Mechanism and distribution of meiotic recombination initiation in mouse

描述(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1-2kb的“热点”内形成，SPO11蛋白在此形成双链断裂(DSB)。了解热点的活性和分布对于理解重组在染色体分离中的作用是重要的。小鼠的减数分裂是这些研究的理想系统，因为它在进化上与人类的减数分裂相似。我们将对影响小鼠热点活性的因素进行表征，并开发出一种识别重组热点的新方法。具体目标是：1.找出导致交叉热点活动变化的因素。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处重组频率的影响。2.测试交叉与非交叉决策中的性别特异性和染色体区域特异性变异。在减数分裂细胞中，交叉只占重组产物的10%-大多数DSB被修复以提供非交叉产物。我们已经提出，非随机的交叉放置部分是由于程序化偏离基因组平均交叉：非交叉比率，并且这种偏离解释了雄性和雌性之间的交叉位置的差异。我们将通过比较男性和女性在染色体区域热点上的交叉和非交叉的相对频率和性二态交叉率来检验这一假设。类似地，我们将测试雄性不同基因组位置的热点是否会随着交换：非交换比率的不同而变化。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共价连接到DSB的52个末端。我们最近证明了这些蛋白质相关的DSB是通过内切核溶解机制来处理的，该机制释放共价结合到短寡核苷酸上的SPO11。我们将利用这一发现，通过对SPO11相关DNA进行测序来确定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，并将这种方法扩展到高通量方法，以绘制和量化整个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。公共卫生相关性卵子或精子中的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 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.