Mechanism of meiotic recombination initiation in yeast

描述（由申请人提供）：本研究的目的是了解减数分裂重组的机制，并确定该过程如何与减数分裂前期的其他事件协调。这些研究的重点是酵母Spo11（一种产生双链断裂（DSBs）并启动减数分裂重组的蛋白质）和与其相互作用的蛋白质。在新的资助期内，我们将继续研究DSB的形成和修复机制。具体目标是：1。通过Mer2的修饰，确定调控DSB形成的机制。两种细胞周期调节激酶，周期蛋白依赖性激酶和Cdc7，聚集在Mer2蛋白上控制DSB的形成。这些激酶对Mer2调控的分子细节将通过确定Mer2在染色体上的定位模式和确定Mer2磷酸化发生的时间和地点来确定。这些研究还将验证Cdc7调节DSB形成时间与DNA复制的假设。2. 对DSB蛋白和多蛋白复合物进行生物化学表征。Spo11需要9种蛋白的活性才能产生减数分裂dsb。根据它们之间的成对相互作用、染色体定位模式、对染色质关联的依赖性和其他行为，这些DSB蛋白可以分为几个不同的功能亚群。然而，这些亚群的生化性质以及它们之间的相互作用尚不清楚。为了更详细地了解这些问题，我们将从减数分裂提取物中亲和纯化DSB蛋白，鉴定相关因子，并表征稳定的多蛋白复合物的结构和生化活性。对DSB蛋白亚群之一的Rec102-Rec104也提出了结构-功能分析。3. 定义交叉稳态的遗传和区域特异性控制。当DSB频率降低时，细胞有能力维持高水平的交叉。这种现象被称为“交叉稳态”，并被认为与控制交叉数量和分布的干扰和其他过程有机制关系。这一假设将通过确定在交叉干扰中已知缺陷的突变体是否在交叉稳态中表现出平行缺陷来验证。研究还提出了验证交叉稳态和交叉-非交叉决策在染色体上的不同位置不同的假设，并开发不需要产生可行减数分裂产物的交叉和非交叉重组的新方法。

DESCRIPTION (provided by applicant): The objectives of this research are to understand the mechanism of meiotic recombination and to determine how this process is coordinated with other events of meiotic prophase. The studies focus on yeast Spo11 (the protein that makes the double-strand breaks (DSBs) that initiate meiotic recombination) and the proteins that interact with it. In the new grant period, experiments are proposed to continue studying the mechanisms of DSB formation and repair. The specific aims are: 1. To determine mechanisms that regulate DSB formation via modification of Mer2. Two cell cycle regulatory kinases, cyclin-dependent kinase and Cdc7, converge on the Mer2 protein to control DSB formation. Molecular details of Mer2 regulation by these kinases will be defined by determining patterns of Mer2 localization on chromosomes and determining when and where Mer2 phosphorylation occurs. These studies will also test the hypothesis that regulation by Cdc7 coordinates timing of DSB formation with DNA replication. 2. To biochemically characterize DSB proteins and multiprotein complexes. Spo11 requires activities of nine proteins in order to generate meiotic DSBs. These DSB proteins can be divided into several distinct functional subgroups based on pairwise interactions among them, chromosomal localization patterns, dependencies for chromatin association, and other behaviors. The biochemical nature of these subgroups and the interactions among them remain unclear, however. To provide more detailed understanding of these issues, DSB proteins will be affinity-purified from meiotic extracts, associated factors will be identified, and the architecture and biochemical activities of stable multiprotein complexes will be characterized. Structure- function analysis is also proposed for Rec102-Rec104, one of the DSB protein subgroups. 3. To define the genetic and region-specific control of crossover homeostasis. Cells have the ability to maintain high levels of crossovers when DSB frequencies are reduced. This phenomenon is referred to as "crossover homeostasis" and has been proposed to be mechanistically related to interference and other processes that control the number and distribution of crossovers. This hypothesis will be tested by determining whether mutants with known defects in crossover interference show parallel defects in crossover homeostasis. Studies are also proposed to test the hypothesis that crossover homeostasis and the crossover-noncrossover decision vary from location to location on chromosomes, and to develop new assays for crossover and noncrossover recombination that do not require the generation of viable meiotic products. Abnormal chromosome numbers in eggs or sperm cause developmental disabilities or spontaneous abortion. These abnormalities often arise because of improper separation of chromosomes caused by defects in meiotic homologous recombination. This project will address fundamental questions about the mechanism and control of recombination.