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 mutation mre11-58. 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 new mre11 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.The genes of the RAD52 epistasis group in Saccharomyces cerevisiae are necessary for repair of double-strand DNA breaks (DSBs) during mitosis and meiosis (35). Mutants resulting from mutations of these genes are classified into two subgroups according to their recombination abilities and meiotic DSB formation properties. One subgroup comprises rad51, -52, -54, -55, and -57 mutants. These are defective in mating-type switching and both mitotic and meiotic recombination (34,35,43). In these mutants, meiosis-specific DSBs form at recombination hot spots but are left unrepaired with extensive processing (34,42,47). Mutants resulting from mutations in these genes are also defective in viable spore formation, and spore inviability is not alleviated by introducing an additional spo13 mutation, which eliminates meiotic reductional division (24). The other subgroup consists of mre11, xrs2, and rad50 null mutants, which are proficient in mating-type switching and show spontaneous recombination at a high frequency during mitosis (1,14,18,30). In rad50 and xrs2 null mutants, processing of DSB ends is reduced and formation of recombinant is delayed (18,20,45). During meiosis, however, these three mutants are deficient in formation of meiosisspecific DSBs, induction of meiotic recombination, and viable spore formation (1,5,18,21), and their viable spore formation deficiency is alleviated by the introduction of a spo13 mutation (35). A mutant resulting from a non-null mutation of RAD50, called rad50S, accumulates unprocessed DSBs, and its spore inviability is not rescued by introducing a spo13 mutation (3). Therefore, MRE11, XRS2, and RAD50 appear to be involved in two distinct processes: (i) DSB repair during mitosis and (ii) DSB formation and processing from DSB ends during meiosis (5,18,21).Recently, a new mutation with ...

Exo1 Roles for Repair of DNA Double-Strand Breaks and Meiotic Crossing Over inSaccharomyces cerevisiae

“…In the exo1 mutant, the kinetics of MAT switching is indistinguishable from that of wild type (Figure 3, D, F, and G). In the mre11 mutant, however, the 0.7-kb band persists after 3 h in glucose medium, and the appearance of the 0.9-kb MATa band is delayed about 2 h (Tsubouchi and Ogawa, 1998) (Figure 3, C, F, and G). In the exo1 mre11 double mutant, ϳ10% of the 0.7-kb HO-cut fragment still remains at 9 h, and the appearance of the 0.9-kb MATa band is further delayed compared with the mre11 single mutant (Figure 3, E, F, and G).…”

“…A lack of any one of these proteins makes cells highly sensitive to DSBs (Game and Mortimer, 1974;Ivanov et al, 1992;Ajimura et al, 1993) and reduces processing of DSB ends produced by the HO endonuclease during mating-type switching (Ivanov et al, 1994;Tsubouchi and Ogawa, 1998). As a consequence, the kinetics of recombinant formation is slow, but the amount of recombinant DNA ultimately formed is the same as in wild type (Ivanov et al, 1994;Tsubouchi and Ogawa, 1998).…”

“…All media used in this study were detailed previously (Tsubouchi and Ogawa, 1998). YPLac contains 1% yeast extract, 2% bacto peptone, and 2% sodium lactate (pH 5.5), and YPGal is identical except that it contains 2% galactose instead of lactate.…”

“…The XRS2 gene was disrupted by transforming strains with pHT59 after SphI and BamHI double digestion. Disruption of MRE11, RAD50, RAD52, and DMC1 was carried out as described previously (Alani et al, 1989;Bishop et al, 1992;Johzuka and Ogawa, 1995;Tsubouchi and Ogawa, 1998).…”

“…Induction of HO break and detection of mating-type switch process by Southern blot analysis have been described (Tsubouchi and Ogawa, 1998). Genomic DNA samples obtained at indicated times were cut with StyI (Eco130I), separated on 0.7% agarose gels, and subjected to Southern blot analysis with a 1.0-kb NdeI-HindIII fragment of pHT46 as a probe.…”