Biochemistry of recombination in meiosis

 描述(申请人提供)：在减数分裂中，同源重组促进了遗传多样性，并确保了第一次减数分裂中染色体的适当分离。重组缺陷会引发异常的染色体分离，是导致自然妊娠丢失(约占临床公认妊娠的35%)、唐斯综合征等先天性出生缺陷(约1/300活产)和智力残疾的主要原因。这个项目的总体目标是确定减数分裂重组的机制，这对减数分裂非整倍体的病因学、连锁图谱和基因组的进化动力学具有重要意义。在减数分裂过程中诱导的拓扑异构酶II样蛋白Rec12(Spo11)催化DNA双链断裂(DSB)的形成，从而启动重组。有趣的是，重组优先聚集在调节其在基因组中的频率和分布的热点。裂殖酵母以其高度同步的减数分裂和明确的热点(再加上优秀的遗传学、分子生物学和蛋白质生物化学)为剖析重组机制提供了强大的系统。在前一个(第一个)资助期，我们定义了Rec12的结构和功能；我们描述了一个包含Rec12的大的多亚单位减数分裂重组复合体(MRC)；我们进一步定义了调控重组的途径机制。在第二个资助期，我们将重点研究将Rec12启动的重组导向热点的机制。一种新的观点认为，组蛋白的翻译后修饰(PTM)在调节不同物种中的重组热点方面发挥着关键作用。然而，有100多种不同的组蛋白PTM，甚至很少有人被询问在重组中可能扮演的角色。我们开发并验证了一种名为微型染色体亲和纯化与质谱学(MiniCAP-MS)的方法，该方法允许我们丰富和表征染色质的离散片段的成分。我们将利用这项革命性的技术，以一种公正的方式系统地识别调节热点激活的组蛋白PTM和蛋白质。我们将应用这项技术来匹配热点和基础控制等位基因，以识别热点特异结合蛋白和组蛋白PTM。基因、分子和芯片序列方法的组合已经到位，以确定功能意义。我们还开发并验证了一种将热点激活蛋白系在染色体上的方法。除了确认单个成分(例如，组蛋白修饰酶)的顺式作用特异性外，该系统还将用于上位性分析，以阐明调节减数分裂重组的染色质重塑途径中的功能顺序。

 DESCRIPTION (provided by applicant): In meiosis, homologous recombination promotes genetic diversity and ensures the proper segregation of chromosomes in the first meiotic division. Defects in recombination trigger aberrant chromosome segregation and are the primary cause of spontaneous pregnancy loss (~35% of clinically recognized pregnancies), congenital birth defects like Downs syndrome (~1/300 live births) and intellectual disability. The overall goa of this project is to define mechanisms of meiotic recombination, which has implications for the etiology of meiotic aneuploidies, for linkage mapping, and for the evolutionary dynamics of genomes. The meiotically induced, topoisomerase II-like protein Rec12 (Spo11) catalyzes the formation of DNA double- strand breaks (DSBs) that initiate recombination. Intriguingly, recombination is clustered preferentially at hotspots that regulate its frequency and distribution in the genome. The fission yeast Schizosaccharomyces pombe, with its highly synchronous meiosis and well-defined hotspots (coupled with excellent genetics, molecular biology and protein biochemistry) provides a powerful system for dissecting mechanisms of recombination. In the previous (first) funding period, we defined the structure and function of Rec12; we characterized a large, multisubunit meiotic recombination complex (MRC) that contains Rec12; and we further defined pathway mechanisms that regulate recombination. In the second funding period, we will focus on mechanisms that direct Rec12-initiated recombination to hotspots. An emerging view is that post-translational modifications (PTMs) of histones have a key role in regulating recombination hotspots in diverse species. However, there are more than a hundred different histone PTMs and few have even been interrogated for a possible role in recombination. We developed and validated an approach called Mini-Chromosome Affinity Purification with Mass Spectrometry (MiniCAP-MS) that allows us to enrich and characterize the constituents of a discrete segment of chromatin. We will use this revolutionary technology to identify systematically, in an unbiased way, histone PTMs and proteins that regulate hotspot activation. We shall apply this technology to matching hotspot and basal control alleles to identify hotspot- specific binding proteins and histone PTMs. A combination of genetic, molecular and ChIP-seq methods are in place to determine functional significance. We also developed and validated a way to tether hotspot-activating proteins to the chromosome. In addition to confirming cis-acting specificity of individual components (e.g., histone modifying enzymes), this system will be used for epistasis analyses to elucidate order of function within pathways of chromatin remodeling that regulate meiotic recombination.