Mechanism and distribution of meiotic recombination initiation in mouse

本研究的目的是了解哺乳动物减数分裂的机制 重组交叉在基因组中非随机发生，优先在1-2内形成 kb“热点”，其中SPO 11蛋白形成双链断裂（DSB）。了解热点 活性和分布对于理解染色体中重组的作用是重要的 种族隔离小鼠减数分裂是一个理想的系统，因为它的进化 与人类减数分裂相似。有助于小鼠中热点活动的因素将是 特征，并将开发用于鉴定重组热点的新方法。的 具体目标是： 1.确定导致交叉热点活性变化的因素。之甚少 已知哺乳动物中热点功能的分子决定因素。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 重组频率在选定的热点。 2.为了测试交叉与交叉中的性别特异性和染色体区域特异性变异， 非交叉决策在减数分裂的重组产物中， 单元-大多数DSB被修复以得到非交叉产物。我们建议 非随机的交叉放置部分地由基因组的程序性偏离引起- 平均交叉：非交叉比率，这种偏差解释了 男性和女性之间的交叉位置。我们将通过比较 热点地区雌雄间交叉和非交叉的相对频率 在染色体区域内具有性二态交叉率。我们将以同样的方式测试 男性不同基因组位置上的热点是否会随 交叉：非交叉比率。 3.开发一种新的方法，直接识别DSB热点使用SPO 11相关 寡核苷酸序列。SPO 11蛋白通过拓扑异构酶样反应切割DNA， 使SPO 11共价连接到DSB的52末端。我们最近证明， 这些与蛋白质相关的DSB通过核酸内切机制进行加工， SPO 11与短寡核苷酸共价结合。我们将利用这一发现来确定DSB 通过对SPO 11相关DNA进行测序来确定热点。我们将使用酵母减数分裂作为模型 并将该方法扩展到高通量方法， 绘制并量化基因组中大量的热点。然后我们将这些 研究以识别小鼠中的DSB热点。

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.