The hop2 mutant of Saccharomyces cerevisiae arrests in meiosis with extensive synaptonemal complex (SC) formation between nonhomologous chromosomes. A screen for multicopy suppressors of a hop2-ts allele identified the MND1 gene. The mnd1-null mutant arrests in meiotic prophase, with most double-strand breaks (DSBs) unrepaired. A low level of mature recombinants is produced, and the Rad51 protein accumulates at numerous foci along chromosomes. SC formation is incomplete, and homolog pairing is severely reduced. The Mnd1 protein localizes to chromatin throughout meiotic prophase, and this localization requires Hop2. Unlike recombination enzymes such as Rad51, Mnd1 localizes to chromosomes even in mutants that fail to initiate meiotic recombination. The Hop2 and Mnd1 proteins coimmunoprecipitate from meiotic cell extracts. These results suggest that Hop2 and Mnd1 work as a complex to promote meiotic chromosome pairing and DSB repair. The identification of Hop2 and Mnd1 homologs in other organisms suggests that the function of this complex is conserved among eukaryotes.Meiosis is a special type of cell division cycle that produces haploid gametes from diploid parental cells. At the first nuclear division of meiosis, sister chromatids remain associated, while homologous chromosomes segregate to opposite poles of the spindle apparatus. This reductional chromosome segregation is preceded by a lengthy prophase during which homologous chromosomes pair with each other, undergo high levels of genetic recombination, and engage in synaptonemal complex (SC) formation. These interactions between homologs are necessary prerequisites to the correct segregation of chromosomes at meiosis I.Meiotic recombination in Saccharomyces cerevisiae and other organisms initiates with double-strand breaks (DSBs), which are catalyzed by a topoisomerase-like protein known as Spo11 (21). DSBs are processed to expose single-stranded tails with 3Ј termini (6, 44), which invade homologous sequences in nonsister chromatids (5,31,40). Strand invasion results in the formation of joint molecules, whose resolution gives rise to crossover and noncrossover products (2, 18, 39). In budding yeast, several proteins are required for efficient strand invasion, but the RecA homolog Rad51 is thought to provide the principal strand-exchange activity (45; reviewed in reference 30). Rad51 and accessory proteins accumulate at discrete foci on meiotic chromosomes, during the period of DSB repair (4,14).Meiotic cell cycle progression is tightly coupled to the status of meiotic recombination events. If DSBs are formed but their subsequent repair is prevented by mutation, then a cell cycle checkpoint (the pachytene checkpoint) causes cells to arrest in mid-meiotic prophase (reviewed in reference 3). This arrest can be alleviated by a second mutation that prevents DSB formation; for example, a spo11 mutation restores sporulation in dmc1 and hop2 strains that normally arrest with hyperresected but unrepaired DSBs (6, 23). Thus, recombination intermediates appear to act a...

DNA binding/double-strand break repair/DSB formation/Mre11/nuclease

In budding yeast, there are two RecA homologs: Rad51 and Dmc1. While Rad51 is involved in both mitotic and meiotic recombination, Dmc1 participates specifically in meiotic recombination. Here, we describe a meiosis-specific protein (Hed1) with a novel Rad51 regulatory function. Several observations indicate that Hed1 attenuates Rad51 activity when Dmc1 is absent. First, although double-strand breaks are normally poorly repaired in the dmc1 mutant, repair becomes efficient when Hed1 is absent, and this effect depends on Rad51. Second, Rad51 and Hed1 colocalize as foci on meiotic chromosomes, and chromosomal localization of Hed1 depends on Rad51. Third, production of Hed1 in vegetative cells inhibits Rad51-dependent recombination events. Fourth, the Hed1 protein shows an interaction with Rad51 in the yeast two-hybrid protein system. We propose that Hed1 provides a mechanism to ensure the coordinated action of Rad51 and Dmc1 during meiosis, by down-regulating Rad51 activity when Dmc1 is unavailable.[Keywords: Dmc1; Rad51; RecA homologs; crossing over; meiosis; recombination] Supplemental material is available at http://www.genesdev.org.

The MRE11, RAD50, and XRS2 genes of Saccharomyces cerevisiae are involved in the repair of DNA double-strand breaks (DSBs) produced by ionizing radiation and by radiomimetic chemicals such as methyl methanesulfonate (MMS). In these mutants, single-strand DNA degradation in a 5Ј to 3Ј direction from DSB ends is reduced. Multiple copies of the EXO1 gene, encoding a 5Ј to 3Ј double-strand DNA exonuclease, were found to suppress the high MMS sensitivity of these mutants. The exo1 single mutant shows weak MMS sensitivity. When an exo1 mutation is combined with an mre11 mutation, both repair of MMS-induced damage and processing of DSBs are more severely reduced than in either single mutant, suggesting that Exo1 and Mre11 function independently in DSB processing. During meiosis, transcription of the EXO1 gene is highly induced. In meiotic cells, the exo1 mutation reduces the processing of DSBs and the frequency of crossing over, but not the frequency of gene conversion. These results suggest that Exo1 functions in the processing of DSB ends and in meiotic crossing over.

During meiosis, homologous chromosomes pair at close proximity to form the synaptonemal complex (SC). This association is mediated by transverse filament proteins that hold the axes of homologous chromosomes together along their entire length. Transverse filament proteins are highly aggregative and can form an aberrant aggregate called the polycomplex that is unassociated with chromosomes. Here, we show that the Ecm11-Gmc2 complex is a novel SC component, functioning to facilitate assembly of the yeast transverse filament protein, Zip1. Ecm11 and Gmc2 initially localize to the synapsis initiation sites, then throughout the synapsed regions of paired homologous chromosomes. The absence of either Ecm11 or Gmc2 substantially compromises the chromosomal assembly of Zip1 as well as polycomplex formation, indicating that the complex is required for extensive Zip1 polymerization. We also show that Ecm11 is SUMOylated in a Gmc2-dependent manner. Remarkably, in the unSUMOylatable ecm11 mutant, assembly of chromosomal Zip1 remained compromised while polycomplex formation became frequent. We propose that the Ecm11-Gmc2 complex facilitates the assembly of Zip1 and that SUMOylation of Ecm11 is critical for ensuring chromosomal assembly of Zip1, thus suppressing polycomplex formation.

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 ...

Two RecA orthologs, Rad51 and Dmc1, mediate homologous recombination in meiotic cells. During budding yeast meiosis, Hed1 coordinates the actions of Rad51 and Dmc1 by down-regulating Rad51 activity. It is thought that Hed1-dependent attenuation of Rad51 facilitates formation of crossovers that are necessary for the correct segregation of chromosomes at the first meiotic division. We purified Hed1 in order to elucidate its mechanism of action. Hed1 binds Rad51 with high affinity and specificity. We show that Hed1 does not adversely affect assembly of the Rad51 presynaptic filament, but it specifically prohibits interaction of Rad51 with Rad54, a Swi2/Snf2-like factor that is indispensable for Rad51-mediated recombination. In congruence with the biochemical results, Hed1 prevents the recruitment of Rad54 to a site-specific DNA double-strand break in vivo but has no effect on the recruitment of Rad51. These findings shed light on the function of Hed1 and, importantly, unveil a novel mechanism for the regulation of homologous recombination.[Keywords: Regulation of meiotic recombination; interhomolog crossovers; Rad51 recombinase; double-strand break repair; homologous recombination] Supplemental material is available at http://www.genesdev.org.

In budding yeast, absence of the Hop2 protein leads to extensive synaptonemal complex (SC) formation between nonhomologous chromosomes, suggesting a crucial role for Hop2 in the proper alignment of homologous chromosomes during meiotic prophase. Genetic analysis indicates that Hop2 acts in the same pathway as the Rad51 and Dmc1 proteins, two homologs of E. coli RecA. Thus, the hop2 mutant phenotype demonstrates the importance of the recombination machinery in promoting accurate chromosome pairing. We propose that the Dmc1/Rad51 recombinases require Hop2 to distinguish homologous from nonhomologous sequences during the homology search process. Thus, when Hop2 is absent, interactions between nonhomologous sequences become inappropriately stabilized and can initiate SC formation. Overexpression of RAD51 largely suppresses the meiotic defects of the dmc1 and hop2 mutants. We conclude that Rad51 is capable of carrying out a homology search independently, whereas Dmc1 requires additional factors such as Hop2.