Role of the nuclease activity of Saccharomyces cerevisiae Mre11 in repair of DNA double-strand breaks in mitotic cells.
The Rad50:Mre11:Xrs2 (RMX) complex functions in repair of DNA double-strand breaks (DSBs) by recombination and nonhomologous end-joining (NHEJ) and is also required for telomere stability. The Mre11 subunit exhibits nuclease activities in vitro, but the role of these activities in repair in mitotic cells has not been established. In this study we have performed a comparative study of three mutants (mre11-D16A, -D56N, and -H125N) previously shown to have reduced nuclease activities in vitro. In ends-in and ends-out chromosome recombination assays using defined plasmid and oligonucleotide DNA substrates, mre11-D16A cells were as deficient as mre11 null strains, but defects were small in mre11-D56N and -H125N mutants. mre11-D16A cells, but not the other mutants, also displayed strong sensitivity to ionizing radiation, with residual resistance largely dependent on the presence of the partially redundant nuclease Exo1. mre11-D16A mutants were also most sensitive to the S-phase-dependent clastogens hydroxyurea and methyl methanesulfonate but, as previously observed for D56N and H125N mutants, were not defective in NHEJ. Importantly, the affinity of purified Mre11-D16A protein for Rad50 and Xrs2 was indistinguishable from wild type and the mutant protein formed complexes with equivalent stoichiometry. Although the role of the nuclease activity has been questioned in previous studies, the comparative data presented here suggest that the nuclease function of Mre11 is required for RMX-mediated recombinational repair and telomere stabilization in mitotic cells.
Lewis
L Kevin
LK
Department of Chemistry and Biochemistry, Texas State University, San Marcos, Texas 78666, USA. lll8@txstate.edu
Storici
Francesca
F
Van Komen
Stephen
S
Calero
Shanna
S
Sung
Patrick
P
Resnick
Michael A
MA
D004247:DNA / Q000378:metabolism*; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000378:metabolism; D003851:Deoxyribonucleases / Q000378:metabolism*; D004706:Endodeoxyribonucleases / Q000235:genetics / Q000378:metabolism*; D005090:Exodeoxyribonucleases / Q000235:genetics / Q000378:metabolism*; D005785:Gene Conversion; D008938:Mitosis / Q000235:genetics*; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics; D009841:Oligonucleotides; D010957:Plasmids / Q000235:genetics; D011995:Recombination, Genetic / Q000235:genetics; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D016615:Telomere / Q000235:genetics
D004268:DNA-Binding Proteins; D009841:Oligonucleotides; C086930:RAD50 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; C086929:XRS2 protein, S cerevisiae; D004247:DNA; D003851:Deoxyribonucleases; D004706:Endodeoxyribonucleases; D005090:Exodeoxyribonucleases; C094191:MRE11 protein, S cerevisiae