Role of Spo11 and recombination in mouse meiosis

描述(申请人提供)：减数分裂过程中的同源重组对于正确的染色体分离和整倍体配子的形成是必不可少的。减数分裂重组是由SPO11蛋白引起的双链断裂(DSB)启动的。这项建议以小鼠为模型系统，研究哺乳动物DSB形成和重组的分子机制。目标是：1.确定确保X-Y重组的机制。性染色体对雄性减数分裂细胞构成了巨大的挑战，因为X和Y只有一个很小的同源性区域(伪常染色体区域，或PAR)，在这个区域内，DSB的形成和重组必须在每一次减数分裂中发生。这笔资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，将在雌性减数分裂和雄性减数分裂中检查PAR的高级结构，这些减数分裂具有更长的PAR。这些研究将确定哪些属性是PAR的内在属性。PAR重组的遗传控制也将被研究，重点是SPO11亚型和DNA损伤反应激酶ATM的作用。2.阐明减数分裂重组的机制及其影响因素。重组导致交叉和非交叉的形成；人们对酵母中导致这些产物的途径知之甚少，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，该方法允许对单个减数分裂重组事件中涉及的所有染色单体进行表征。利用这个小鼠的“四分体”实验，将描绘出野生型精母细胞中的重组模式。还将研究在没有自动柜员机功能的情况下的重组。3.确定ATM如何控制DSB的数量和分布。ATM通过负反馈环控制DSB数量，但这种调节的分子基础尚不清楚。为了解决这个问题，将确定ATM响应SPO11产生的DSB而被激活的机制，重点是Mre11-RAD51-NBS1复合体的作用。此外，一种新的全基因组DSB定位方法将被应用于ATM-/-精母细胞，以深入了解ATM对DSB在基因组中分布的影响。

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.