title: Mutations in Mre11 phosphoesterase motif I that impair Saccharomyces cerevisiae Mre11-Rad50-Xrs2 complex stability in addition to nuclease activity.
abstract: The Mre11-Rad50-Xrs2 complex is involved in DNA double-strand break repair, telomere maintenance, and the intra-S phase checkpoint. The Mre11 subunit has nuclease activity in vitro, but the role of the nuclease in DNA repair and telomere maintenance remains controversial. We generated six mre11 alleles with substitutions of conserved residues within the Mre11-phosphoesterase motifs and compared the phenotypes conferred, as well as exonuclease activity and complex formation, by the mutant proteins. Substitutions of Asp16 conferred the most severe DNA repair and telomere length defects. Interactions between Mre11-D16A or Mre11-D16N and Rad50 or Xrs2 were severely compromised, whereas the mre11 alleles with greater DNA repair proficiency also exhibited stable complex formation. At all of the targeted residues, alanine substitution resulted in a more severe defect in DNA repair compared to the more conservative asparagine substitutions, but all of the mutant proteins exhibited <2% of the exonuclease activity observed for wild-type Mre11. Our results show that the structural integrity of the Mre11-Rad50-Xrs2 complex is more important than the catalytic activity of the Mre11 nuclease for the overall functions of the complex in vegetative cells.
authors.affiliation: Institute of Cancer Research and Department of Microbiology, Columbia University Medical Center, 701 W. 168th Street, New York, NY 10032, USA.
authors.forename: Berit O
authors.initials: BO
authors.lastname: Krogh
authors.forename: Bertrand
authors.initials: B
authors.lastname: Llorente
authors.forename: Alicia
authors.initials: A
authors.lastname: Lam
authors.forename: Lorraine S
authors.initials: LS
authors.lastname: Symington
mesh_terms: D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000378:metabolism; D004706:Endodeoxyribonucleases / Q000235:genetics*; D005090:Exodeoxyribonucleases / Q000235:genetics*; D005092:Exonucleases / Q000378:metabolism; D005720:Gamma Rays; D006918:Hydroxyurea; D047468:Immunoprecipitation; D008741:Methyl Methanesulfonate; D046912:Multiprotein Complexes / Q000235:genetics / Q000378:metabolism*; D009154:Mutation / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics* / Q000378:metabolism; D016615:Telomere / Q000235:genetics
chemical_list: D004268:DNA-Binding Proteins; D046912:Multiprotein Complexes; C086930:RAD50 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; C086929:XRS2 protein, S cerevisiae; D008741:Methyl Methanesulfonate; D004706:Endodeoxyribonucleases; D005090:Exodeoxyribonucleases; D005092:Exonucleases; C094191:MRE11 protein, S cerevisiae; D006918:Hydroxyurea