Isolation of COMI, a New Gene Required to Complete Meiotic Double-Strand Break-Induced Recombination in Saccharomyces cereuisiae

We have designed a screen to isolate mutants defective during a specific part of meiotic prophase I of the yeast Saccharomyces cereuisiae. Genes required for the repair of meiotic double-strand breaks or for the separation of recombined chromosomes are targets of this mutant hunt. The specificity is achieved by selecting for mutants that produce viable spores when recombination and reductional segregation are prevented by mutations in SPOlI and SP013 genes, but fail to yield viable spores during a normal Rec+ meiosis. We have identified and characterized a mutation c o d I , which blocks processing of meiotic double-strand breaks and which interferes with synaptonemal complex formation, homologous pairing and, as a consequence, spore viability after induction of meiotic recombination. The COMI/ SAE2 gene was cloned by complementation, and the deletion mutant has a phenotype similar to coml1. coml/sae2 mutants closely resemble the phenotype of radSUS, as assayed by phase-contrast microscopy for spore formation, physical and genetic analysis of recombination, fluorescence in situ hybridization to quantify homologous pairing and immunofluorescence and electron microscopy to determine the capability to synapse axial elements. I N order to allow repeated conjugation and thus exchange of genetic material between different individuals, an organism needs to be able to separate again the two complete genomes that contributed to the diploid. Eukaryotes generally use a process called meiosis, which consists of two subsequent cell divisions preceded by only a single round of DNA replication to generate haploid cells for sexual reproduction. Both divisions resemble mitosis in many aspects, but during the first (reductional) division typically homologous parental chromosomes are recognized, paired and recombined and parental centromeres are segregated to opposite poles, whereas the sister centromeres do not come apart before the second (equational) meiotic division. Thus the reductional division seems to encompass the newly acquired meiosis-specific functions for chrcmosome sorting and genetic recombination and accordingly has been the focus of many investigations. The yeast Saccharomyces cereuisiae has served as a remarkable model organism, helping to dissect meiotic mechanisms through the use of genetic screens combined with biochemical and cytological methods. Genetic screens have become more and more specific, concentrating on defined aspects of the meiotic process, e.g., recombination or homologue interaction (ESPOSITO and E s POSITO 1974; ROCKMILL and ROEDER 1988; HOL LINGSWORTH and BYERS 1989; AJIMURA et al. 1993; HOLLINCSWORTH et al. 1995). Corresponding author: Franz Klein, Department of Cytology and Genetics, Institute of Botany, University of Vienna, Rennweg 14, 1030 Wien, Austria. E-mail: [email protected] Genetics 146 781-795 (July, 1997) In yeast meiotic recombination and synapsis are initiated by meiosis-specific DNA double-strand breaks (DSBs) (NICOLAS et al. 1989; SUN et al. 1989; CAO et al. 1990). These breaks are central to meiosis and all genes acting before DSB formation can be regarded as early prophase I genes. A class of mutations that eliminate DSBs have been characterized by a genetic screen, selecting for suppressors of spore lethality caused by unrepaired DSBs in sporulating haploid rad52, spol3 double mutants (MALONE et al. 1991). Such mutants are generally deficient for the initiation of meiotic recombination and thus can be rescued by relieving the requirement for reductional segregation in spol3. Other mutations, like hop1 (HOLLINGSWORTH and BYERS 1989) and red1 ( ROCKMILL and ROEDER 1990), can also be rescued by spol3, although some DSBs are formed (ROCKMILL and ROEDER 1990; SCHWACHA and KLECKNER 1994). This suggests that DSBs can be fully repaired in these mutants without ensuring correct reductional segregation. A number of mutations that cannot be rescued by spol3 have been characterized and some of these interfere with processing of the DSBs. A class of nonnull alleles of rad50 designated rad50S are defective at the earliest stage of DSB repair, namely in the processing of 5' ends at the initiation site to form the 3' singlestranded ends required for strand invasion (ALANI et al. 1990). This is different from the phenotype of the rad50null mutant, which does not initiate meiotic recombination and therefore is rescued by spol3, demonstrating the important role of RADS0 both for the initiation and processing of meiotic DSBs. Recently, covalent 782 S. Prinz, A. Amon and F. Klein attachment of an unidentified protein to the unresected 5’ ends in rad50S mutants has been demonstrated (DE MASSY et al. 1995; KEENEY and KLECKNER 1995; LIU et al. 1995). These attachments are very likely to prevent efficient repair of DSBs during meiosis, causing a block or at least a delay before the first meiotic division. The unrepaired lesions, rather than absence of recombination, may be the reason for lethality of the diploid spores produced by spol3, rad5OS double mutants. Dmclp, Rad5lp, Rad55p and Rad57p all show homology to the bacterial recombination protein recA. Consequently their function is implicated in the strand invasion step of DSB repair, which is supported by their phenotypes. dmcl, rad51, rad55, rad57, rad52 and seplnull mutants accumulate DSBs at least transiently, but, in contrast to rad50S, produce 3‘ single-stranded tails even longer than wild type (reviewed in SHINOHARA and OGAWA 1995; TISHKOFF et al. 1995). According to results obtained by SUGAWARA et al. (1995) the role of RAD51, RAD55 and RAD57 is rather indirect, namely to provide access for other recombination enzymes to the homologous donor copy. A recently characterized member of the group of genes required for DSB repair is RAD58 (XRS4) (CHEPURNAYA et al. 1995). Some of these mutants (e.g., dmcl, rad51 and sepl) have been shown to cause an arrest or delay in pachytene in addition to a strong ascospore formation defect. Inactivation of SP013 can neither alleviate this block nor restore spore viability, at least in the SK1 strain background (TISHKOFF et al. 1991; BISHOP et al. 1992). In contrast, by blocking meiotic recombination (e.g., in a spol 1 mutant), the arrest or delay of dmcl or rad51 cells can be abolished. However in a strain background where dmcl does not arrest, restoration of wild-type levels of spore viability after inactivation of SPO13 alone has been reported (ROCKMILL et al. 1995). The role of the SEPl gene is less clear. I n vitro DNA strand exchange activity (KOLODNER et al. 1987), as well as in vitro microtubule polymerizing activity (INTERTHAL et al. 1995), has been reported for the Sepl protein, stimulating diverse interpretations for the role of this important factor. However since the defects of sepl mutants are not suppressed by rad50, spol3 (TISHKOFF et al. 1991), it can be concluded that a pathway not related to DSBinduced recombination is affected. The list of genes known to date to be involved in repair of meiotic DSBs is certainly not complete. Mutations in ZIP1, TOP2 and ME15 are not believed to directly impede processing of meiotic DSBs, yet they cause the cells to arrest or delay in pachytene independently of the SP013 function. Ziplp (SYM et al. 1993; SYM and ROEDER 1994) is a component of the synaptonemal complex (SC) required for complete synapsis. Separation of recombined homologues during meiosis I was shown to be dependent on topoisomerase I1 with the use of a cold-sensitive allele of TOP2 (ROSE et al. 1990a). IMEI5, a recently isolated meiosis-specific gene, is probably required for disassembly of the SC since mei5 mutant strains arrest at pachytene with complete SCs (M. MODESTI and C. GIROUX, personal communication). However, the meiotic arrest of a mei5 mutant strain is relieved in the presence of a spol l mutation, which eliminates DSB formation and synapsis (GIROUX et al. 1993). These mutants affect processes subsequent to but dependent on meiotic recombination, as in each case the elimination of recombination eliminates also the mutant phenotype. It is likely that this interesting class of mutations will expand as more genes involved in SC formation and chromosome separation are identified. Mutations in genes exerting meiotic cell cycle control also are not expected to be influenced by spo13. cdc28, cdc34 and cdc39 cells arrest (or delay) in the pachytene stage of meiotic prophase, although they block at Start during the vegetative cell cycle (SHUSTER and BYERS 1989). NLlT80 (XU et al. 1995) is a meiosis-specific gene required for exit from meiotic pachytene, but in contrast to mei5, ndt80 cells arrest even in a spol l , spol3 background. Thus ATIT80 is required for processes independent of meiotic recombination, possibly being involved in meiotic cell cycle control. In this article we introduce a genetic screen that permits the identification of mutations that prevent the formation of viable spores in a spol3 background, but that permit successful sporulation in spol l , sf1013 strains. We describe the identification and characterization of a new gene COMl/SAE2 that is required for the processing and the repair of meiotic DSBs. MATERIALS AND METHODS Media, growth and sporulation conditions: Cells were grown in YPD or synthetic complete medium supplemented with appropriate amino acids (ROSE et al. 1990b). Selection of G418 resistant transformants was performed on YPD plates containing 200 mg/liter of G418 (geneticin; GIBCO BRL, Gaithersburg, MD). Sporulation conditions as well as spheroplasting were as described previously (LOIDL et al. 1991). Sporulation of strains Y164 and Y219 on solid medium was performed on SPM+ (0.25% yeast extract, 0.1% glucose, 1.5% potassium acetate plus 1/5 of amino acid or base supplement of a regular synthetic complete medium). Plasmid constructions: To construct plasmid p21 the SPOl? gene was cloned as a 2.2-kilobase (kb) BamHI-NruI fragment derived from pTWl5 (a gift from R. E. ESPOSITO) into pRS316 (SIKORSKY and HIETER 1989) cut with HindIII, blunted and cut with BamHI. For the construction of p155 the SalI-SmaI fragment of YCp50, containing URA?, was deleted. Into the BamHI site of the resulting vector the ADE2 gene was cloned as a BamHI cassette. SPOlI was introduced as a ClaI fragment derived from pGB429 (a gift from C. N. GIROUX). Plasmid p259, which can be easily lost due to an unstable centromere EN?, was obtained by first subcloning SPOl? as a 2.2-kb BamHI-NmI fragment into pIC19H from which it was inserted into pNC161. The original plasmid complementing coml-1 containing an -6kb insert, was isolated from a yeast genomic library (the shuttle vector M111 carrying the TRPl gene and CEN?, containing partial Sau3AIdigested genomic yeast DNA inserted into its BamHI site; a gift from G. AMMERER) as described below and was designated p260. Isolation of COMl 783