We have previously shown that cells mutant for TOP3, a gene encoding a prokaryotic-like type I topoisomerase in Saccharomyces cerevisiae, display a pleiotropic phenotype including slow growth and genome instability. We identified a mutation, sgsl (slow growth suppressor), that suppresses both the growth defect and the increased genomic instability of top3 mutants. Here we report the independent isolation of the SGS1 gene in a screen for proteins that interact with Top3. DNA sequence analysis reveals that the putative Sgsl protein is highly homologous to the helicase encoded by the Escherichia coli recQ gene. These results imply that Sgsl creates a deleterious topological substrate that Top3 preferentially resolves. The interaction of the Sgsl helicase homolog and the Top3 topoisomerase is reminiscent of the recently described structure of reverse gyrase from Sulfolobus acidocaldarius, in which a type I DNA topoisomerase and a helicase-like domain are fused in a single polypeptide.Topoisomerases are ubiquitous enzymes essential for many aspects of DNA metabolism, including replication, transcriptional activation or repression, chromosome segregation, and genome stability (2,5,6,15,22,26,28,41,49). Changes in the expression of topoisomerases result in a pleiotropic phenotype in bacteria and yeast cells (33,50). In bacteria, there are four known topoisomerases. Mutations in three of these, topA, gyrAlgyrB, and parC/parE, cause changes in superhelical structure of DNA that affect the growth of the cell, transcription, transposition of transposon elements and replication as well as segregation of plasmids and daughter chromosomes (21,43,50). The fourth topoisomerase (topB) has been characterized in vitro (10,11,44) and shown to be involved in repetitive sequence stability in vivo (40).In Saccharomyces cerevisiae, topoisomerase mutations result in defects in nuclear division, transcription, recombination, and the supercoiling of plasmids. The TOP2 gene product is essential during mitosis and meiosis for the proper segregation of daughter chromosomes (12,19,20,36

Toxic recombination events are detected in vegetative Saccharomyces cerevisiae cells through negative growth interactions between certain combinations of mutations. For example, mutations affecting both the Srs2 and Sgs1 helicases result in extremely poor growth, a phenotype suppressed by mutations in genes that govern early stages of recombination. Here, we identify a similar interaction involving double mutations affecting Sgs1 or Top3 and Mus81 or Mms4. We also find that the primary DNA structures that initiate these toxic recombination events cannot be double-strand breaks and thus are likely to be single-stranded DNA. We interpret our results in the context of the idea that replication stalling leaves single-stranded DNA, which can then be processed by two competing mechanisms: recombination and nonrecombination gap-filling. Functions involved in preventing toxic recombination would either avoid replicative defects or act on recombination intermediates. Our results suggest that Srs2 channels recombination intermediates back into the gap-filling route, whereas Sgs1/Top3 and Mus81/Mms4 are involved in recombination and/or in replication to allow replication restart

DNA helicases are involved in many aspects of DNA metabolism, including transcription, replication, recombination and repair. In the yeast Saccharomyces cerevisiae, the absence of the Sgs1 helicase results in genomic instability and accelerated ageing. In human cells, mutations in orthologues of SGS1 lead to Bloom (BS), Werner (WS) or Rothmund-Thomson (RTS) syndromes, which are rare, autosomal recessive diseases characterized by genetic instability associated with cancer predisposition. Although data concerning these human diseases are accumulating, there is still no clear idea of the function of the proteins involved. Here we show that sgs1Delta mutants are deficient in DNA repair and are defective for induced recombination events that involve homologous chromosomes. The role of homologous recombination is further evidenced in haploid cells in which both Sgs1p and Srs2p are absent. Yeast SRS2 encodes another DNA helicase involved in the maintenance of genome integrity. Our data suggest that some defects observed in BS, WS or RTS are the consequence of unrestrained recombination.

The RecQ helicases are conserved from bacteria to humans and play a critical role in genome stability. In humans, loss of RecQ gene function is associated with cancer predisposition and/or premature aging. Recent data have shown that the RecQ helicases function during two distinct steps during DNA repair; DNA end resection and resolution of double Holliday junctions (dHJs). RecQ functions in these different processing steps has important implications for its role in repair of double-strand breaks (DSBs) that occur during DNA replication, meiosis and at specific genomic loci such as telomeres.

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.

DNA polymerases play a central role during homologous recombination (HR), but the identity of the enzyme(s) implicated remains elusive. The pol3-ct allele of the gene encoding the catalytic subunit of DNA polymerase ␦ (Pol␦) has highlighted a role for this polymerase in meiotic HR. We now address the ubiquitous role of Pol␦ during HR in somatic cells. We find that pol3-ct affects gene conversion tract length during mitotic recombination whether the event is initiated by single-strand gaps following UV irradiation or by site-specific double-strand breaks. We show that the pol3-ct effects on gene conversion are completely independent of mismatch repair, indicating that shorter gene conversion tracts in pol3-ct correspond to shorter extensions of primed DNA synthesis. Interestingly, we find that shorter repair tracts do not favor synthesis-dependent strand annealing at the expense of double-strand-break repair. Finally, we show that the DNA polymerases that have been previously suspected to mediate HR repair synthesis (Pol and Pol) do not affect gene conversion during induced HR, including in the pol3-ct background. Our results argue strongly for the preferential recruitment of Pol␦ during HR.Homologous recombination (HR) is a process that allows genetic exchange between DNA sequences sharing homology and leads to gene conversion or crossovers (COs). The ingenuity of this process is underscored by its conservation from bacteria to humans and its implication in a variety of unrelated nuclear processes. HR is implicated in the restart of stalled replication forks (24). It serves in the repair of DNA damage, such as single-strand gaps, double-strand breaks (DSBs) and interstrand cross-links. It is implicated in mating type switching in yeast strains and in the diversification of immunoglobulinvariable genes in vertebrates (6). In meiosis, the primary function of HR is to establish a physical connection between homologous chromosomes to ensure their correct disjunction at the first meiotic division. In addition, meiotic HR contributes to diversity by creating new linkage arrangements between genes or parts of genes (35).In the past few years, it has become evident that these multiple roles of HR are achieved with variations in the process. Two primary models allow for gene conversion as it is observed in different contexts (Fig. 1). In the seminal DSB repair (DSBR) model of Szostak et al. (41), supported mainly by molecular meiotic studies in yeast, the formation of DSBs is followed by exonucleolytic degradation of the 5Ј ends of the broken duplex to expose single-stranded tails with 3Ј termini (40) (Fig. 1). DSB formation and 5Ј-end resection are followed by the invasion of an intact nonsister chromatid by only one of the two single-stranded tails (13). Single-end invasion (SEI) results in hybrid DNA (hDNA), in which the two strands in a duplex are of different parental origin. If the two parental duplexes are genetically different within the region of strand exchange, the resulting hDNA contains mismatched base pairs...

Yeast cells mutant for TOP3, the gene encoding the evolutionary conserved type I-5Ј topoisomerase, display a wide range of phenotypes including altered cell cycle, hyper-recombination, abnormal gene expression, poor mating, chromosome instability and absence of sporulation. In this report, an analysis of the role of TOP3 in the meiotic process indicates that top3Δ mutants enter meiosis and complete the initial steps of recombination. However, reductional division does not occur. Deletion of the SPO11 gene, which prevents recombination between homologous chromosomes in meiosis I division, allows top3Δ mutants to form viable spores, indicating that Top3 is required to complete recombination successfully. A topoisomerase activity is involved in this process, since expression of bacterial TopA in yeast top3Δ mutants permits sporulation. The meiotic block is also partially suppressed by a deletion of SGS1, a gene encoding a helicase that interacts with Top3. We propose an essential role for Top3 in the processing of molecules generated during meiotic recombination.

In vegetative cells, most recombination intermediates are metabolized without an association with a crossover (CO). The avoidance of COs allows for repair and prevents genomic rearrangements, potentially deleterious if the sequences involved are at ectopic locations. We have designed a system that permits to screen spontaneous intragenic recombination events in Saccharomyces cerevisiae and to investigate the CO outcome in different genetic contexts. We have analyzed the CO outcome in the absence of the Srs2 and Sgs1 helicases, DNA damage checkpoint proteins as well as in a mutant proliferating cell nuclear antigen (PCNA) and found that they all contribute to genome stability. Remarkably high effects on COs are mediated by srs2D, mrc1D and a pol30-RR mutation in PCNA. Our results support the view that Mrc1 plays a specific role in DNA replication, promoting the Srs2 recruitment to PCNA independently of checkpoint signaling. Srs2 would prevent formation of double Holliday junctions (dHJs) and thus CO formation. Sgs1 also negatively regulates CO formation but through a different process that resolves dHJs to yield non-CO products.