This proposal focuses on chromosome synapsis and cell cycle regulation during meiosis in budding yeast. Chromosome synapsis and meiotic recombination are required for proper meiotic chromosome segregation and thus for the generation of euploid gametes. Meiotic checkpoints block nuclear division in response to defects in synapsis and recombination and thus prevent the production of aneuploid offspring. Zipi is a major structural component of the synaptonemal complex. Zip 1-interacting proteins will be identified and characterized. The significance of the DNA-binding activity of Zipi will be explored. A multi-protein ZipfMsh complex acts at sites of synapsis initiation to trigger Zipi polymerization. The structure and assembly of this complex will be investigated using the two-hybrid protein system and indirect immunofluoresence. To test the hypothesis that synapsis initiates at crossover sites, Zip/Msh complexes will be recovered by chromatin immunoprecipitation and the frequency of crossing over in the associated DNA will be measured. The role of the Zip/Msh complex in regulating crossover distribution will be investigated by (i) determining the effect of zip4 on crossover interference, and (ii) determining the effects of various zip/msh mutations on the rates of crossing over on chromosomes of different sizes. The Ssp2 and Ssp3 proteins appear to negatively regulate meiotic cell cycle progression at or prior to the initiation of recombination. ssp2 and ssp3 will be examined for their effects on (i) the lengths of meiotic S-phase and prophase, (ii) the DNA replication and homolog pairing checkpoints, and (iii) the coupling between DNA replication and recombination initiation. Ssp2 and Ssp3 will be immunolocalized. The Redi and Ddcl proteins undergo Meki-dependent phosphorylation in response to the initiation of recombination. The importance of Redi phosphorylation will be determined by identifying and mutating the phosphorylated residues. The relative importance of Redi and Ddcl phosphorylation in arresting the cell cycle at pachytene will be assessed by separating these events by mutation. Proteins that coimmunoprecipitate with Meki will be identified in order to find novel Meki substrates and regulators. The meiosis-specific Pch2 localizes to the nucleolus and is required for the pachytene checkpoint. Activity of the Cdcl4 phosphatase is regulated by sequestration in the nucleolus. The possibility that Pch2 regulates Cdcl4 activity will be tested by examining the effects of Cdcl4 inactivation and overproduction.
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