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个范围内形成 Kb“热点”，SPO11蛋白形成双链断裂(DSB)。了解热点 活性和分布对于理解重组在染色体中的作用很重要。 种族隔离。小鼠减数分裂是这些研究的理想系统，因为它是进化的。 与人类减数分裂相似。影响鼠标热点活动的因素将是 并将开发一种新的识别重组热点的方法。这个 具体目标是： 1.找出导致交叉热点活动变化的因素。小才是 已知哺乳动物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 选定热点的重组频率。 2.测试杂交后代的性别特异性和染色体区域特异性变异。 非交叉决策。杂交只占减数分裂中重组产物的10%左右 电池-大多数DSB都经过修复，以提供非交叉产品。我们已经提议 非随机的交叉放置在一定程度上是由于程序偏离了基因组- 平均交叉率：非交叉率，这一偏差解释了 雄性和雌性之间的交叉位置。我们将通过比较来检验这一假说 热点地区男性与女性的交叉和非交叉的相对频率 在有性二态交叉率的染色体区域内。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因 交叉：非交叉比率。 3.建立了一种利用SPO11基因直接识别DSB热点的新方法 寡核苷酸序列。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 这些与蛋白质相关的双链断裂蛋白由一种核酸内切机制处理，释放出 SPO11与短的寡核苷酸共价结合。我们将利用这一发现来识别DSB 通过对SPO11相关DNA进行测序来确定热点。我们将以酵母减数分裂为模型 用于原则证明的系统，并将该方法扩展到高通量方法，以 绘制并量化整个基因组中的大量热点。然后我们将延长这些 研究以确定小鼠的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.