Role of Spo11 and recombination in mouse meiosis

描述（由申请人提供）：减数分裂过程中的同源重组对于染色体分离和整倍体配子的形成至关重要。减数分裂重组是由SPO11蛋白产生的双链断裂（DSBs）引发的。本研究以小鼠为模型系统，探讨了哺乳动物DSB形成和重组的分子机制。目标是：1；定义确保X-Y重组的机制。性染色体对男性减数分裂细胞构成重大挑战，因为X和Y只共享一个小的同源区域（假常染色体区域，或PAR），在这个区域内，每次减数分裂都必须发生DSB的形成和重组。该基金支持的研究揭示了小鼠PAR的独特结构特性和动态行为，这似乎对正确的X-Y分离至关重要。为了确定这些特性的分子基础，我们将在雌性减数分裂和具有更长的PAR的雄性减数分裂中检查PAR的高阶结构。这些研究将确定PAR的内在特性。PAR重组的遗传控制也将被检查，重点是SPO11同工型和DNA损伤反应激酶ATM的作用。2. 目的：阐明减数分裂重组的机制及相关因素。重组导致交叉和非交叉的形成；我们对酵母中产生这些产物的途径了解很多，但对哺乳动物中的这些机制知之甚少。这些问题将使用一种新的分析方法来解决，该方法允许所有染色单体参与单个减数分裂重组事件。使用这种小鼠“四分体”试验，将描述野生型精母细胞的重组模式。在没有ATM功能的情况下重组也将被检查。3. 确定ATM如何控制dsb的数量和分布。ATM通过负反馈回路稳态控制DSB数量，但这种调控的分子基础尚不清楚。为了解决这一问题，将确定ATM在响应SPO11产生的dsb时被激活的机制，重点研究MRE11-RAD51-NBS1复合物的作用。此外，一种新的全基因组DSB定位方法将应用于Atm-/-精母细胞，以深入了解Atm对DSB在整个基因组分布的影响。

DESCRIPTION (provided by applicant): Homologous recombination during meiosis is essential for proper chromosome segregation and thus for formation of euploid gametes. Meiotic recombination is initiated by double-strand breaks (DSBs) made by the SPO11 protein. This proposal addresses molecular mechanisms underlying mammalian DSB formation and recombination, using mouse as a model system. Aims are: 1. To define mechanisms that ensure X-Y recombination. Sex chromosomes pose significant challenges to male meiotic cells because the X and Y share only a small region of homology (the pseudoautosomal region, or PAR) within which DSB formation and recombination must occur in every meiosis. Studies supported by this grant uncovered unique structural properties and dynamic behaviors of the mouse PAR that appear critical for proper X-Y segregation. To define the molecular basis of these properties, the higher order structure of the PAR will be examined in female meiosis and in meiosis of males bearing a much longer PAR. These studies will determine what properties are intrinsic to the PAR. The genetic control of PAR recombination will also be examined, focusing on roles of SPO11 isoforms and the DNA damage response kinase ATM. 2. To elucidate mechanisms of meiotic recombination and the factors involved. Recombination leads to formation of both crossovers and noncrossovers; much is known about the pathways leading to these products in yeasts, but comparatively little is known about these mechanisms in mammals. These issues will be addressed using a novel assay that allows all chromatids involved in a single meiotic recombination event to be characterized. Using this mouse "tetrad" assay, recombination patterns in wild-type spermatocytes will be delineated. Recombination in the absence of ATM function will also be examined. 3. To determine how ATM controls the number and distribution of DSBs. ATM homeostatically controls DSB numbers via a negative feedback loop, but the molecular basis of this regulation is unclear. To address this issue, the mechanism by which ATM is activated in response to SPO11- generated DSBs will be determined, focusing on the role of the MRE11-RAD51-NBS1 complex. In addition, a new method for genome-wide DSB mapping will be applied to Atm-/- spermatocytes to provide insight into the influence of ATM on the distribution of DSBs across the genome.