Chromosome Synapsis and Meiotic Cell Cycle Regulation

描述（申请人提供）：本提案主要针对染色体 芽殖酵母减数分裂过程中突触和细胞周期调控。染色体 正确减数分裂染色体需要联会和减数分裂重组 分离，从而产生整倍体配子。减数分裂检查点 阻止细胞核分裂，以应对突触和重组中的缺陷， 从而防止非整倍体后代的产生。 Zipi是联会复合体的主要结构成分。Zip 1-相互作用的蛋白质将被鉴定和表征。意义 Zipi的DNA结合活性将被探索。 一种多蛋白ZipfMsh复合物作用于突触起始位点， 紫皮聚合。这个综合体的结构和组装将是 研究使用双杂交蛋白质系统和间接 免疫荧光为了验证突触在交叉时启动的假设 位点，Zip/Msh复合物将通过染色质免疫沉淀回收， 测量相关DNA中的交换频率。的作用 Zip/Msh复合物在调节交叉分布中的作用将是 通过（i）确定ZIP 4对交叉干扰的影响， 和（ii）确定各种zip/msh突变对以下速率的影响： 在不同大小的染色体上进行交换。 Ssp 2和Ssp 3蛋白似乎负调控减数分裂细胞周期 在重组开始时或之前的进展。SSP 2和SSP 3将 检查它们对（i）减数分裂S期长度和 前期，（ii）DNA复制和同源物配对检查点，和（iii） DNA复制和重组起始之间的耦合。SSP 2和 Ssp 3将被免疫定位。 Redi和Ddcl蛋白在应答中经历Meki依赖性磷酸化， 重组的开始。Redi磷酸化的重要性 通过鉴定和突变磷酸化残基来确定。的 Redi和Ddcl磷酸化在细胞停滞中的相对重要性 将通过突变分离这些事件来评估粗线期的周期。 与Meki共免疫沉淀的蛋白质将被鉴定，以找到 新的Meki底物和调节剂。 减数分裂特异性Pch 2定位于核仁，并且是细胞分裂所必需的。 粗线期检查点CdCl 4磷酸酶的活性受 在核仁中的隔离。Pch 2调节CdCl 4的可能性 活性将通过检查CdCl 4失活的影响来测试， 生产过剩。

DESCRIPTION (provided by applicant): 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.