title: Genetic and biochemical evidences reveal novel insights into the mechanism underlyingSaccharomyces cerevisiaeSae2-mediated abrogation of DNA replication stress.
abstract: In Saccharomyces cerevisiae, the Mre11-Rad50-Xrs2 (MRX) protein complex plays pivotal roles in double-strand break (DSB) repair, replication stress and telomere length maintenance. Another protein linked to DSB repair is Sae2, which regulates MRX persistence at DSBs. However, very little is known about its role in DNA replication stress and repair. Here, we reveal a crucial role for Sae2 in DNA replication stress. We show that different mutant alleles of SAE2 cause hypersensitivity to genotoxic agents, and when combined with Δmre11 or nuclease-defective mre11 mutant alleles, the double mutants are considerably more sensitive suggesting that the sae2 mutations synergize with mre11 mutations. Biochemical studies demonstrate that Sae2 exists as a dimer in solution, associates preferentially with single-stranded and branched DNA structures, exhibits structure-specific endonuclease activity and cleaves these substrates from the 5' end. Furthermore, we show that the nuclease activity is indeed intrinsic to Sae2. Interestingly, sae2G270D protein possesses DNA-binding activity, but lacks detectable nuclease activity. Altogether, our data suggest a direct role for Sae2 nuclease activity in processing of the DNA structures that arise during replication and DNA damage and provide insights into the mechanism underlying Mre11-Sae2-mediated abrogation of replication stressrelated defects in S. cerevisiae.
authors.affiliation: Department of Biochemistry, Indian Institute of Science, Bangalore 560 012, India.
authors.forename: Indrajeet
authors.initials: I
authors.lastname: Ghodke
authors.forename: K
authors.initials: K
authors.lastname: Muniyappa
mesh_terms: D053903:DNA Breaks, Double-Stranded; D004260:DNA Repair / Q000235:genetics*; D004261:DNA Replication / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000235:genetics; D004706:Endodeoxyribonucleases / Q000096:biosynthesis / Q000235:genetics*; D004720:Endonucleases / Q000096:biosynthesis* / Q000235:genetics; D005090:Exodeoxyribonucleases / Q000096:biosynthesis / Q000235:genetics*; D046912:Multiprotein Complexes / Q000235:genetics; D009154:Mutation; D012441:Saccharomyces cerevisiae / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000096:biosynthesis* / Q000235:genetics*; D059505:Telomere Homeostasis / Q000235:genetics
chemical_list: D004268:DNA-Binding Proteins; D046912:Multiprotein Complexes; C086930:RAD50 protein, S cerevisiae; C107849:SAE2 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; C086929:XRS2 protein, S cerevisiae; D004706:Endodeoxyribonucleases; D004720:Endonucleases; D005090:Exodeoxyribonucleases; C094191:MRE11 protein, S cerevisiae