Molecular Dissection of Mitotic Recombination in the Yeast Saccharomyces cerevisiae

Abstract

Recombination plays a central role in the repair of broken chromosomes in all eukaryotes. We carried out a systematic study of mitotic recombination. Using several assays, we established the chronological sequence of events necessary to repair a single double-strand break. Once a chromosome is broken, yeast cells become immediately committed to recombinational repair. Recombination is completed within an hour and exhibits two kinetic gaps. By using this kinetic framework we also characterized the role played by several proteins in the recombinational process. In the absence of Rad52, the broken chromosome ends, both 5 and 3, are rapidly degraded. This is not due to the inability to recombine, since the 3 single-stranded DNA ends are stable in a strain lacking donor sequences. Rad57 is required for two consecutive strand exchange reactions. Surprisingly, we found that the Srs2 helicase also plays an early positive role in the recombination process.The process of recombination plays an essential role during meiosis and in DNA repair during vegetative growth. Doublestrand breaks (DSBs) arise frequently as a consequence of exposure to external insults or as a direct result of natural cell metabolism. Recombinational repair of DSBs is important in solving collapsed replication forks during DNA replication (20). If left unrepaired, DSBs result in broken chromosomes, genetic alterations, or cell death. Repair of DSBs (DSBR) takes place in eukaryotes by two competing processes: nonhomologous end joining and homologous recombination. In yeast cells, homologous recombination is the prevalent mechanism used (reviewed in reference 36).In a classic model for DSB-initiated recombination (53) (Fig. 1A), single-stranded degradation of the broken DNA molecule generates protruding 3Ј-OH ends that can invade homologous regions, creating a D-loop. The invasion process yields regions of heteroduplex DNA (hDNA) that may contain mismatches. The invading 3Ј end is then used to prime DNA synthesis. Eventually, the displaced donor strand pairs with single-stranded DNA (ssDNA) from the opposing end of the DSB, also serving as a template for DNA synthesis. Ligation results in the formation of a structure containing two Holliday junctions, which can be resolved to yield either crossover or noncrossover products. Mismatch repair of the hDNA may result in gene conversion events (Fig. 1A, left).An alternative model for gene conversion, termed the synthesis-dependent strand-annealing (SDSA) model (32), proposes that after DSB formation and resection, a single 3Ј single-stranded end invades the intact homologous template. DNA synthesis is followed by reannealing of the newly synthesized DNA with the opposite broken arm. In the basic version of this model (Fig. 1A, center), only gene conversion, and not crossover, can be obtained, although variations allowing crossing-over have been also proposed (reviewed in reference 36).Homologous recombination is catalyzed by a number of proteins encoded mostly by genes of the RAD52 epistasis group (36, 51). ...

Unloading of homologous recombination factors is required for restoring double‐stranded DNA at damage repair loci

“…Similarly, break processing and strand‐invasion operate during DSB repair by homologous recombination, but on two DNA ends. Not surprisingly, Srs2 is also required for DSB repair involving both ends (Vaze et al , 2002; Aylon et al , 2003) as re‐synthesis of both processed DNA ends would be required to complete the repair.…”

Role of ATP Hydrolysis in the Antirecombinase Function of Saccharomyces cerevisiae Srs2 Protein

“…Recently, reports from Haber and co-workers (20) and Kupiec and co-workers (19) showed that Srs2 is also needed for the repair of a site-specific double-strand break by synthesis-dependent single-strand annealing or double-ended synthesis-dependent single-strand annealing, the major pathway of gene conversion in mitotic yeast cells. In one of these published studies, the srs2 K41A allele was found to be defective in synthesis-dependent single-strand annealing (20).…”

“…In addition to the recombination-related phenotypes, the srs2⌬ mutant is partially deficient in the activation of the intra-S DNA damage checkpoint in response to treatment with methylmethane sulfonate (MMS) (18). Additionally, two independent studies (19,20) have implicated SRS2 in DNA double-strand break repair by the synthesis-dependent single-strand annealing mechanism as well. Srs2 also appears to influence the efficiency of single-strand annealing (17).…”

Regulation of Recombination and Genomic Maintenance

“…Interestingly, both yeast Srs2 and mammalian RECQ5 have been implicated in antirecombination (see above) and anticrossover pathways (Aylon et al 2003;Ira et al 2003;Paliwal et al 2014). Although the mechanisms involved still need to be established, it is possible that removal of Rad51 from ssDNA is involved in both.…”