title: Heteroduplex DNA position defines the roles of the Sgs1, Srs2, and Mph1 helicases in promoting distinct recombination outcomes.
abstract: The contributions of the Sgs1, Mph1, and Srs2 DNA helicases during mitotic double-strand break (DSB) repair in yeast were investigated using a gap-repair assay. A diverged chromosomal substrate was used as a repair template for the gapped plasmid, allowing mismatch-containing heteroduplex DNA (hDNA) formed during recombination to be monitored. Overall DSB repair efficiencies and the proportions of crossovers (COs) versus noncrossovers (NCOs) were determined in wild-type and helicase-defective strains, allowing the efficiency of CO and NCO production in each background to be calculated. In addition, the products of individual NCO events were sequenced to determine the location of hDNA. Because hDNA position is expected to differ depending on whether a NCO is produced by synthesis-dependent-strand-annealing (SDSA) or through a Holliday junction (HJ)-containing intermediate, its position allows the underlying molecular mechanism to be inferred. Results demonstrate that each helicase reduces the proportion of CO recombinants, but that each does so in a fundamentally different way. Mph1 does not affect the overall efficiency of gap repair, and its loss alters the CO-NCO by promoting SDSA at the expense of HJ-containing intermediates. By contrast, Sgs1 and Srs2 are each required for efficient gap repair, strongly promoting NCO formation and having little effect on CO efficiency. hDNA analyses suggest that all three helicases promote SDSA, and that Sgs1 and Srs2 additionally dismantle HJ-containing intermediates. The hDNA data are consistent with the proposed role of Sgs1 in the dissolution of double HJs, and we propose that Srs2 dismantles nicked HJs.
authors.affiliation: Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, North Carolina, United States of America.
authors.forename: Katrina
authors.initials: K
authors.lastname: Mitchel
authors.forename: Kevin
authors.initials: K
authors.lastname: Lehner
authors.forename: Sue
authors.initials: S
authors.lastname: Jinks-Robertson
mesh_terms: D015825:Chromosomes, Fungal / Q000235:genetics; D003434:Crossing Over, Genetic; D053487:DEAD-box RNA Helicases / Q000235:genetics*; D053903:DNA Breaks, Double-Stranded; D004265:DNA Helicases / Q000235:genetics*; D004260:DNA Repair / Q000235:genetics; D045566:DNA, Cruciform / Q000235:genetics; D008938:Mitosis / Q000235:genetics; D009692:Nucleic Acid Heteroduplexes / Q000235:genetics; D051135:Rad51 Recombinase / Q000235:genetics; D053484:RecQ Helicases / Q000235:genetics*; D011995:Recombination, Genetic; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics*
chemical_list: D045566:DNA, Cruciform; D009692:Nucleic Acid Heteroduplexes; D029701:Saccharomyces cerevisiae Proteins; C061376:SRS2 protein, S cerevisiae; D051135:Rad51 Recombinase; C491240:MPH1 protein, S cerevisiae; C090472:SGS1 protein, S cerevisiae; D004265:DNA Helicases; D053484:RecQ Helicases; D053487:DEAD-box RNA Helicases