Alteration of N-terminal phosphoesterase signature motifs inactivates Saccharomyces cerevisiae Mre11.
Saccharomyces cerevisiae Mre11, Rad50, and Xrs2 function in a protein complex that is important for nonhomologous recombination. Null mutants of MRE11, RAD50, and XRS2 are characterized by ionizing radiation sensitivity and mitotic interhomologue hyperrecombination. We mutagenized the four highly conserved phosphoesterase signature motifs of Mre11 to create mre11-11, mre11-2, mre11-3, and mre11-4 and assessed the functional consequences of these mutant alleles with respect to mitotic interhomologue recombination, chromosome loss, ionizing radiation sensitivity, double-strand break repair, and protein interaction. We found that mre11 mutants that behaved as the null were sensitive to ionizing radiation and deficient in double-strand break repair. We also observed that these null mutants exhibited a hyperrecombination phenotype in mitotic cells, consistent with previous reports, but did not exhibit an increased frequency of chromosome loss. Differential ionizing radiation sensitivities among the hypomorphic mre11 alleles correlated with the trends observed in the other phenotypes examined. Two-hybrid interaction testing showed that all but one of the mre11 mutations disrupted the Mre11-Rad50 interaction. Mutagenesis of the phosphoesterase signatures in Mre11 thus demonstrated the importance of these conserved motifs for recombinational DNA repair.
Laboratory of Genetics, University of Wisconsin Medical School, Madison, Wisconsin 53706, USA.
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Bressan
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Olivares
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Nelms
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D000595:Amino Acid Sequence / Q000502:physiology*; D015825:Chromosomes, Fungal / Q000235:genetics; D017124:Conserved Sequence / Q000235:genetics; D004260:DNA Repair / Q000235:genetics*; D004706:Endodeoxyribonucleases; D004950:Esterases / Q000235:genetics*; D005090:Exodeoxyribonucleases; D005656:Fungal Proteins / Q000235:genetics* / Q000502:physiology; D005720:Gamma Rays; D008969:Molecular Sequence Data; D009006:Monosomy; D016297:Mutagenesis, Site-Directed; D011995:Recombination, Genetic; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000502:physiology / Q000528:radiation effects; D029701:Saccharomyces cerevisiae Proteins
D005656:Fungal Proteins; D029701:Saccharomyces cerevisiae Proteins; D004706:Endodeoxyribonucleases; D004950:Esterases; D005090:Exodeoxyribonucleases; C094191:MRE11 protein, S cerevisiae