Chromosome Synapsis and Meiotic Cell Cycle Regulation

描述(申请者提供)：本提案重点关注染色体 芽殖酵母减数分裂过程中的突触与细胞周期调控。染色体 正常的减数分裂染色体需要联会和减数分裂重组 分离，从而导致整倍体配子的产生。减数分裂检查点 阻断核分裂对突触和重组缺陷的反应 从而防止产生非整倍体后代。 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鉴定。它的意义 Zipi的DNA结合活性的研究将被探索。 一个多蛋白ZipfMsh复合体在突触起始位置作用于触发 Zipi聚合。这个综合体的结构和组装将是 利用双杂交蛋白质系统和间接研究 免疫荧光。检验突触在交叉时启动的假设 ，Zip/MSH复合体将通过染色质免疫沉淀和 相关DNA的交叉频率将被测量。角色 Zip/MSH复合体在调节交叉分布方面的作用将是 通过(I)确定ZIP4对交叉干扰的影响来调查， 以及(Ii)确定各种ZIP/MSH突变对 在不同大小的染色体上杂交。 SSP2和SSP3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。SSP2和SSP3将 观察它们对(I)S减数分裂相长度和 前期，(Ii)DNA复制和同源配对检查点，以及(Iii) DNA复制和重组启动之间的耦合。Ssp2和 SSP3将被免疫定位。 作为响应，Redi和DdCL蛋白经历了依赖于Meki的磷酸化 到重新组合的开始。Redi磷酸化的重要性将 通过鉴定和突变磷酸化残基来确定。这个 REDi和DdCL磷酸化在细胞停滞中的相对重要性 粗线期的周期将通过突变分离这些事件来评估。 将鉴定与Meki免疫共沉淀的蛋白质，以便找到 新颖的Meki底物和调节剂。 减数分裂特异的Pch2定位于核仁，是 粗线期检查站。CdCl4磷酸酶的活性受 核仁中的隔离。Pch2调控氯化镉的可能性 活性将通过检测氯化镉灭活和 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.