Mechanism and distribution of meiotic recombination initiation in mouse

描述（由申请人提供）：本研究的目的是了解哺乳动物减数分裂重组的机制。交叉在基因组中非随机发生，优先在SPO11蛋白形成双链断裂（DSBs）的1- 2kb“热点”内形成。了解热点的活动和分布对理解重组在染色体分离中的作用具有重要意义。由于小鼠减数分裂与人类减数分裂的进化相似性，因此小鼠减数分裂是这些研究的理想系统。对小鼠热点活动的影响因素进行表征，并开发一种识别重组热点的新方法。具体目标是：1。确定导致交叉热点活动变化的因素。我们对哺乳动物热点功能的分子决定因素知之甚少。为了解决这个问题，我们将研究性别、菌株背景和局部序列差异对选定热点地区重组频率的影响。2. 在交叉与非交叉决策中检测性别特异性和染色体区域特异性变异。在减数分裂细胞中，交叉产物仅占重组产物的约10%，大多数dsb被修复后产生非交叉产物。我们提出，非随机交叉位置部分源于基因组的程序性偏差——平均交叉：非交叉比率，这种偏差解释了雄性和雌性之间交叉位置的差异。我们将通过比较染色体区域内具有两性二态交叉率的热点区域的雄性和雌性交叉和非交叉的相对频率来检验这一假设。我们将类似地测试雄性不同基因组位置的热点是否因交叉：非交叉比率而不同。3. 建立一种利用spo11相关寡核苷酸序列直接识别DSB热点的新方法。SPO11蛋白通过拓扑异构酶样反应切割DNA，使SPO11共价附着在DSB的52末端。我们最近证明，这些蛋白质相关的dsb是通过核内溶解机制加工的，该机制释放与短寡核苷酸共价结合的SPO11。我们将利用这一发现，通过测序spo11相关的DNA来确定DSB热点。我们将使用酵母减数分裂作为原理证明的模型系统，并将该方法扩展到高通量方法，以绘制和量化基因组中的大量热点。然后，我们将扩展这些研究，以确定小鼠的DSB热点。卵子或精子染色体数目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缺陷引起的染色体分离不当引起的。该项目将解决有关重组机制和控制的基本问题。

DESCRIPTION (provided by applicant): The objective of this research is to understand the mechanism of mammalian meiotic recombination. Crossovers occur nonrandomly in the genome, forming preferentially within 1-2 kb "hotspots" where SPO11 protein forms double-strand breaks (DSBs). Understanding hotspot activity and distribution is important for understanding the role of recombination in chromosome segregation. Mouse meiosis is an ideal system for these studies because of its evolutionary similarity to human meiosis. Factors that contribute to hotspot activity in mouse will be characterized, and a new method for identifying recombination hotspots will be developed. The specific aims are: 1. To identify factors that contribute to variation in crossover hotspot activity. Little is known about the molecular determinants of hotspot function in mammals. To address this issue, we will examine effects of sex, strain background, and local sequence differences on recombination frequencies at selected hotspots. 2. To test for sex-specific and chromosome region-specific variation in the crossover vs. noncrossover decision. Crossovers are only ~10% of the recombination products in a meiotic cell-the majority of DSBs are repaired to give noncrossover products. We have proposed that non random crossover placement arises in part from programmed deviation from the genome- average crossover:noncrossover ratio and that this deviation accounts for differences in crossover position between males and females. We will test this hypothesis by comparing relative frequencies of crossovers and noncrossovers between male and female at hotspots within chromosomal regions with sexually dimorphic crossover rates. We will similarly test whether hotspots at different genomic positions in males vary with respect to the crossover:noncrossover ratio. 3. To develop a new method to directly identify DSB hotspots using SPO11-associated oligonucleotide sequences. SPO11 protein cleaves DNA via a topoisomerase-like reaction that leaves SPO11 covalently attached to the 52 termini of the DSB. We recently demonstrated that these protein-associated DSBs are processed by an endonucleolytic mechanism that releases SPO11 covalently bound to a short oligonucleotide. We will exploit this finding to identify DSB hotspots by sequencing the SPO11-associated DNA. We will use yeast meiosis as a model system for proof of principle and to extend this methodology to high-throughput methods to map and quantify large numbers of hotspots across the genome. We will then extend these studies to identify DSB hotspots in the mouse. PUBLIC HEALTH RELEVANCE 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.