The Saccharomyces cerevisiae MUM 2 Gene Interacts With the DNA Replication Machinery and Is Required for Meiotic Levels of Double Strand Breaks

The Saccharomyces cerevisiae MUM2 gene is essential for meiotic, but not mitotic, DNA replication and thus sporulation. Genetic interactions between MUM2 and a component of the origin recognition complex and polymerase a-primase suggest that MUM2 influences the function of the DNA replication machinery. Early meiotic gene expression is induced to a much greater extent in mum2 cells than in meiotic cells treated with the DNA synthesis inhibitor hydroxyurea. This result indicates that the mum2 meiotic arrest is downstream of the arrest induced by hydroxyurea and suggests that DNA synthesis is initiated in the mutant. Genetic analyses indicate that the recombination that occurs in mum2 mutants is dependent on the normal recombination machinery and on synaptonemal complex components and therefore is not a consequence of lesions created by incompletely replicated DNA. Both meiotic ectopic and allelic recombination are similarly reduced in the mum2 mutant, and the levels are consistent with the levels of meiosisspecific DSBs that are generated. Cytological analyses of mum2 mutants show that chromosome pairing and synapsis occur, although at reduced levels compared to wild type. Given the near-wild-type levels of meiotic gene expression, pairing, and synapsis, we suggest that the reduction in DNA replication is directly responsible for the reduced level of DSBs and meiotic recombination. MEIOSIS is a specialized cell cycle that enables dipthe machines that carry out DNA synthesis (reviewed in Baker and Bell 1998). Genetic analyses in the yeast loid organisms to reproduce sexually by generating haploid gametes through two successive divisions. Saccharomyces cerevisiae indicate that the replicative machinery used to synthesize DNA in vegetative cells is also At meiosis I, also known as the reductional division, homologous chromosomes disjoin from each other. At required for the duplication of chromosomes in meiosis (Budd et al. 1989). In contrast, the regulatory controls meiosis II, as at mitosis, sister chromatids separate and move to opposite poles. The meiotic divisions, in turn, for these two replication modes appear distinct (Schild and Byers 1978; Hollingsworth and Sclafani 1993; are tightly linked to gamete differentiation. Fusion of gametes at fertilization restores the diploid chromoDirick et al. 1998; Stuart and Wittenberg 1998). During prophase, the duplicated homologous chrosome number and initiates zygotic development. High fidelity of meiotic chromosome segregation is essential mosomes align, synapse, and recombine. Alignment refers to the presynaptic association of the homologous for the propagation of all sexually reproducing organisms. chromosomes (Loidl et al. 1994; Weiner and Kleckner 1994), while chromosome synapsis is the intimate associThe replication of chromosomes is the first detectable cytological event in meiosis. The coordinated synthesis ation of homologous chromosomes in the context of the synaptonemal complex (SC). The SC is a meiosisof genomic DNA requires multiple levels of regulation and a large number of gene products. The origin recogspecific structure that is elaborated along the lengths of the chromosomes (reviewed in von Wettstein et al. nition complex (ORC) and the multi-subunit minichromosome maintenance (MCM) complex, among others, 1984). Several genes that encode structural proteins of the SC have been identified; genetic analyses indicate are essential in initiating DNA replication (reviewed in Dutta and Bell 1997). Primase and the replicative that the proper assembly of the SC is essential for chromosome segregation (reviewed in Kupiec et al. 1997). polymerases, themselves multi-protein complexes, are All meiotic recombination in yeast (and presumably in other organisms as well) that has been studied to Corresponding author: JoAnne Engebrecht, Department of Pharmacodate is initiated by double strand breaks (DSBs; Sun et logical Sciences, State University of New York, Stony Brook, NY 11794al. 1989; Cao et al. 1990; Cervantes et al. 2000). DSBs 8651. E-mail: joanne@pharm.sunysb.edu occur before the SC is fully formed, while mature recom1Present address: Fred Hutchinson Cancer Research Center, Seattle, WA 98109-1024. binants do not occur until chromosome synapsis is comGenetics 157: 1179–1189 (March 2001) 1180 L. Davis et al. mated. Mitotic segregants that had lost the plasmid were isoplete (Padmore et al. 1991). In most organisms reciprolated, thus generating isogenic MUM2 and mum2 strains homocal exchange is essential for proper chromosome segzygous for the arg4 alleles. sae2D derivatives were made by regation at the reductional division (Hawley 1987). transforming pME1210 (see below) into Y1244 and Y1245 Studies in S. cerevisiae have been instrumental in elucidatderivatives. orc and pol temperature-sensitive alleles were introduced into these same strains. ing the requirement for chromosome pairing, synapsis, hop1::LEU2, rec104::LEU2, red1::LEU2, and spo11::TRP1 deand genetic recombination in ensuring proper chromorivatives of JB128 (Bharagava et al. 1992) were made by some segregation at the meiosis I division (reviewed in transforming Y898 (JB128 MUM2/mum2::LYS2) with pNH37Kupiec et al. 1997). However, very little attention has been 2 (Hollingsworth and Byers 1989), pNH131-10, pNH119devoted to the role of DNA replication in meiosis. Recent 8 (Hollingsworth and Johnson 1993), and pGB324 (Giroux et al. 1989), respectively. Transformants were induced to studies indicate that DNA replication is important for meisporulate and the resulting tetrads were dissected to generate otic recombination (Borde et al. 2000). Further, SPO11 homozygous deletions. (Klapholz et al. 1985) and REC8 (Klein et al. 1999), two Plasmids: SAE2 sequences were isolated by PCR amplificayeast genes originally identified for their roles in recombition using primers P68, 59-TCC AAG CTT TTG CAC GTCnation and sister chromatid cohesion, respectively, have 39, and P69, 59-GTT CCC GTG GTA GAA ATG-39. The PCR product was digested with SacII and SalI and the resulting subsequently been shown to play distinct roles in meiotic 1.9-kb product was inserted into the SacII and XhoI sites of DNA replication (Cha et al. 2000). However, the connecBluescript SK1 (Stratagene, La Jolla, CA) to generate tion between DNA replication and homologous chromopME1214. The 1.1-kb HindIII-HpaI URA3 fragment from some interaction remains unknown. Yep352 (Hill et al. 1986) was inserted into the HindIII-EcoRV The yeast MUM2 gene was identified in a screen for sites of pME1214 to generate the sae2D::URA3 deletion allele in pME1220. Plasmid ME1220 was digested with SacI and HpaI meiotic mutants and is required specifically for meiotic to allow substitution of the SAE2 locus in yeast. Sequences DNA replication (Engebrecht et al. 1998). The Mum2 near THR4 (YCR47c), which contain a meiotic hotspot (Wu protein is not similar to other proteins; however, the C and Lichten 1994), were isolated by PCR amplification using terminus is predicted to form a coiled coil. Coiled-coil primers P66, 59-GAC TAC ATG GAC ACT GAG-39, and P67, domains are found in diverse classes of proteins including 59-CTA GAG TTC AAC TGC AAG G-39. The PCR product was molecular motors and SC components. Synthetic interacdigested with HindIII and the resulting 0.9-kb product was inserted in the HindIII site of Bluescript SK1 to generate tions with a component of ORC and polymerase a-primase pME1210. The 618-bp DraI-EcoRI SPO11 fragment from suggest that MUM2 influences the functioning of the replipGB320 (Giroux et al. 1989) was inserted into the EcoRV and cation machinery. Genetic analyses of mum2 mutants in EcoRI sites of Bluescript SK1 to generate pME1845. meiosis indicate that perturbation of DNA replication Analysis of growth: Strains harboring conditional alleles modestly affects chromosome pairing and synapsis, while with and without MUM2 were grown overnight to saturation at 258. Serial dilutions were spotted onto rich medium and meiotic recombination is greatly impaired. Further, these incubated at the indicated temperature for 3 days. Viability studies reveal a direct correlation between the level of was monitored by plating cells in triplicate at 258 after the meiotic DSBs and DNA replication and suggest that DNA indicated times at different temperatures; doubling times were replication is an essential prerequisite to DSB formation. calculated as described (Engebrecht et al. 1998). Flow cytometric analysis: Cultures in log phase were split and propagated at 258, 338, and 368 for the indicated times and prepared for flow cytometric analysis as described (EngeMATERIALS AND METHODS brecht et al. 1998). At least 10,000 cells were analyzed per sample. Yeast strains and media: Routine growth and manipulation of S. cerevisiae strains were performed as described (Rose et al. RNA isolation and analysis: Strains were cultured in YPAcetate, introduced into sporulation medium, and assessed by 1990). Yeast strains were transformed using the lithium acetate procedure (Ito et al. 1983). All integrative transformants were 496-diamidino-2-phenylindole (DAPI) staining as described (Engebrecht et al. 1998). Hydroxyurea (HU, 200 mm; Aldrich verified by Southern blot analysis or polymerase chain reaction (PCR). Gene disruptions were performed by one-step gene reChemicals, Milwaukee) was added after 2.5 hr in sporulation medium to a portion of the culture. RNA was prepared and placement; orc2 and pol1 alleles were introduced by two-step gene replacement (Rothstein 1991) using plasmids JR1267 (orc2-1) Northern blot analysis was performed as described (Rose et al. 1995). Radioactive RNA probes were synthesized by in vitro and YIp5-pol1-17 (Budd and Campbell 1987). Yeast strains used in this study are listed in Table 1. transcription (Krieg and Melton 1987) of plasmids ME1845 (SPO11, see above) digested with HindIII using T3 polymerase Diploid strains harboring the arg4-bgl and arg4-nsp allel