An essential DNA strand‐exchange activity is conserved in the divergent N‐termini of BLM orthologs

The gene mutated in Bloom's syndrome, BLM, encodes a member of the RecQ family of DNA helicases that is needed to suppress genome instability and cancer predisposition. BLM is highly conserved and all BLM orthologs, including budding yeast Sgs1, have a large N‐terminus that binds Top3–Rmi1 but has no known catalytic activity. In this study, we describe a sub‐domain of the Sgs1 N‐terminus that shows in vitro single‐strand DNA (ssDNA) binding, ssDNA annealing and strand‐exchange (SE) activities. These activities are conserved in the human and Drosophila orthologs. SE between duplex DNA and homologous ssDNA requires no cofactors and is inhibited by a single mismatched base pair. The SE domain of Sgs1 is required in vivo for the suppression of hyper‐recombination, suppression of synthetic lethality and heteroduplex rejection. The top3Δ slow‐growth phenotype is also SE dependent. Surprisingly, the highly divergent human SE domain functions in yeast. This work identifies SE as a new molecular function of BLM/Sgs1, and we propose that at least one role of SE is to mediate the strand‐passage events catalysed by Top3–Rmi1.     

Sgs1 regulates gene conversion tract lengths and crossovers independently of its helicase activity

RecQ helicases maintain genome stability and suppress tumors in higher eukaryotes through roles in replication and DNA repair. The yeast RecQ homolog Sgs1 interacts with Top3 topoisomerase and Rmi1. In vitro, Sgs1 binds to and branch migrates Holliday junctions (HJs) and the human RecQ homolog BLM, with Top3alpha, resolves synthetic double HJs in a noncrossover sense. Sgs1 suppresses crossovers during the homologous recombination (HR) repair of DNA double-strand breaks (DSBs). Crossovers are associated with long gene conversion tracts, suggesting a model in which Sgs1 helicase catalyzes reverse branch migration and convergence of double HJs for noncrossover resolution by Top3. Consistent with this model, we show that allelic crossovers and gene conversion tract lengths are increased in sgs1Delta. However, crossover and tract length suppression was independent of Sgs1 helicase activity, which argues against helicase-dependent HJ convergence. HJs may converge passively by a "random walk," and Sgs1 may play a structural role in stimulating Top3-dependent resolution. In addition to the new helicase-independent functions for Sgs1 in crossover and tract length control, we define three new helicase-dependent functions, including the suppression of chromosome loss, chromosome missegregation, and synthetic lethality in srs2Delta. We propose that Sgs1 has helicase-dependent functions in replication and helicase-independent functions in DSB repair by HR.     

Yeast Rmi1/Nce4 controls genome stability as a subunit of the Sgs1-Top3 complex

Genome stability requires a set of RecQ-Top3 DNA helicase-topoisomerase complexes whose sole budding yeast homolog is encoded by SGS1-TOP3. RMI1/NCE4 was identified as a potential intermediate in the SGS1-TOP3 pathway, based on the observation that strains lacking any one of these genes require MUS81 and MMS4 for viability. This idea was tested by confirming that sgs1 and rmi1 mutants display the same spectrum of synthetic lethal interactions, including the requirements for SLX1, SLX4, SLX5, and SLX8, and by demonstrating that rmi1 mus81 synthetic lethality is dependent on homologous recombination. On their own, mutations in RMI1 result in phenotypes that mimic those of sgs1 or top3 strains including slow growth, hyperrecombination, DNA damage sensitivity, and reduced sporulation. And like top3 strains, most rmi1 phenotypes are suppressed by mutations in SGS1. We show that Rmi1 forms a heteromeric complex with Sgs1-Top3 in yeast and that these proteins interact directly in a recombinant system. The Rmi1-Top3 complex is stable in the absence of the Sgs1 helicase, but the loss of either Rmi1 or Top3 in yeast compromises its partner's interaction with Sgs1. Biochemical studies demonstrate that recombinant Rmi1 is a structure-specific DNA binding protein with a preference for cruciform structures. We propose that the DNA binding specificity of Rmi1 plays a role in targeting Sgs1-Top3 to appropriate substrates.     

RMI1/NCE4, a suppressor of genome instability, encodes a member of the RecQ helicase/Topo III complex

SGS1 encodes a DNA helicase whose homologues in human cells include the BLM, WRN, and RECQ4 genes, mutations in which lead to cancer-predisposition syndromes. Clustering of synthetic genetic interactions identified by large-scale genetic network analysis revealed that the genetic interaction profile of the gene RMI1 (RecQ-mediated genome instability, also known as NCE4 and YPL024W) was highly similar to that of SGS1 and TOP3, suggesting a functional relationship between Rmi1 and the Sgs1/Top3 complex. We show that Rmi1 physically interacts with Sgs1 and Top3 and is a third member of this complex. Cells lacking RMI1 activate the Rad53 checkpoint kinase, undergo a mitotic delay, and display increased relocalization of the recombination repair protein Rad52, indicating the presence of spontaneous DNA damage. Consistent with a role for RMI1 in maintaining genome integrity, rmi1Delta cells exhibit increased recombination frequency and increased frequency of gross chromosomal rearrangements. In addition, rmi1Delta strains fail to fully activate Rad53 upon exposure to DNA-damaging agents, suggesting that Rmi1 is also an important part of the Rad53-dependent DNA damage response.     

Resolution by Unassisted Top3 Points to Template Switch Recombination Intermediates during DNA Replication

The evolutionarily conserved Sgs1/Top3/Rmi1 (STR) complex plays vital roles in DNA replication and repair. One crucial activity of the complex is dissolution of toxic X-shaped recombination intermediates that accumulate during replication of damaged DNA. However, despite several years of study the nature of these X-shaped molecules remains debated. Here we use genetic approaches and two-dimensional gel electrophoresis of genomic DNA to show that Top3, unassisted by Sgs1 and Rmi1, has modest capacities to provide resistance to MMS and to resolve recombination-dependent X-shaped molecules. The X-shaped molecules have structural properties consistent with hemicatenane-related template switch recombination intermediates (Rec-Xs) but not Holliday junction (HJ) intermediates. Consistent with these findings, we demonstrate that purified Top3 can resolve a synthetic Rec-X but not a synthetic double HJ in vitro. We also find that unassisted Top3 does not affect crossing over during double strand break repair, which is known to involve double HJ intermediates, confirming that unassisted Top3 activities are restricted to substrates that are distinct from HJs. These data help illuminate the nature of the X-shaped molecules that accumulate during replication of damaged DNA templates, and also clarify the roles played by Top3 and the STR complex as a whole during the resolution of replication-associated recombination intermediates.     

Mapping the DNA topoisomerase III binding domain of the Sgs1 DNA helicase

Several members of the RecQ family of DNA helicases are known to interact with DNA topoisomerase III (Top3). Here we show that the Saccharomyces cerevisiae Sgs1 and Top3 proteins physically interact in cell extracts and bind directly in vitro. Sgs1 and Top3 proteins coimmunoprecipitate from cell extracts under stringent conditions, indicating that Sgs1 and Top3 are present in a stable complex. The domain of Sgs1 which interacts with Top3 was identified by expressing Sgs1 truncations in yeast. The results indicate that the NH(2)-terminal 158 amino acids of Sgs1 are sufficient for the high affinity interaction between Sgs1 and Top3. In vitro assays using purified Top3 and NH(2)-terminal Sgs1 fragments demonstrate that at least part of the interaction is through direct protein-protein interactions with these 158 amino acids. Consistent with these physical data, we find that mutant phenotypes caused by a point mutation or small deletions in the Sgs1 NH(2) terminus can be suppressed by Top3 overexpression. We conclude that Sgs1 and Top3 form a tight complex in vivo and that the first 158 amino acids of Sgs1 are necessary and sufficient for this interaction. Thus, a primary role of the Sgs1 amino terminus is to mediate the Top3 interaction.     

Phosphoproteomics reveals a distinctive Mec1/ATR signaling response upon DNA end hyper‐resection

The Mec1/ATR kinase is crucial for genome maintenance in response to a range of genotoxic insults, but it remains unclear how it promotes context‐dependent signaling and DNA repair. Using phosphoproteomic analyses, we uncovered a distinctive Mec1/ATR signaling response triggered by extensive nucleolytic processing (resection) of DNA ends. Budding yeast cells lacking Rad9, a checkpoint adaptor and an inhibitor of resection, exhibit a selective increase in Mec1‐dependent phosphorylation of proteins associated with single‐strand DNA (ssDNA) transactions, including the ssDNA‐binding protein Rfa2, the translocase/ubiquitin ligase Uls1, and the Sgs1‐Top3‐Rmi1 (STR) complex that regulates homologous recombination (HR). Extensive Mec1‐dependent phosphorylation of the STR complex, mostly on the Sgs1 helicase subunit, promotes an interaction between STR and the DNA repair scaffolding protein Dpb11. Fusion of Sgs1 to phosphopeptide‐binding domains of Dpb11 strongly impairs HR‐mediated repair, supporting a model whereby Mec1 signaling regulates STR upon hyper‐resection to influence recombination outcomes. Overall, the identification of a distinct Mec1 signaling response triggered by hyper‐resection highlights the multi‐faceted action of this kinase in the coordination of checkpoint signaling and HR‐mediated DNA repair.     

The genetic consequences of ablating helicase activity and the Top3 interaction domain of Sgs1

Sgs1, the RecQ helicase homolog, and Top3, the type-IA topoisomerase, physically interact and are required for genomic stability in budding yeast. Similarly, topoisomerase III genes physically pair with homologs of SGS1 in humans that are involved in the cancer predisposition and premature aging diseases Bloom, Werner, and Rothmund-Thompson syndromes. In the absence of Top1 activity, sgs1 mutants are severely growth impaired. Here, we investigate the role of Sgs1 helicase activity and its N-terminal Top3 interaction domain by using an allele-replacement technique to integrate mutant alleles at the native SGS1 genomic locus. We compare the phenotype of helicase-defective (sgs1-hd) and N-terminal deletion (sgs1-NDelta) strains to wild-type and sgs1 null strains. Like the sgs1 null, sgs1-hd mutations suppress top3 slow growth, cause a growth defect in the absence of Srs2 helicase, and impair meiosis. However, for recombination and the synthetic interaction with top1Delta mutations, loss of helicase activity exhibits a less severe phenotype than the null. Interestingly, deletion of the Top3 interaction domain of Sgs1 causes a top3-like phenotype, and furthermore, this effect is dependent on helicase activity. These results suggest that the protein-protein interaction between these two DNA-metabolism enzymes, even in the absence of helicase activity, is important for their function in catalyzing specific changes in DNA topology.     

Mechanistically distinct roles for Sgs1p in checkpoint activation and replication fork maintenance

The RecQ helicase Sgs1p forms a complex with the type 1 DNA topoisomerase Top3p that resolves double Holliday junctions resulting from Rad51-mediated exchange. We find, however, that Sgs1p functions independently of both Top3p and Rad51p to stimulate the checkpoint kinase Rad53p when replication forks stall due to dNTP depletion on hydroxyurea. Checkpoint activation does not require Sgs1p function as a helicase, and correlates with its ability to bind the Rad53p kinase FHA1 motif directly. On the other hand, Sgs1p's helicase activity is required together with Top3p and the strand-exchange factor Rad51p, to help stabilise DNA polymerase epsilon at stalled replication forks. In this function, the Sgs1p/Top3p complex acts in parallel to the Claspin-related adaptor, Mrc1p, although the sgs1 and mrc1 mutations are epistatic for Rad53p activation. We thus identify two distinct pathways through which Sgs1p contributes to genomic integrity: checkpoint kinase activation requires Sgs1p as a noncatalytic Rad53p-binding site, while the combined Top3p/Sgs1p resolvase activity contributes to replisome stability and recovery from arrested replication forks.     

Pathways for Holliday junction processing during homologous recombination in Saccharomyces cerevisiae

The Saccharomyces cerevisiae Rmi1 protein is a component of the highly conserved Sgs1-Top3-Rmi1 complex. Deletion of SGS1, TOP3, or RMI1 is synthetically lethal when combined with the loss of the Mus81-Mms4 or Slx1-Slx4 endonucleases, which have been implicated in Holliday junction (HJ) resolution. To investigate the causes of this synthetic lethality, we isolated a temperature-sensitive mutant of the RMI1 strain, referred to as the rmi1-1 mutant. At the restrictive temperature, this mutant phenocopies an rmi1Δ strain but behaves like the wild type at the permissive temperature. Following a transient exposure to methyl methanesulfonate, rmi1-1 mutants accumulate unprocessed homologous recombination repair (HRR) intermediates. These intermediates are slowly resolved at the restrictive temperature, revealing a redundant resolution activity when Rmi1 is impaired. This resolution depends on Mus81-Mms4 but not on either Slx1-Slx4 or another HJ resolvase, Yen1. Similar results were also observed when Top3 function was impaired. We propose that the Sgs1-Top3-Rmi1 complex constitutes the main pathway for the processing of HJ-containing HRR intermediates but that Mus81-Mms4 can also resolve these intermediates.     

Essential functions of C terminus of Drosophila Topoisomerase IIIα in double holliday junction dissolution

Topoisomerase IIIα (Top3α) is an essential component of the double Holliday junction (dHJ) dissolvasome complex in metazoans, along with Blm and Rmi1/2. This important anti-recombinogenic function cannot be performed by Top3β, the other type IA topoisomerase present in metazoans. The two share a catalytic core but diverge in their tail regions. To understand this difference in function, we investigated the role of the unique C terminus of Top3α. The Drosophila C terminus contains an insert region not conserved among metazoans. This insert contributes an independent interaction with Blm, which may account for the absence of Rmi1 in Drosophila. Mutant Top3α lacking this insert maintains the ability to perform dHJ dissolution but only partially rescues a top3α null fly line, indicating an in vivo role for the insert. Truncation of the C terminus has a minimal effect on the type IA relaxation activity of Top3α; however, dHJ dissolution is greatly reduced. The Top3α C terminus was found to strongly interact with both Blm and DNA, which are critical to the dissolution reaction; these interactions are greatly reduced in the truncated enzyme. The truncation mutant also cannot rescue the viability of top3α null flies, indicating an essential in vivo role. Our data therefore suggest that the Top3α C terminus has an important role in dHJ dissolution (by providing an interaction interface for Blm and DNA) and an essential function in vivo.     

Involvement of topoisomerase III in telomere-telomere recombination

Telomere maintenance is required for chromosome stability, and telomeres are typically replicated by the action of telomerase. In both mammalian tumor and yeast cells that lack telomerase, telomeres are maintained by an alternative (ALT) recombination mechanism. In yeast, Sgs1p and its associated type IA topoisomerase, Top3p, may work coordinately in removing Holliday junction intermediates from a crossover-producing recombination pathway. Previous studies have also indicated that Sgs1 helicase acts in a telomere recombination pathway. Here we show that topoisomerase III is involved in telomere-telomere recombination. The recovery of telomere recombination-dependent survivors in a telomerase-minus yeast strain was dependent on Top3p catalytic activity. Moreover, the RIF1 and RIF2 genes are required for the establishment of TOP3/SGS1-dependent telomere-telomere recombination. In human Saos-2 ALT cells, human topoisomerase IIIalpha (hTOP3alpha) also contributes to telomere recombination. Strikingly, the telomerase activity is clearly enhanced in surviving si-hTOP3alpha Saos-2 ALT cells. Altogether, the present results suggest a potential role for hTOP3alpha in dissociating telomeric structures in telomerase-deficient cells, providing therapeutic implications in human tumors.     

Sgs1 helicase and two nucleases Dna2 and Exo1 resect DNA double-strand break ends

Formation of single-strand DNA (ssDNA) tails at a double-strand break (DSB) is a key step in homologous recombination and DNA-damage signaling. The enzyme(s) producing ssDNA at DSBs in eukaryotes remain unknown. We monitored 5'-strand resection at inducible DSB ends in yeast and identified proteins required for two stages of resection: initiation and long-range 5'-strand resection. We show that the Mre11-Rad50-Xrs2 complex (MRX) initiates 5' degradation, whereas Sgs1 and Dna2 degrade 5' strands exposing long 3' strands. Deletion of SGS1 or DNA2 reduces resection and DSB repair by single-strand annealing between distant repeats while the remaining long-range resection activity depends on the exonuclease Exo1. In exo1Deltasgs1Delta double mutants, the MRX complex together with Sae2 nuclease generate, in a stepwise manner, only few hundred nucleotides of ssDNA at the break, resulting in inefficient gene conversion and G2/M damage checkpoint arrest. These results provide important insights into the early steps of DSB repair in eukaryotes.     

Processing of homologous recombination repair intermediates by the Sgs1-Top3-Rmi1 and Mus81-Mms4 complexes

Homologous recombination repair (HRR) is an evolutionarily conserved cellular process that is important for the maintenance of genome stability during S phase. Inactivation of the Saccharomyces cerevisiae Sgs1-Top3-Rmi1 complex leads to the accumulation of unprocessed, X-shaped HRR intermediates (X structures) following replicative stress. Further characterization of these X structures may reveal why loss of BLM (the human Sgs1 ortholog) leads to the human cancer predisposition disorder, Bloom syndrome. In two recent complementary studies, we examined the nature of the X structures arising in yeast strains lacking Sgs1, Top3 or Rmi1 by identifying which proteins could process these structures in vivo. We revealed that the unprocessed X structures that accumulate in these strains could be resolved by the ectopic overexpression of two different Holliday junction (HJ) resolvases, and that the endogenous Mus81-Mms4 endonuclease could also remove them, albeit slowly. In this review, we discuss the implications of these results and review the putative roles for the Sgs1-Top3-Rmi1 and Mus81-Mms4 complexes in the processing of various types of HRR intermediates during S phase.     

The absence of Top3 reveals an interaction between the Sgs1 and Pif1 DNA helicases in Saccharomyces cerevisiae

RecQ DNA helicases and Topo III topoisomerases have conserved genetic, physical, and functional interactions that are consistent with a model in which RecQ creates a recombination-dependent substrate that is resolved by Topo III. The phenotype associated with Topo III loss suggests that accumulation of a RecQ-created substrate is detrimental. In yeast, mutation of the TOP3 gene encoding Topo III causes pleiotropic defects that are suppressed by deletion of the RecQ homolog Sgs1. We searched for gene dosage suppressors of top3 and identified Pif1, a DNA helicase that acts with polarity opposite to that of Sgs1. Pif1 overexpression suppresses multiple top3 defects, but exacerbates sgs1 and sgs1 top3 defects. Furthermore, Pif1 helicase activity is essential in the absence of Top3 in an Sgs1-dependent manner. These data clearly demonstrate that Pif1 helicase activity is required to counteract Sgs1 helicase activity that has become uncoupled from Top3. Pif1 genetic interactions with the Sgs1-Top3 pathway are dependent upon homologous recombination. We also find that Pif1 is recruited to DNA repair foci and that the frequency of these foci is significantly increased in top3 mutants. Our results support a model in which Pif1 has a direct role in the prevention or repair of Sgs1-induced DNA damage that accumulates in top3 mutants.     

Release of Ku and MRN from DNA ends by Mre11 nuclease activity and Ctp1 is required for homologous recombination repair of double-strand breaks

The multifunctional Mre11-Rad50-Nbs1 (MRN) protein complex recruits ATM/Tel1 checkpoint kinase and CtIP/Ctp1 homologous recombination (HR) repair factor to double-strand breaks (DSBs). HR repair commences with the 5'-to-3' resection of DNA ends, generating 3' single-strand DNA (ssDNA) overhangs that bind Replication Protein A (RPA) complex, followed by Rad51 recombinase. In Saccharomyces cerevisiae, the Mre11-Rad50-Xrs2 (MRX) complex is critical for DSB resection, although the enigmatic ssDNA endonuclease activity of Mre11 and the DNA-end processing factor Sae2 (CtIP/Ctp1 ortholog) are largely unnecessary unless the resection activities of Exo1 and Sgs1-Dna2 are also eliminated. Mre11 nuclease activity and Ctp1/CtIP are essential for DSB repair in Schizosaccharomyces pombe and mammals. To investigate DNA end resection in Schizo. pombe, we adapted an assay that directly measures ssDNA formation at a defined DSB. We found that Mre11 and Ctp1 are essential for the efficient initiation of resection, consistent with their equally crucial roles in DSB repair. Exo1 is largely responsible for extended resection up to 3.1 kb from a DSB, with an activity dependent on Rqh1 (Sgs1) DNA helicase having a minor role. Despite its critical function in DSB repair, Mre11 nuclease activity is not required for resection in fission yeast. However, Mre11 nuclease and Ctp1 are required to disassociate the MRN complex and the Ku70-Ku80 nonhomologous end-joining (NHEJ) complex from DSBs, which is required for efficient RPA localization. Eliminating Ku makes Mre11 nuclease activity dispensable for MRN disassociation and RPA localization, while improving repair of a one-ended DSB formed by replication fork collapse. From these data we propose that release of the MRN complex and Ku from DNA ends by Mre11 nuclease activity and Ctp1 is a critical step required to expose ssDNA for RPA localization and ensuing HR repair.     

Characterization of the slow-growth phenotype of S. cerevisiae Whip/Mgs1 Sgs1 double deletion mutants

RecQ DNA helicases from many organisms have been indicated to function in the maintenance of genomic stability. In human cells, mutation in the WRN helicase, a RecQ-like DNA helicase, results in the Werner syndrome (WS), a genetic disorder characterized by genomic instability and premature ageing. Similarly, mutation in SGS1, the RECQ homologue in budding yeast, results in genomic instability and accelerated ageing. We previously demonstrated that mouse WRN interacts physically with a novel, highly conserved protein that we named WHIP, and that in budding yeast cells, simultaneous deletion of WHIP/MGS1 and SGS1 results in slow growth and shortened life span. Here we show by using genetic analysis in Saccharomyces cerevisiae that mgs1Delta sgs1Delta cells have increased rates of terminal G2/M arrest, and show elevated rates of spontaneous sister chromatid recombination (SCR) and rDNA array recombination. Finally, we report that complementation of the synthetic relationship between SGS1 and WHIP/MGS1 requires both the helicase and Top3-binding activities of Sgs1, as well as the ATPase activity of Mgs1. Our results suggest that Whip/Mgs1 is implicated in DNA metabolism, and is required for normal growth and cell cycle progression in the absence of Sgs1.     

Saccharomyces cerevisiae Mre11/Rad50/Xrs2 and Ku proteins regulate association of Exo1 and Dna2 with DNA breaks

Single‐stranded DNA constitutes an important early intermediate for homologous recombination and damage‐induced cell cycle checkpoint activation. In Saccharomyces cerevisiae, efficient double‐strand break (DSB) end resection requires several enzymes; Mre11/Rad50/Xrs2 (MRX) and Sae2 are implicated in the onset of 5′‐strand resection, whereas Sgs1/Top3/Rmi1 with Dna2 and Exo1 are involved in extensive resection. However, the molecular events leading to a switch from the MRX/Sae2‐dependent initiation to the Exo1‐ and Dna2‐dependent resection remain unclear. Here, we show that MRX recruits Dna2 nuclease to DSB ends. MRX also stimulates recruitment of Exo1 and antagonizes excess binding of the Ku complex to DSB ends. Using resection assay with purified enzymes in vitro, we found that Ku and MRX regulate the nuclease activity of Exo1 in an opposite way. Efficient loading of Dna2 and Exo1 requires neither Sae2 nor Mre11 nuclease activities. However, Mre11 nuclease activity is essential for resection in the absence of extensive resection enzymes. The results provide new insights into how MRX catalyses end resection and recombination initiation.     

Functional and physical interaction between Sgs1 and Top3 and Sgs1-independent function of Top3 in DNA recombination repair

A mutant allele of SGS1 of Saccharomyces cerevisiae was identified as a suppressor of the slow-growth phenotype of top3 mutants. We previously reported the involvement of Top3 via the interaction with the N-terminal region of Sgs1 in the complementation of methylmethanesulfonate (MMS) sensitivity and the suppression of hyper recombination of a sgs1 mutant. In this study, we found that several amino acids residues in the N-terminal region of Sgs1 between residues 4 and 33 were responsible for binding to Top3 and essential for complementing the sensitivity to MMS of sgsl cells. Two-hybrid assays suggested that the region of Top3 responsible for the binding to Sgs1 was bipartite, with portion in the N- and C-terminal domains. Although disruption of the SGS1 gene suppressed the semi-lethality of the top3 mutant of strain MR, the sgsl-top3 double mutant grew more slowly and was more sensitive to MMS than the sgsl single mutant, indicating that Top3 plays some role independently of Sgs1. The DNA topoisomerase activity of Top3 was required for the Top3 function to repair DNA damages induced by MMS, as shown by the fact that the TOP3 gene carrying a mutation (Phe for Tyr) at the amino acid residue essential for its activity (residue 356) failed to restore the MMS sensitivity of sgs1-top3 to the level of that of the sgs1 single mutant. Epistatic analysis using the sgs1-top3 double mutant, rad52 mutant and sgs1-top3-rad52 triple mutant indicated that TOP3 belongs to the RAD52 recombinational repair pathway.     

Sgs1 function in the repair of DNA replication intermediates is separable from its role in homologous recombinational repair

Mutations in human homologues of the bacterial RecQ helicase cause diseases leading to cancer predisposition and/or shortened lifespan (Werner, Bloom, and Rothmund–Thomson syndromes). The budding yeast Saccharomyces cerevisiae has one RecQ helicase, Sgs1, which functions with Top3 and Rmi1 in DNA repair. Here, we report separation‐of‐function alleles of SGS1 that suppress the slow growth of top3Δ and rmi1Δ cells similar to an SGS1 deletion, but are resistant to DNA damage similar to wild‐type SGS1. In one allele, the second acidic region is deleted, and in the other, only a single aspartic acid residue 664 is deleted. sgs1‐D664Δ, unlike sgs1Δ, neither disrupts DNA recombination nor has synthetic growth defects when combined with DNA repair mutants. However, during S phase, it accumulates replication‐associated X‐shaped structures at damaged replication forks. Furthermore, fluorescent microscopy reveals that the sgs1‐D664Δ allele exhibits increased spontaneous RPA foci, suggesting that the persistent X‐structures may contain single‐stranded DNA. Taken together, these results suggest that the Sgs1 function in repair of DNA replication intermediates can be uncoupled from its role in homologous recombinational repair.