigate the function of the Rrp proteins, we studied the DNA damage sensitivities and genetic interactions of null mutants with known DNA repair mutants. Single Δrrp1 and Δrrp2 mutants were not sensitive to CPT, 4NQO, CDPP, MMS, HU, UV or IR. The double mutants Δrrp1 Δrhp51 and Δrrp2 Δrhp51 plus the triple Δrrp1 Δrrp2 Δrhp51 mutant did not display significant additional sensitivity. However, the double mutants Δrrp1 Δrhp57 and Δrrp2 Δrhp57 were significantly more sensitive to MMS, CPT, HU and IR than the Δrhp57 single mutant. The checkpoint response in these strains was functional. In S. pombe, Rhp55/57 acts in parallel with a second mediator complex, Swi5/Sfr1, to facilitate Rhp51-dependent DNA repair. Δrrp1 Δsfr1 and Δrrp2 Δsfr1 double mutants did not show significant additional sensitivity, suggesting a function for Rrp proteins in the Swi5/Sfr1 pathway of DSB repair. Consistent with this, Δrrp1 Δrhp57 and Δrrp2 Δrhp57 mutants, but not Δrrp1 Δsfr1 or Δrrp2 Δsfr1 double mutants, exhibited slow growth and aberrations in cell and nuclear morphology that are typical of Δrhp51.     

Multiple Regulation of Rad51-Mediated Homologous Recombination by Fission Yeast Fbh1

Fbh1, an F-box helicase related to bacterial UvrD, has been proposed to modulate homologous recombination in fission yeast. We provide several lines of evidence for such modulation. Fbh1, but not the related helicases Srs2 and Rqh1, suppressed the formation of crossover recombinants from single HO-induced DNA double-strand breaks. Purified Fbh1 in complex with Skp1 (Fbh1-Skp1 complex) inhibited Rad51-driven DNA strand exchange by disrupting Rad51 nucleoprotein filaments in an ATP-dependent manner; this disruption was alleviated by the Swi5-Sfr1 complex, an auxiliary activator of Rad51. In addition, the reconstituted SCF^Fbh1 complex, composed of purified Fbh1-Skp1 and Pcu1-Rbx1, displayed ubiquitin-ligase E3 activity toward Rad51. Furthermore, Fbh1 reduced the protein level of Rad51 in stationary phase in an F-box-dependent, but not in a helicase domain-independent manner. These results suggest that Fbh1 negatively regulates Rad51-mediated homologous recombination via its two putative, unrelated activities, namely DNA unwinding/translocation and ubiquitin ligation. In addition to its anti-recombinase activity, we tentatively suggest that Fbh1 might also have a pro-recombination role in vivo, because the Fbh1-Skp1 complex stimulated Rad51-mediated strand exchange in vitro after strand exchange had been initiated.     

Sws1 is a conserved regulator of homologous recombination in eukaryotic cells

Rad52-dependent homologous recombination (HR) is regulated by the antirecombinase activities of Srs2 and Rqh1/Sgs1 DNA helicases in fission yeast and budding yeast. Functional analysis of Srs2 in Schizosaccharomyces pombe led us to the discovery of Sws1, a novel HR protein with a SWIM-type Zn finger. Inactivation of Sws1 suppresses the genotoxic sensitivity of srs2Delta and rqh1Delta mutants and rescues the inviability of srs2Delta rqh1Delta cells. Sws1 functions at an early step of recombination in a pro-recombinogenic complex with Rlp1 and Rdl1, two RecA-like proteins that are most closely related to the human Rad51 paralogs XRCC2 and RAD51D, respectively. This finding indicates that the XRCC2-RAD51D complex is conserved in lower eukaryotes. A SWS1 homolog exists in human cells. It associates with RAD51D and ablating its expression reduces the number of RAD51 foci. These studies unveil a conserved pathway for the initiation and control of HR in eukaryotic cells.     

Rad51/Dmc1 paralogs and mediators oppose DNA helicases to limit hybrid DNA formation and promote crossovers during meiotic recombination

During meiosis programmed DNA double-strand breaks (DSBs) are repaired by homologous recombination using the sister chromatid or the homologous chromosome (homolog) as a template. This repair results in crossover (CO) and non-crossover (NCO) recombinants. Only CO formation between homologs provides the physical linkages guiding correct chromosome segregation, which are essential to produce healthy gametes. The factors that determine the CO/NCO decision are still poorly understood. Using Schizosaccharomyces pombe as a model we show that the Rad51/Dmc1-paralog complexes Rad55-Rad57 and Rdl1-Rlp1-Sws1 together with Swi5-Sfr1 play a major role in antagonizing both the FANCM-family DNA helicase/translocase Fml1 and the RecQ-type DNA helicase Rqh1 to limit hybrid DNA formation and promote Mus81-Eme1-dependent COs. A common attribute of these protein complexes is an ability to stabilize the Rad51/Dmc1 nucleoprotein filament, and we propose that it is this property that imposes constraints on which enzymes gain access to the recombination intermediate, thereby controlling the manner in which it is processed and resolved.     

A postsynaptic role for Rhp55/57 that is responsible for cell death in Deltarqh1 mutants following replication arrest in Schizosaccharomyces pombe

Following replication arrest, multiple cellular responses are triggered to maintain genomic integrity. In fission yeast, the RecQ helicase, Rqh1, plays a critical role in this process. This is demonstrated in Deltarqh1 cells that, following treatment with hydroxyurea (HU), undergo an aberrant mitosis leading to cell death. Previous data suggest that Rqh1 functions with homologous recombination (HR) in recovery from replication arrest. We have found that loss of the HR genes rhp55(+) or rhp57(+), but not rhp51(+) or rhp54(+), suppresses the HU sensitivity of Deltarqh1 cells. Much of this suppression requires Rhp51 and Rhp54. In addition, this suppression is partially dependent on swi5(+). In budding yeast, overexpressing Rad51 (the Rhp51 homolog) minimized the need for Rad55/57 (Rhp55/57) in nucleoprotein filament formation. We overexpressed Rhp51 in Schizosaccharomyces pombe and found that it greatly reduced the requirement for Rhp55/57 in recovery from DNA damage. However, overexpressing Rhp51 did not change the Deltarhp55 suppression of the HU sensitivity of Deltarqh1, supporting an Rhp55/57 function during HR independent of nucleoprotein filament formation. These results are consistent with Rqh1 playing a role late in HR following replication arrest and provide evidence for a postsynaptic function for Rhp55/57.     

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.     

Rad51 presynaptic filament stabilization function of the mouse Swi5-Sfr1 heterodimeric complex

Homologous recombination (HR) represents a major error-free pathway to eliminate pre-carcinogenic chromosomal lesions. The DNA strand invasion reaction in HR is mediated by a helical filament of the Rad51 recombinase assembled on single-stranded DNA that is derived from the nucleolytic processing of the primary lesion. Recent studies have found that the human and mouse Swi5 and Sfr1 proteins form a complex that influences Rad51-mediated HR in cells. Here, we provide biophysical evidence that the mouse Swi5-Sfr1 complex has a 1:1 stoichiometry. Importantly, the Swi5-Sfr1 complex, but neither Swi5 nor Sfr1 alone, physically interacts with Rad51 and stimulates Rad51-mediated homologous DNA pairing. This stimulatory effect stems from the stabilization of the Rad51-ssDNA presynaptic filament. Moreover, we provide evidence that the RSfp (rodent Sfr1 proline rich) motif in Sfr1 serves as a negative regulatory element. These results thus reveal an evolutionarily conserved function in the Swi5-Sfr1 complex and furnish valuable information as to the regulatory role of the RSfp motif that is specific to the mammalian Sfr1 orthologs.     

Role for the mammalian Swi5-Sfr1 complex in DNA strand break repair through homologous recombination

In fission yeast, the Swi5-Sfr1 complex plays an important role in homologous recombination (HR), a pathway crucial for the maintenance of genomic integrity. Here we identify and characterize mammalian Swi5 and Sfr1 homologues. Mouse Swi5 and Sfr1 are nuclear proteins that form a complex in vivo and in vitro. Swi5 interacts in vitro with Rad51, the DNA strand-exchange protein which functions during HR. By generating Swi5(-/-) and Sfr1(-/-) embryonic stem cell lines, we found that both proteins are mutually interdependent for their stability. Importantly, the Swi5-Sfr1 complex plays a role in HR when Rad51 function is perturbed in vivo by expression of a BRC peptide from BRCA2. Swi5(-/-) and Sfr1(-/-) cells are selectively sensitive to agents that cause DNA strand breaks, in particular ionizing radiation, camptothecin, and the Parp inhibitor olaparib. Consistent with a role in HR, sister chromatid exchange induced by Parp inhibition is attenuated in Swi5(-/-) and Sfr1(-/-) cells, and chromosome aberrations are increased. Thus, Swi5-Sfr1 is a newly identified complex required for genomic integrity in mammalian cells with a specific role in the repair of DNA strand breaks.     

Unwinding of synthetic replication and recombination substrates by Srs2

The budding yeast Srs2 protein possesses 3′ to 5′ DNA helicase activity and channels untimely recombination to post-replication repair by removing Rad51 from ssDNA. However, it also promotes recombination via a synthesis-dependent strand-annealing pathway (SDSA). Furthermore, at the replication fork, Srs2 is required for fork progression and prevents the instability of trinucleotide repeats. To better understand the multiple roles of the Srs2 helicase during these processes, we analysed the ability of Srs2 to bind and unwind various DNA substrates that mimic structures present during DNA replication and recombination. While leading or lagging strands were efficiently unwound, the presence of ssDNA binding protein RPA presented an obstacle for Srs2 translocation. We also tested the preferred directionality of unwinding of various substrates and studied the effect of Rad51 and Mre11 proteins on Srs2 helicase activity. These biochemical results help us understand the possible role of Srs2 in the processing of stalled or blocked replication forks as a part of post-replication repair as well as homologous recombination (HR).     

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.     

The Yeast Shu Complex Utilizes Homologous Recombination Machinery for Error-free Lesion Bypass via Physical Interaction with a Rad51 Paralogue

DNA-damage tolerance (DDT) is defined as a mechanism by which eukaryotic cells resume DNA synthesis to fill the single-stranded DNA gaps left by replication-blocking lesions. Eukaryotic cells employ two different means of DDT, namely translesion DNA synthesis (TLS) and template switching, both of which are coordinately regulated through sequential ubiquitination of PCNA at the K164 residue. In the budding yeast Saccharomyces cerevisiae, the same PCNA-K164 residue can also be sumoylated, which recruits the Srs2 helicase to prevent undesired homologous recombination (HR). While the mediation of TLS by PCNA monoubiquitination has been extensively characterized, the method by which K63-linked PCNA polyubiquitination leads to template switching remains unclear. We recently identified a yeast heterotetrameric Shu complex that couples error-free DDT to HR as a critical step of template switching. Here we report that the Csm2 subunit of Shu physically interacts with Rad55, an accessory protein involved in HR. Rad55 and Rad57 are Rad51 paralogues and form a heterodimer to promote Rad51-ssDNA filament formation by antagonizing Srs2 activity. Although Rad55-Rad57 and Shu function in the same pathway and both act to inhibit Srs2 activity, Shu appears to be dedicated to error-free DDT while the Rad55-Rad57 complex is also involved in double-strand break repair. This study reveals the detailed steps of error-free lesion bypass and also brings to light an intrinsic interplay between error-free DDT and Srs2-mediated inhibition of HR.     

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.     

Role for the fission yeast RecQ helicase in DNA repair in G2

Members of the RecQ helicase subfamily are mutated in several human genomic instability syndromes, such as Bloom, Werner, and Rothmund-Thomson syndromes. We show that Rqh1, the single Schizosaccharomyces pombe homologue, is a 3'-to-5' helicase and exists with Top3 in a high-molecular-weight complex. top3 deletion is inviable, and this is suppressed by concomitant loss of rqh1 helicase activity or loss of recombination functions. This is consistent with RecQ helicases in other systems. By using epistasis analysis of the UV radiation sensitivity and by analyzing the kinetics of Rhp51 (Rad51 homologue), Rqh1, and Top3 focus formation in response to UV in synchronized cells, we identify the first evidence of a function for Rqh1 and Top3 in the repair of UV-induced DNA damage in G(2). Our data provide evidence that Rqh1 functions after Rad51 focus formation during DNA repair. We also identify a function for Rqh1 upstream of recombination in an Rhp18-dependent (Rad18 homologue) pathway. The model that these data allow us to propose helps to reconcile different interpretations of RecQ family helicase function that have arisen between work based on the S. pombe system and models based on studies of Saccharomyces cerevisiae SGS1 suggesting that RecQ helicases act before Rad51.     

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.     

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.     

Rad51 Paralog Complexes BCDX2 and CX3 Act at Different Stages in the BRCA1-BRCA2-Dependent Homologous Recombination Pathway

The Rad51 paralogs are required for homologous recombination (HR) and the maintenance of genomic stability. The molecular mechanisms by which the five vertebrate Rad51 paralogs regulate HR and genomic integrity remain unclear. The Rad51 paralogs associate with one another in two distinct complexes: Rad51B-Rad51C-Rad51D-XRCC2 (BCDX2) and Rad51C-XRCC3 (CX3). We find that the BCDX2 and CX3 complexes act at different stages of the HR pathway. In response to DNA damage, the BCDX2 complex acts downstream of BRCA2 recruitment but upstream of Rad51 recruitment. In contrast, the CX3 complex acts downstream of Rad51 recruitment but still has a marked impact on the measured frequency of homologous recombination. Both complexes are epistatic with BRCA2 and synthetically lethal with Rad52. We conclude that human Rad51 paralogs facilitate BRCA2-Rad51-dependent homologous recombination at different stages in the pathway and function independently of Rad52.     

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.     

Mechanistic insights into the activation of Rad51-mediated strand exchange from the structure of a recombination activator, the Swi5-Sfr1 complex

Rad51 forms a helical filament on single-stranded DNA and promotes strand exchange between two homologous DNA molecules during homologous recombination. The Swi5-Sfr1 complex interacts directly with Rad51 and stimulates strand exchange. Here we describe structural and functional aspects of the complex. Swi5 and the C-terminal core domain of Sfr1 form an essential activator complex with a parallel coiled-coil heterodimer joined firmly together via two previously uncharacterized leucine-zipper motifs and a bundle. The resultant coiled coil is sharply kinked, generating an elongated crescent-shaped structure suitable for transient binding within the helical groove of the Rad51 filament. The N-terminal region of Sfr1, meanwhile, has an interface for binding of Rad51. Our data suggest that the snug fit resulting from the complementary geometry of the heterodimer activates the Rad51 filament and that the N-terminal domain of Sfr1 plays a role in the efficient recruitment of the Swi5-Sfr1 complex to the Rad51 filaments.     

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.