Mechanism of meiotic recombination initiation in yeast

描述(申请人提供)：这项研究的目的是了解减数分裂重组的机制，并确定这一过程如何与减数分裂前期的其他事件协调。研究的重点是酵母Spo11(一种使双链断裂(DSB)启动减数分裂重组的蛋白质)及其相互作用的蛋白质。在新的资助期间，建议继续进行实验，以研究DSB的形成和修复机制。其具体目的是：1.确定通过修饰Mer2调控DSB形成的机制。两种细胞周期调节蛋白--细胞周期蛋白依赖性激酶和CDC7，聚集在Mer2蛋白上，控制DSB的形成。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，将确定这些激酶调控Mer2的分子细节。这些研究还将检验这样一种假设，即CDC7的调节协调了DSB形成与DNA复制的时机。2.对DSB蛋白和多蛋白复合体进行生化表征。Spo11需要9种蛋白质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、染色质结合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仍不清楚。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，确定相关因素，并表征稳定的多蛋白复合体的结构和生化活性。对DSB蛋白亚群之一Rec102-Rec104进行了结构-功能分析。3.明确交叉内稳态的遗传和区域特异性控制。当DSB频率降低时，小区有能力维持高水平的交叉。这种现象被称为“交叉稳态”，已被提出与干扰和其他控制交叉数量和分布的过程有关。这一假设将通过确定在交叉干扰中具有已知缺陷的突变体是否在交叉稳态中显示平行缺陷来检验。还建议进行研究，以检验染色体上不同位置的交换动态平衡和交换-非交换决策不同的假设，并开发不需要产生可行的减数分裂产物的交换和非交换重组的新方法。 卵子或精子中的染色体数量异常会导致发育障碍或自然流产。这些异常通常是由于减数分裂同源重组中的缺陷导致的染色体分离不当所致。这个项目将解决关于重组的机制和控制的基本问题。

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.