The Genetic Analysis of a Chromosome-Specific Meiotic Mutant That Permits a Premature Separation of Sister Chromatids in DROSOPHILA MELANOGASTER

mei-G87 is a recessive meiotic mutant that increases second chromosome nondisjunction in both males and females. A significant proportion of the diplo-2 exceptions are equational. In females, diplo-2 reductional exceptions are usually noncrossovers, but, in equational exceptions, crossover frequency and distribution are the same as that found in the haplo-2 controls. The frequencies of nondisjunction are relatively low: 0.6% in females and 1.3% in males. Nondisjunction frequency is affected by environmental conditions (possibly humidity). The defect in mei-G87, as in other "second division" mutants, appears to be a failure to maintain sister-chromatid cohesion. mei-G87 increases nondisjunction of only the second chromosome. This may indicate either a weak mutant with only the second chromosome being sensitive enough to misbehave or it may indicate that chromosome-specific regions responsible for sister-chromatid cohesion exist.     

Precocious Meiotic Centromere Separation of a Novel Yeast Chromosome

In Saccharomyces cerevisiae, a reciprocal translocation between chromosome II and a linear plasmid carrying a centromere (CEN6) has split chromosome II into two fragments: one, approximately 530 kilobase pairs (kbp) in size, has the left arm and part of the right arm of chromosome II; the other, a telocentric fragment approximately 350 kbp in size, has CEN6 and the rest of the right arm of chromosome II. A cross of this yeast strain with a strain containing a complete chromosome II exhibits a high frequency of precocious centromere separation (separation of sister chromatids during meiosis I) of the telocentric fragment. Precocious centromere separation is not due to the position of the centromere per se, since diploids that are homozygous for both fragments of chromosome II segregate the telocentric fragment with normal meiotic behavior. The precocious centromere separation described here differs from previously described examples in that pairing and synapsis of this telocentric chromosome seem to be normal. One model of how centromeres function in meiosis is that replication of the centromere is delayed until the second meiotic division. Data presented in this paper indicate that replication of the centromere is complete before the first meiotic division. The precocious separation of the centromere described here may be due to improper synapsis of sequences flanking the centromere.     

Pds5 Prevents the PolySUMO-Dependent Separation of Sister Chromatids

1. Download : Download high-res image (267KB)
2. Download : Download full-size image

     

The Cohesion Protein MEI-S332 Localizes to Condensed Meiotic and Mitotic Centromeres until Sister Chromatids Separate

The Drosophila MEI-S332 protein has been shown to be required for the maintenance of sister-chromatid cohesion in male and female meiosis. The protein localizes to the centromeres during male meiosis when the sister chromatids are attached, and it is no longer detectable after they separate. Drosophila melanogaster male meiosis is atypical in several respects, making it important to define MEI-S332 behavior during female meiosis, which better typifies meiosis in eukaryotes. We find that MEI-S332 localizes to the centromeres of prometaphase I chromosomes in oocytes, remaining there until it is delocalized at anaphase II. By using oocytes we were able to obtain sufficient material to investigate the fate of MEI-S332 after the metaphase II–anaphase II transition. The levels of MEI-S332 protein are unchanged after the completion of meiosis, even when translation is blocked, suggesting that the protein dissociates from the centromeres but is not degraded at the onset of anaphase II. Unexpectedly, MEI-S332 is present during embryogenesis, localizes onto the centromeres of mitotic chromosomes, and is delocalized from anaphase chromosomes. Thus, MEI-S332 associates with the centromeres of both meiotic and mitotic chromosomes and dissociates from them at anaphase.     

Heteroduplex DNA Formation and Homolog Pairing in Yeast Meiotic Mutants

Previous studies of Saccharomyces cerevisiae have identified several meiosis-specific genes whose products are required for wild-type levels of meiotic recombination and for normal synaptonemal complex (SC) formation. Several of these mutants were examined in a physical assay designed to detect heteroduplex DNA (hDNA) intermediates in meiotic recombination. hDNA was not detected in the rec102, mei4 and hop1 mutants; it was observed at reduced levels in red1, mek1 and mer1 strains and at greater than the wild-type level in zip1. These results indicate that the REC102, MEI4, HOP1, RED1, MEK1 and MER1 gene products act before hDNA formation in the meiotic recombination pathway, whereas ZIP1 acts later. The same mutants assayed for hDNA formation were monitored for meiotic chromosome pairing by in situ hybridization of chromosome-specific DNA probes to spread meiotic nuclei. Homolog pairing occurs at wild-type levels in the zip1 and mek1 mutants, but is substantially reduced in mei4, rec102, hop1, red1 and mer1 strains. Even mutants that fail to recombine or to make any SC or sc precursors undergo a significant amount of meiotic chromosome pairing. The in situ hybridization procedure revealed defects in meiotic chromatin condensation in mer1, red1 and hop1 strains.     

Mek1 Suppression of Meiotic Double-Strand Break Repair Is Specific to Sister Chromatids,Chromosome Autonomous and Independent of Rec8 Cohesin Complexes

During meiosis, recombination is directed to occur between homologous chromosomes to create connections necessary for proper segregation at meiosis I. Partner choice is determined at the time of strand invasion and is mediated by two recombinases: Rad51 and the meiosis-specific Dmc1. In budding yeast, interhomolog bias is created in part by the activity of a meiosis-specific kinase, Mek1, which is localized to the protein cores of condensed sister chromatids. Analysis of meiotic double-strand break (DSB) repair in haploid and disomic haploid strains reveals that Mek1 suppresses meiotic intersister DSB repair by working directly on sister chromatids. Rec8 cohesin complexes are not required, however, either for suppression of intersister DSB repair or for the repair itself. Regulation of DSB repair in meiosis is chromosome autonomous such that unrepaired breaks on haploid chromosomes do not prevent interhomolog repair between disomic homologs. The pattern of DSB repair in haploids containing Dmc1 and/or Rad51 indicates that Mek1 acts on Rad51-specific recombination processes.IN eukaryotes, meiosis is a specialized type of cell division that produces the gametes required for sexual reproduction. In meiosis, one round of DNA replication is followed by two rounds of chromosome segregation, termed meiosis I and II. As a result of the two divisions, four haploid cells are produced, each containing half the number of chromosomes as the diploid parent. Proper segregation at meiosis I requires connections between homologous chromosomes that are created by a combination of sister chromatid cohesion and recombination (Petronczki et al. 2003). In vegetative cells, cohesion is mediated by multisubunit ring-shaped complexes that are removed by proteolysis of the kleisin subunit, Mcd1/Scc1 (Onn et al. 2008). In meiotic cells, introduction of a meiosis-specific kleisin subunit, Rec8, allows for a two-step removal of cohesion with loss of arm cohesion at anaphase I and centromere cohesion at anaphase II (Klein et al. 1999). Missegregation of chromosomes during meiosis causes abnormal chromosome numbers in gametes that may lead to infertility and genetic disorders such as trisomy 21 or Down''s syndrome.In mitotically dividing budding yeast cells, recombination is mediated by an evolutionarily conserved RecA-like recombinase, Rad51, and occurs preferentially between sister chromatids (Kadyk and Hartwell 1992). In contrast, recombination during meiosis is initiated by the deliberate formation of double-strand breaks (DSBs) by an evolutionarily conserved, topoisomerase-like protein, Spo11, and occurs preferentially between homologous chromosomes (Jackson and Fink 1985; Schwacha and Kleckner 1997; Keeney 2001). After DSB formation, the 5′ ends on either side of the breaks are resected, resulting in 3′ single stranded (ss) tails. Rad51, and the meiosis-specific recombinase Dmc1, bind to the 3′ ssDNA tails to form protein/DNA filaments that promote strand invasion of homologous chromosomes. DNA synthesis and ligation result in the formation of double Holliday junctions, which are then preferentially resolved into crossovers (Allers and Lichten 2001; Hunter 2007).The precise roles that the Rad51 and Dmc1 recombinase activities play in meiotic recombination have been unclear because experiments have indicated both overlapping and distinct functions for the two proteins (Sheridan and Bishop 2006; Hunter 2007). While both rad51Δ and dmc1Δ mutants reduce interhomolog recombination, other studies suggest that Rad51, in complex with the accessory protein Rad54, is involved primarily in intersister DSB repair. In contrast, Dmc1, in conjunction with the accessory protein Rdh54/Tid1 (a paralog of Rad54), effects DSB repair in meiotic cells by invasion of nonsister chromatids (Dresser et al. 1997; Schwacha and Kleckner 1997; Shinohara et al. 1997a,b; Arbel et al. 1999; Bishop et al. 1999; Hayase et al. 2004; Sheridan and Bishop 2006).The preference for recombination to occur between homologous chromosomes during meiosis is created in part by Dmc1. DSBs accumulate in dmc1Δ diploids due to a failure in strand invasion (Bishop et al. 1992; Hunter and Kleckner 2001). In the efficiently sporulating SK1 strain background, these unrepaired breaks trigger the meiotic recombination checkpoint, resulting in prophase arrest (Lydall et al. 1996; Roeder and Bailis 2000). In dmc1Δ mutants, Rad51 is present at DSBs, yet there is no strand invasion of sister chromatids (Bishop 1994; Shinohara et al. 1997a). These results suggest that in addition to Dmc1 promoting interhomolog strand invasion, Rad51 activity must also be suppressed.Recent studies have shown that during meiosis Rad51 recombinase activity is inhibited by two different mechanisms that decrease the formation of Rad51/Rad54 complexes: (1) binding of the meiosis-specific Hed1 protein to Rad51, thereby excluding interaction with Rad54, and (2) reduction in the affinity of Rad54 for Rad51 due to phosphorylation of Rad54 by Mek1 (Tsubouchi and Roeder 2006; Busygina et al. 2008; Niu et al. 2009). Mek1 is a meiosis-specific kinase that is activated in response to DSBs (Niu et al. 2005, 2007; Carballo et al. 2008). In addition to phosphorylating Rad54, Mek1 phosphorylation of an as yet undetermined substrate is required to suppress Rad51/Rad54-mediated strand invasion of sister chromatids (Niu et al. 2009).To dissect the mechanism by which Mek1 suppresses meiotic intersister DSB repair, we took advantage of the ability of yeast cells to undergo haploid meiosis. The lack of homologous chromosomes in haploid cells makes it possible to examine sister-chromatid-specific events in the absence of interhomolog recombination. De Massy et al. (1994) previously observed a delay in DSB repair in haploid cells and proposed that this delay was due to a constraint in using sister chromatids. We have shown that this delay is dependent on MEK1 and utilized the haploid system to determine various biological parameters required to suppress meiotic intersister DSB repair. Our results indicate that Rad51 and Dmc1 recombinase activities have distinct roles during meiosis and that interhomolog bias is established specifically on sister chromatids through regulation of Rad51, not Dmc1. rec8Δ diploids exhibit defects in meiotic DSB repair (Klein et al. 1999; Brar et al. 2009). Given that cohesin complexes are specific for sister chromatids, we investigated the role of REC8 in intersister DSB repair and found it is required neither for suppressing intersister DSB repair during meiosis nor for the repair itself.     

The Rec102 Mutant of Yeast Is Defective in Meiotic Recombination and Chromosome Synapsis

A mutation at the REC102 locus was identified in a screen for yeast mutants that produce inviable spores. rec102 spore lethality is rescued by a spo13 mutation, which causes cells to bypass the meiosis I division. The rec102 mutation completely eliminates meiotically induced gene conversion and crossing over but has no effect on mitotic recombination frequencies. Cytological studies indicate that the rec102 mutant makes axial elements (precursors to the synaptonemal complex), but homologous chromosomes fail to synapse. In addition, meiotic chromosome segregation is significantly delayed in rec102 strains. Studies of double and triple mutants indicate that the REC102 protein acts before the RAD52 gene product in the meiotic recombination pathway. The REC102 gene was cloned based on complementation of the mutant defect and the gene was mapped to chromosome XII between CDC25 and STE11.     

A New Mapping Method Employing a Meiotic Rec- Mutant of Yeast

A rapid new mapping method has been developed for localizing a dominant or recessive mutation to a particular chromosome of yeast. The procedure utilizes the ability of strains homozygous for the spo11-1 mutation to undergo chromosome segregation without appreciable recombination during sporulation. The level of sporulation in spo11-1/spo11-1 diploids is reduced and asci are often immature or abnormal in appearance; spore viability is less than 1%. The first step of the mapping procedure is the construction of a haploid spo11-1 strain carrying a recessive drug-resistance marker and the unmapped mutation(s). This strain is crossed to a set of three spo11-1 mapping tester strains containing, among them, a recessive marker on each chromosome. The resulting spo11-1/spo11-1 diploids are sporulated and plated on drug-containing medium. Viable meiotic products that express the drug-resistance marker due to chromosome haploidization are selectively recovered. These meiotic products are haploid for most, but generally not all, chromosomes. The level of disomy for individual chromosomes averages 19%. Each of the recessive chromosomal markers is expressed in approximately a third of the drug-resistant segregants. Ninety-eight percent of these segregants show no evidence of intergenic recombination. Thus, two markers located on the same chromosome, but on different homologs, are virtually never expressed in the same drug-resistant clone. The utility of this mapping procedure is demonstrated by confirming the chromosomal location of seven known markers, as well as by the assignment of a previously unmapped mutation, spo12-1, to chromosome VIII. In addition, the analysis of the products of spo11-1 meiosis indicates that several markers previously assigned to either chromosome XIV or chromosome XVII are actually on the same chromosome.     

Meiotic Nondisjunction and Aneuploids in Intersyngenic Hybrids of PARAMECIUM CAUDATUM

Artificially induced intersyngenic crosses in Paramecium caudatum can produce viable and fertile hybrids. When F₁ hybrids of double E mating type (Mt¹/Mt³ or Mt¹²/Mt³) were crossed with mating type O (mt/mt), aberrant segregants of double E and single O type were produced. This segregation was not explained by ordinary equal or unequal crossing over. Breeding analyses of these segregants by using linkage between Mt and cnrA (a behavioral mutant) revealed that they were produced by meiotic nondisjunction of bivalent chromosomes carrying Mt genes, and thus the double E and single O segregants were aneuploids: trisomics ( Mt¹/Mt³/mt or Mt ¹²/Mt³/mt) and monosomics (mt), respectively. An aberrant segregant was also obtained for another locus, tnd 2, independent of both Mt and cnrA, suggesting the occurrence of meiotic nondisjunction throughout hybrid genomes. These aneuploids will be useful for genetic study in this species. The occurrence of meiotic nondisjunction in the intersyngenic hybrids also suggests that syngens of P. caudatum have been reproductively isolated for long enough to develop chromosomal incompatibility in their meiotic process.     

Mitotic Chromosome Structure: Reproducibility of Folding and Symmetry between Sister Chromatids

Mitotic chromosome structure and pathways of mitotic condensation remain unknown. The limited amount of structural data on mitotic chromosome structure makes it impossible to distinguish between several mutually conflicting models. Here we used a Chinese hamster ovary cell line with three different lac operator-tagged vector insertions distributed over an ∼1 μm chromosome arm region to determine positioning reproducibility, long-range correlation in large-scale chromatin folding, and sister chromatid symmetry in minimally perturbed, metaphase chromosomes. The three-dimensional positions of these lac operator-tagged spots, stained with lac repressor, were measured in isolated metaphase chromosomes relative to the central chromatid axes labeled with antibodies to topoisomerase II. Longitudinal, but not axial, positioning of spots was reproducible but showed intrinsic variability, up to ∼300 nm, between sister chromatids. Spot positions on the same chromatid were uncorrelated, and no correlation or symmetry between the positions of corresponding spots on sister chromatids was detectable, showing the absence of highly ordered, long-range chromatin folding over tens of mega-basepairs. Our observations are in agreement with the absence of any regular, reproducible helical, last level of chromosome folding, but remain consistent with any hierarchical folding model in which irregularity in folding exists at one or multiple levels.     

酵母DNA修复基因RAD16温度敏感突变株的分离及人类互补基因的鉴定

用PCR介导的基因重组法敲除酵母RAD16基因, 用羟胺处理酵母RAD16基因表达质粒, 获得RAD16基因突变库,并将其转化RAD16基因敲除的酵母细胞. 用平皿复制法获得温度敏感突变株 rad16-ts2,经互补试验证实,该突变表型为RAD16基因突变所致.将人cDNA表达文库转化rad16-ts2后筛选温度敏感拯救(rescue)表型, 回收拯救表型酵母细胞中的质粒. 测序结果表明,所获得的人cDNA克隆为HLTF/Zbu1/SMACA3基因.比较分析显示,人类该基因编码蛋白质氨基酸序列与酵母Rad16的同一性为32%,相似性则达50%.人HLTF/Zbu1/MACA3基因在HeLa细胞中过表达,可显著抑制过氧化氢损伤和UV照射诱导的细胞凋亡.     

The Yeast Red1 Protein Localizes to the Cores of Meiotic Chromosomes

Mutants in the meiosis-specific RED1 gene of S. cerevisiae fail to make any synaptonemal complex (SC) or any obvious precursors to the SC. Using antibodies that specifically recognize the Red1 protein, Red1 has been localized along meiotic pachytene chromosomes. Red1 also localizes to the unsynapsed axial elements present in a zip1 mutant, suggesting that Red1 is a component of the lateral elements of mature SCs. Anti-Red1 staining is confined to the cores of meiotic chromosomes and is not associated with the loops of chromatin that lie outside the SC. Analysis of the spo11 mutant demonstrates that Red1 localization does not depend upon meiotic recombination. The localization of Red1 has been compared with two other meiosisspecific components of chromosomes, Hop1 and Zip1; Zip1 serves as a marker for synapsed chromosomes. Double labeling of wild-type meiotic chromosomes with anti-Zip1 and anti-Red1 antibodies demonstrates that Red1 localizes to chromosomes both before and during pachytene. Double labeling with anti-Hop1and anti-Red1 antibodies reveals that Hop1 protein localizes only in areas that also contain Red1, and studies of Hop1 localization in a red1 null mutant demonstrate that Hop1 localization depends on Red1 function. These observations are consistent with previous genetic studies suggesting that Red1 and Hop1 directly interact. There is little or no Hop1 protein on pachytene chromosomes or in synapsed chromosomal regions.     

Meiotic Nondisjunction and Recombination of Chromosome III and Homologous Fragments in Saccharomyces Cerevisiae

A yeast strain, in which nondisjunction of chromosome III at the first-meiotic division could be assayed, was constructed. Using chromosome fragmentation plasmids, chromosomal fragments (CFs) were derived in isogenic strains from six sites along chromosome III and one site on chromosome VII. Whereas the presence of the CFs derived from chromosome III increased considerably the meiosis I nondisjunction of that chromosome, the CF derived from chromosome VII had no effect on chromosome III segregation. The effects of the chromosome III-derived fragments were not linearly related to fragment length. Two regions, one of 12 kb in size located at the left end of the chromosome, and the other of 5 kb, located at the center of the right arm, were found to have profound effects on chromosome III nondisjunction. Most disomics arising from meioses in strains containing chromosome III CFs did not contain the CF; thus it appears that the two chromosome III homologs had segregated away from the CF. Among the disomics, recombination between the homologous chromosomes III was lower than expected from the genetic distance, while recombination between one of the chromosomes III and the fragment was frequent. We suggest that there are sites along the chromosome that are more involved than others in the pairing of homologous chromosomes and that the pairing between fragment and homologs involves recombination among these latter elements.     

The Exonuclease Homolog OsRAD1 Promotes Accurate Meiotic Double-Strand Break Repair by Suppressing Nonhomologous End Joining

During meiosis, programmed double-strand breaks (DSBs) are generated to initiate homologous recombination, which is crucial for faithful chromosome segregation. In yeast, Radiation sensitive1 (RAD1) acts together with Radiation sensitive9 (RAD9) and Hydroxyurea sensitive1 (HUS1) to facilitate meiotic recombination via cell-cycle checkpoint control. However, little is known about the meiotic functions of these proteins in higher eukaryotes. Here, we characterized a RAD1 homolog in rice (Oryza sativa) and obtained evidence that O. sativa RAD1 (OsRAD1) is important for meiotic DSB repair. Loss of OsRAD1 led to abnormal chromosome association and fragmentation upon completion of homologous pairing and synapsis. These aberrant chromosome associations were independent of OsDMC1. We found that classical nonhomologous end-joining mediated by Ku70 accounted for most of the ectopic associations in Osrad1. In addition, OsRAD1 interacts directly with OsHUS1 and OsRAD9, suggesting that these proteins act as a complex to promote DSB repair during rice meiosis. Together, these findings suggest that the 9-1-1 complex facilitates accurate meiotic recombination by suppressing nonhomologous end-joining during meiosis in rice.Meiosis comprises two successive cell divisions after a single S phase, generating four haploid products. To ensure proper chromosome segregation at the first meiotic division, crossovers (COs) are formed between homologous chromosomes (Kleckner, 2006). CO formation requires faithful repair of programmed DNA double-strand breaks (DSBs) introduced by the protein SPO11 (Keeney et al., 1997; Shinohara et al., 1997).Mitotic cells employ two basic strategies for DSB repair: homologous recombination (HR) and classical nonhomologous end-joining (C-NHEJ; Deriano and Roth, 2013). HR requires an undamaged template sequence for repair, while the C-NHEJ pathway involves direct ligation of the broken ends in a Ku-dependent manner. Both HR and C-NHEJ safeguard genome integrity during mitosis (Ceccaldi et al., 2016; Symington and Gautier, 2011). However, DSBs are preferentially repaired by HR during meiosis, because only this pathway generates COs. C-NHEJ competes with HR and creates de novo mutations in the gametes, indicating that this activity should be restricted during meiotic DSB repair. Previous studies have identified several factors essential for preventing C-NHEJ in meiosis (Goedecke et al., 1999; Martin et al., 2005; Adamo et al., 2010; Lemmens et al., 2013).Although the mechanism inhibiting C-NHEJ during meiosis is still elusive, regulators guaranteeing the success of the HR pathway have been extensively studied. Radiation sensitive1 (RAD1) is an evolutionarily conserved protein whose best-known function is checkpoint signaling. RAD1, a member of the ring-shaped RAD9-RAD1-HUS1 (9-1-1) complex, plays a crucial role in activating the pachytene checkpoint, a surveillance mechanism for monitoring the progression of meiotic HR in many organisms (Lydall et al., 1996; Hong and Roeder, 2002; Eichinger and Jentsch, 2010).In addition to their well-known roles in checkpoint signaling, members of 9-1-1 complex may also play a direct role in facilitating DSB repair and HR during meiosis. RAD1 is associated with both synapsed and unsynapsed chromosomes during prophase I in mouse (Freire et al., 1998). The homolog of RAD1 in Saccharomyces cerevisiae is Rad17, and rad17 mutant exhibits persistent Rad51 foci (Shinohara et al., 2003). Moreover, mutations in Rad17 lead to a reduced frequency of interhomolog recombination, aberrant synapsis, increased rates of ectopic recombination events, and illegitimate repair from the sister chromatids during meiosis (Grushcow et al., 1999). Recently, Rad17 was shown to be necessary for the efficient assembly of ZMM proteins (Shinohara et al., 2015). Apart from RAD1, the other partners of 9-1-1 were also shown to be involved in DSB repair. HUS1 is proved essential for meiotic DSB repair in Drosophila (Peretz et al., 2009). Moreover, Hus1 inactivation in mouse testicular germ cells results in persistent meiotic DNA damage, chromosomal defects, and germ cell depletion (Lyndaker et al., 2013). Nevertheless, little is known about the role of 9-1-1 proteins in higher plants. In Arabidopsis (Arabidopsis thaliana), mutants of RAD9 show increased sensitivity to genotoxic agents and delayed general repair of mitotic DSBs (Heitzeberg et al., 2004). A recent study indicates that HUS1 is involved in DSB repair of both mitotic and meiotic cells in rice (Che et al., 2014).In this study, we showed that OsRAD1 was required for the accurate repair of DSBs in rice during meiosis. Importantly, we demonstrated that the defective meiotic DSB repair in the Osrad1 mutants could be partially suppressed by blocking the C-NHEJ pathway. We also investigated the relationship between OsRAD1 and other key recombination proteins. Together, our findings indicated that the 9-1-1 complex plays a crucial role in the meiotic DSB repair mechanism.     

Yeast Mer1 Mutants Display Reduced Levels of Meiotic Recombination

Mutations at the MER1 locus were identified in a search for meiotic mutants defective in chromosome segregation. mer1 strains show decreased levels of inter- and intrachromosomal meiotic recombination and produce inviable spores. The MER1 gene was cloned by complementation of the spore inviability phenotype. Strains carrying disruptions of the MER1 gene are mitotically viable. The epistatic relationships between MER1 and previously characterized meiotic genes are described.     

The Sep1 Mutant of Saccharomyces Cerevisiae Arrests in Pachytene and Is Deficient in Meiotic Recombination

Strand exchange protein 1 (Sep1) from Saccharomyces cerevisiae promotes homologous pairing of DNA in vitro and sep1 mutants display pleiotropic phenotypes in both vegetative and meiotic cells. In this study, we examined in detail the ability of the sep1 mutant to progress through meiosis I prophase and to undergo meiotic recombination. In meiotic return-to-growth experiments, commitment to meiotic recombination began at the same time in wild type and mutant; however, recombinants accumulated at decreased rates in the mutant. Gene conversion eventually reached nearly wild-type levels, whereas crossing over reached 15-50% of wild type. In an assay of intrachromosomal pop-out recombination, the sep1, dmc1 and rad51 single mutations had only small effects; however, pop-out recombination was virtually eliminated in the sep1 dmc1 and sep1 rad51 double mutants, providing evidence for multiple recombination pathways. Analysis of meiotic recombination intermediates indicates that the sep1 mutant is deficient in meiotic double-strand break repair. In a physical assay, the formation of mature reciprocal recombinants in the sep1 mutant was delayed relative to wild type and ultimately reached only 50% of the wild-type level. Electron microscopic analysis of meiotic nuclear spreads indicates that the sep1δ mutant arrests in pachytene, with apparently normal synaptonemal complex. This arrest is RAD9-independent. We hypothesize that the Sep1 protein participates directly in meiotic recombination and that other strand exchange enzymes, acting in parallel recombination pathways, are able to substitute partially for the absence of the Sep1 protein.