A Novel mre11 Mutation Impairs Processing of Double-Strand Breaks of DNA during Both Mitosis and Meiosis

ABSTRACT

Using complementation tests and nucleotide sequencing, we showed that the rad58-4 mutation was an allele of the MRE11 gene and have renamed the mutationmre11-58. Two amino acid changes from the wild-type sequence were identified; one is located at a conserved site of a phosphodiesterase motif, and the other is a homologous amino acid change at a nonconserved site. Unlike mre11 null mutations, the mre11-58 mutation allowed meiosis-specific double-strand DNA breaks (DSBs) to form at recombination hot spots but failed to process those breaks. DSB ends of this mutant were resistant to lambda exonuclease treatment. These phenotypes are similar to those of rad50S mutants. In contrast to rad50S, however, mre11-58 was highly sensitive to methyl methanesulfonate treatment. DSB end processing induced by HO endonuclease was suppressed in both mre11-58 and the mre11 disruption mutant. We constructed a newmre11 mutant that contains only the phosphodiesterase motif mutation of the Mre11-58 protein and named it mre11-58S. This mutant showed the same phenotypes observed in mre11-58, suggesting that the phosphodiesterase consensus sequence is important for nucleolytic processing of DSB ends during both mitosis and meiosis.

ACKNOWLEDGMENTS

We thank J. Tomizawa, T. Ogawa, A. Shinohara, H. Masukata, and T. Nakagawa for helpful discussion and critical reading of the manuscript; D. K. Bishop and M. Lichten for critical reading of the manuscript; H. Oshiumi for providing strains and plasmids; M. Sekiguchi for technical assistance; and members of the Ogawa laboratory for helpful discussion. We also thank N. Kleckner and J. E. Haber for providing yeast strains and plasmids.

This work was supported by a grant-in aid for Specially Promoted Research (06101003) from the Ministry of Education, Science, Sports and Culture of Japan, by the Howard Hughes Medical Institute and by the Human Frontier Science Program.