The budding yeast Msh4 protein functions in chromosome synapsis and the regulation of crossover distribution.

The budding yeast MSH4 gene encodes a MutS homolog produced specifically in meiotic cells. Msh4 is not required for meiotic mismatch repair or gene conversion, but it is required for wild-type levels of crossing over. Here, we show that a msh4 null mutation substantially decreases crossover interference. With respect to the defect in interference and the level of crossing over, msh4 is similar to the zip1 mutant, which lacks a structural component of the synaptonemal complex (SC). Furthermore, epistasis tests indicate that msh4 and zip1 affect the same subset of meiotic crossovers. In the msh4 mutant, SC formation is delayed compared to wild type, and full synapsis is achieved in only about half of all nuclei. The simultaneous defects in synapsis and interference observed in msh4 (and also zip1 and ndj1/tam1) suggest a role for the SC in mediating interference. The Msh4 protein localizes to discrete foci on meiotic chromosomes and colocalizes with Zip2, a protein involved in the initiation of chromosome synapsis. Both Zip2 and Zip1 are required for the normal localization of Msh4 to chromosomes, raising the possibility that the zip1 and zip2 defects in crossing over are indirect, resulting from the failure to localize Msh4 properly.

Role for the silencing protein Dot1 in meiotic checkpoint control.

During the meiotic cell cycle, a surveillance mechanism called the "pachytene checkpoint" ensures proper chromosome segregation by preventing meiotic progression when recombination and chromosome synapsis are defective. The silencing protein Dot1 (also known as Pch1) is required for checkpoint-mediated pachytene arrest of the zip1 and dmc1 mutants of Saccharomyces cerevisiae. In the absence of DOT1, the zip1 and dmc1 mutants inappropriately progress through meiosis, generating inviable meiotic products. Other components of the pachytene checkpoint include the nucleolar protein Pch2 and the heterochromatin component Sir2. In dot1, disruption of the checkpoint correlates with the loss of concentration of Pch2 and Sir2 in the nucleolus. In addition to its checkpoint function, Dot1 blocks the repair of meiotic double-strand breaks by a Rad54-dependent pathway of recombination between sister chromatids. In vegetative cells, mutation of DOT1 results in delocalization of Sir3 from telomeres, accounting for the impaired telomeric silencing in dot1.

The pachytene checkpoint prevents accumulation and phosphorylation of the meiosis-specific transcription factor Ndt80.

In budding yeast, many mutants defective in meiotic recombination and chromosome synapsis undergo checkpoint-mediated arrest at the pachytene stage of meiotic prophase. We recovered the NDT80 gene in a screen for genes whose overexpression bypasses the pachytene checkpoint. Ndt80 is a meiosis-specific transcription factor that promotes expression of genes required for exit from pachytene and entry into meiosis I. Herein, we show that the Ndt80 protein accumulates and is extensively phosphorylated during meiosis in wild type but not in cells arrested at the pachytene checkpoint. Our results indicate that inhibition of Ndt80 activity is one mechanism used to achieve pachytene arrest.

The pachytene checkpoint.

The pachytene checkpoint prevents meiotic nuclear division in cells that fail to complete meiotic recombination and chromosome synapsis. This control mechanism prevents chromosome missegregation that would lead to the production of aneuploid gametes. The pachytene checkpoint requires a subset of proteins that function in the mitotic DNA damage checkpoint. In budding yeast, the pachytene checkpoint also requires meiosis-specific chromosomal proteins and, unexpectedly, proteins concentrated in the nucleolus. Progress has been made in identifying components of the cell-cycle machinery that are impacted by the checkpoint.

Zip3 provides a link between recombination enzymes and synaptonemal complex proteins.

In budding yeast, absence of the meiosis-specific Zip3 protein (also known as Cst9) causes synaptonemal complex formation to be delayed and incomplete. The Zip3 protein colocalizes with Zip2 at discrete foci on meiotic chromosomes, corresponding to the sites where synapsis initiates. Observations suggest that Zip3 promotes synapsis by recruiting the Zip2 protein to chromosomes and/or stabilizing the association of Zip2 with chromosomes. Zip3 interacts with a number of gene products involved in meiotic recombination, including proteins that act at both early (Mre11, Rad51, and Rad57) and late (Msh4 and Msh5) steps in the exchange process. We speculate that Zip3 is a component of recombination nodules and serves to link the initiation of synapsis to meiotic recombination.

Bypass of a meiotic checkpoint by overproduction of meiotic chromosomal proteins.

The Saccharomyces cerevisiae zip1 mutant, which exhibits defects in synaptonemal complex formation and meiotic recombination, triggers a checkpoint that causes cells to arrest at the pachytene stage of meiotic prophase. Overproduction of either the meiotic chromosomal protein Red1 or the meiotic kinase Mek1 bypasses this checkpoint, allowing zip1 cells to sporulate. Red1 or Mek1 overproduction also promotes sporulation of other mutants (zip2, dmc1, hop2) that undergo checkpoint-mediated arrest at pachytene. In addition, Red1 overproduction antagonizes interhomolog interactions in the zip1 mutant, substantially decreasing double-strand break formation, meiotic recombination, and homologous chromosome pairing. Mek1 overproduction, in contrast, suppresses checkpoint-induced arrest without significantly decreasing meiotic recombination. Cooverproduction of Red1 and Mek1 fails to bypass the checkpoint; moreover, overproduction of the meiotic chromosomal protein Hop1 blocks the Red1 and Mek1 overproduction phenotypes. These results suggest that meiotic chromosomal proteins function in the signaling of meiotic prophase defects and that the correct stoichiometry of Red1, Mek1, and Hop1 is needed to achieve checkpoint-mediated cell cycle arrest at pachytene.

Cloning and characterization of the Kluyveromyces lactis homologs of the Saccharomyces cerevisiae RED1 and HOP1 genes.

The synaptonemal complex (SC) is a meiosis-specific proteinaceous structure that holds homologous chromosomes close together along their length during the pachytene stage of meiotic prophase. The SC is observed in sexually reproducing fungi, plants and animals and is highly conserved at the cytological level. Despite this striking conservation of structure, however, the known protein components of the SC do not appear to be highly conserved across species. In Saccharomyces cerevisiae, the products of the RED1 and HOP1 genes are associated with the lateral elements of the SC. Using a functional complementation strategy, we have isolated homologs of these genes from the related yeast, Kluyveromyces lactis. The predicted K. lactis Red1 protein is 26% identical to the S. cerevisiae Red1 protein, and the K. lactis Hop1 protein is 40% identical to the S. cerevisiae Hop1 protein. The K. lactis RED1 gene fully complements the S. cerevisiae red1 mutant, both when overexpressed and when present in two copies in a diploid. However, the K. lactis HOP1 gene complements a hop1 mutant poorly when overproduced and not at all when present in two copies in a diploid. Unlike the S. cerevisiae RED1 gene, the K. lactis RED1 contains an intron; the transcript of the K. lactis gene is efficiently spliced during meiosis in S. cerevisiae.

Pachytene exit controlled by reversal of Mek1-dependent phosphorylation.

During yeast meiosis, a checkpoint prevents exit from pachytene in response to defects in meiotic recombination and chromosome synapsis. This pachytene checkpoint requires two meiotic chromosomal proteins, Red1 and Mek1; Mek1 is a kinase that phosphorylates Red1. In mutants that undergo checkpoint-mediated pachytene arrest, Mek1 is active and Red1 remains phosphorylated. Activation of Mek1 requires the initiation of meiotic recombination and certain DNA damage checkpoint proteins. Mek1 kinase activity and checkpoint-induced pachytene arrest are counteracted by protein phosphatase type 1 (Glc7). Glc7 coimmunoprecipitates with Red1, colocalizes with Red1 on chromosomes, and dephosphorylates Red1 in vitro. We speculate that phosphorylated Red1 prevents exit from pachytene and that completion of meiotic recombination triggers Glc7-dependent dephosphorylation of Red1.

Organization of the yeast Zip1 protein within the central region of the synaptonemal complex.

The yeast Zip1 protein is a component of the central region of the synaptonemal complex (SC). Zip1 is predicted to form an alpha-helical coiled coil, flanked by globular domains at the NH(2) and COOH termini. Immunogold labeling with domain-specific anti-Zip1 antibodies demonstrates that the NH(2)-terminal domain of Zip1 is located in the middle of the central region of the SC, whereas the COOH-terminal domain is embedded in the lateral elements of the complex. Previous studies have shown that overproduction of Zip1 results in the assembly of two types of aggregates, polycomplexes and networks, that are unassociated with chromatin. Our epitope mapping data indicate that the organization of Zip1 within polycomplexes is similar to that of the SC, whereas the organization of Zip1 within networks is fundamentally different. Zip1 protein purified from bacteria assembles into dimers in vitro, and electron microscopic analysis demonstrates that the two monomers within a dimer are arranged in parallel and in register. Together, these results suggest that two Zip1 dimers, lying head-to-head, span the width of the SC.

Large-scale analysis of the yeast genome by transposon tagging and gene disruption.

Economical methods by which gene function may be analysed on a genomic scale are relatively scarce. To fill this need, we have developed a transposon-tagging strategy for the genome-wide analysis of disruption phenotypes, gene expression and protein localization, and have applied this method to the large-scale analysis of gene function in the budding yeast Saccharomyces cerevisiae. Here we present the largest collection of defined yeast mutants ever generated within a single genetic background--a collection of over 11,000 strains, each carrying a transposon inserted within a region of the genome expressed during vegetative growth and/or sporulation. These insertions affect nearly 2,000 annotated genes, representing about one-third of the 6,200 predicted genes in the yeast genome. We have used this collection to determine disruption phenotypes for nearly 8,000 strains using 20 different growth conditions; the resulting data sets were clustered to identify groups of functionally related genes. We have also identified over 300 previously non-annotated open reading frames and analysed by indirect immunofluorescence over 1,300 transposon-tagged proteins. In total, our study encompasses over 260,000 data points, constituting the largest functional analysis of the yeast genome ever undertaken.

Splicing of the meiosis-specific HOP2 transcript utilizes a unique 5' splice site.

The Saccharomyces cerevisiae HOP2 gene is required to prevent formation of synaptonemal complex between nonhomologous chromosomes during meiosis. The HOP2 gene is expressed specifically in meiotic cells, with the transcript reaching maximum abundance early in meiotic prophase. The HOP2 coding region is interrupted by an intron located near the 5' end of the gene. This intron contains a nonconsensus 5' splice site (GUUAAGU) that differs from the consensus 5' splice signal (GUAPyGU) by the insertion of a nucleotide and by a single nucleotide substitution. Bases flanking the HOP2 5' splice site have the potential to pair with sequences in U1 small nuclear RNA, and mutations disrupting this pairing reduce splicing efficiency. HOP2 pre-mRNA is spliced efficiently in the absence of the Mer1 and Nam8 proteins, which are required for splicing the transcripts of two other meiosis-specific genes.

Pch2 links chromatin silencing to meiotic checkpoint control.

The PCH2 gene of Saccharomyces cerevisiae is required for the meiotic checkpoint that prevents chromosome segregation when recombination and chromosome synapsis are defective. Mutation of PCH2 relieves the checkpoint-induced pachytene arrest of the zip1, zip2, and dmc1 mutants, resulting in chromosome missegregation and low spore viability. Most of the Pch2 protein localizes to the nucleolus, where it represses meiotic interhomolog recombination in the ribosomal DNA, apparently by excluding the meiosis-specific Hop1 protein. Nucleolar localization of Pch2 depends on the silencing factor Sir2, and mutation of SIR2 also bypasses the zip1 pachytene arrest. Under certain circumstances, Sir3-dependent localization of Pch2 to telomeres also provides checkpoint function. These unexpected findings link the nucleolus, chromatin silencing, and the pachytene checkpoint.

The pachytene checkpoint in S. cerevisiae depends on Swe1-mediated phosphorylation of the cyclin-dependent kinase Cdc28.

Mutants defective in meiotic recombination and synaptonemal complex formation undergo checkpoint-mediated arrest in mid-meiotic prophase. In S. cerevisiae, this checkpoint requires Swe1, which phosphorylates and inactivates the cyclin-dependent kinase Cdc28. A swe1 deletion allows mutants that normally arrest in meiotic prophase to sporulate at wild-type levels, though sporulation is delayed. This delay is eliminated by overproducing Clb1, the major cyclin required for meiosis I. The Swe1 protein accumulates and is hyperphosphorylated in checkpoint-arrested cells. Our results suggest that meiotic arrest is mediated both by increasing Swe1 activity and limiting cyclin production, with Swe1 being the primary downstream target of checkpoint control. The requirement for Swe1 distinguishes the pachytene checkpoint from the DNA damage checkpoints operating in vegetative cells.

Synaptonemal complex morphogenesis and sister-chromatid cohesion require Mek1-dependent phosphorylation of a meiotic chromosomal protein.

Development of yeast meiotic chromosome cores into full-length synaptonemal complexes requires the MEK1 gene product, a meiosis-specific protein kinase homolog. The Mek1 protein associates with meiotic chromosomes and colocalizes with the Red1 protein, which is a component of meiotic chromosome cores. Mek1 and Red1 interact physically in meiotic cells, as demonstrated by coimmunoprecipitation and the two-hybrid protein system. Hop1, another protein associated with meiotic chromosome cores, also interacts with Mek1 but only in the presence of Red1. Red1 displays Mek1-dependent phosphorylation, both in vitro and in vivo, and Mek1 kinase activity is necessary for Mek1 function in vivo. Fluorescent in situ hybridization analysis indicates that Mek1-mediated phosphorylation of Red1 is required for meiotic sister-chromatid cohesion, raising the possibility that cohesion is regulated by protein phosphorylation.

The meiosis-specific Hop2 protein of S. cerevisiae ensures synapsis between homologous chromosomes.

The hop2 mutant of S. cerevisiae displays a novel phenotype: meiotic chromosomes form nearly wild-type amounts of synaptonemal complex, but most chromosomes are engaged in synapsis with nonhomologous partners. The meiosis-specific Hop2 protein localizes to chromosomes prior to and during synapsis and in the absence of the double-strand breaks that initiate recombination. hop2 strains sustain a wild-type level of meiotic double-strand breaks, but these breaks remain unrepaired. The hop2 mutant arrests at the pachytene stage of meiotic prophase with the RecA-like protein Dmc1 located at numerous sites along synapsed chromosomes. We propose that the Hop2 protein functions to prevent synapsis between nonhomologous chromosomes.

Telomere-mediated chromosome pairing during meiosis in budding yeast.

Certain haploid strains of Saccharomyces cerevisiae can undergo meiosis, but meiotic prophase progression and subsequent nuclear division are delayed if these haploids carry an extra chromosome (i. e., are disomic). Observations indicate that interactions between homologous chromosomes cause a delay in meiotic prophase, perhaps to allow time for interhomolog interactions to be completed. Analysis of meiotic mutants demonstrates that the relevant aspect of homolog recognition is independent of meiotic recombination and synaptonemal complex formation. A disome in which the extra chromosome is circular sporulates without a delay, indicating that telomeres are important for homolog recognition. Consistent with this hypothesis, fluorescent in situ hybridization demonstrates that a circular chromosome has a reduced capacity to pair with its homolog, and a telomere-associated meiotic protein (Ndj1) is required to delay sporulation in disomes. A circular dimer containing two copies of the same chromosome delays meiosis to the same extent as two linear homologs, implying that physical proximity bypasses the requirement for telomeres in homolog pairing. Analysis of a disome carrying two linear permuted chromosomes suggests that even nonhomologous chromosome ends can promote homolog pairing to a limited extent. We speculate that telomere-mediated chromosome movement and/or telomere clustering promote homolog pairing.

Meiotic chromosome morphology and behavior in zip1 mutants of Saccharomyces cerevisiae.

The yeast Zip1 protein (Zip1p) is a component of the central region of the synaptonemal complex (SC). Zip1p is predicted to form a dimer consisting of a coiled-coil domain flanked by globular domains. To analyze the organization of Zip1p within the SC, in-frame deletions of ZIP1 were constructed and analyzed. The results demonstrate that the C terminus but not the N terminus of Zip1p is required for its localization to chromosomes. Deletions in the carboxy half of the predicted coiled-coil region cause decreases in the width of the SC. Based on these results, a model for the organization of Zip1p within the SC is proposed. zip1 deletion mutations were also examined for their effects on sporulation, spore viability, crossing over, and crossover interference. The results demonstrate that the extent of synapsis is positively correlated with the levels of spore viability, crossing over, and crossover interference. In contrast, the role of Zip1p in synapsis is separable from its role in meiotic cell cycle progression. zip1 mutants display interval-specific effects on crossing over.

Zip2, a meiosis-specific protein required for the initiation of chromosome synapsis.

We describe the identification and characterization of the Saccharomyces cerevisiae ZIP2 gene, which encodes a novel meiosis-specific protein essential for synaptonemal complex formation. In the zip2 mutant, chromosomes are homologously paired but not synapsed. The Zip2 protein localizes to discrete foci on meiotic chromosomes; these foci correspond to sites of convergence between paired homologs that are believed to be sites of synapsis initiation. Localization of Zip2p requires the initiation of meiotic recombination. In a mutant defective in double-strand break repair, Zip2p colocalizes with proteins involved in double-strand break formation and processing. We propose that Zip2p promotes the initiation of chromosome synapsis and that localization of Zip2p to sites of interhomolog recombination ensures synapsis between homologous chromosomes.