Biochemistry of recombination in meiosis

减数分裂重组的生物化学

• 批准号: 8961476
• 负责人: Wayne P Wahls
• 金额: $ 29.46万
• 依托单位: UNIV OF ARKANSAS FOR MED SCIS
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-08-10 至 2019-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8961476
• 关键词: Affinity Chromatography; Alleles; Amino Acids; Aneuploidy; Animal Model; Antibodies; Biochemical; Biochemistry; Biological; Biological Assay; Biological Models; Biology; ChIP-seq; Chromatin; Chromatin Remodeling Factor; Chromosome Mapping; Chromosome Segregation; Chromosome abnormality; Chromosomes; Complex; Congenital Abnormality; Coupled; DNA Double Strand Break; Defect; Diagnostic; Diploidy; Down Syndrome; Ensure; Enzymes; Etiology; Event; Fission Yeast; Frequencies; Funding; Gene Targeting; Genes; Genetic; Genetic Epistasis; Genetic Recombination; Genetic Variation; Genome; Haploidy; Histone Code; Histones; Individual; Intellectual functioning disability; Knowledge; Live Birth; Macromolecular Complexes; Mass Spectrum Analysis; Measures; Meiosis; Meiotic Recombination; Methods; Molecular; Molecular Biology; Molecular Genetics; Organism; Parents; Pathway interactions; Positioning Attribute; Post-Translational Protein Processing; Pregnancy; Pregnancy loss; Protein Biochemistry; Proteins; Regulation; Relative (related person); Resolution; Role; SPO11 gene; Sampling; Scientist; Site; Specificity; Spontaneous abortion; Structure; System; Technology; Testing; Time; Topoisomerase II; Work; base; chromatin remodeling; genome-wide; histone acetyltransferase; histone-binding proteins; homologous recombination; in vivo; mutant; novel; protein protein interaction; public health relevance; reverse genetics; tool

 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.

 描述（由应用提供）：在减数分裂中，同源重组促进遗传多样性，并确保在第一个减数分裂分裂中正确分离染色体。重组缺陷引发异常的染色体分离，是赞助妊娠丧失的主要原因（占临床认可的怀孕的35％），先天性先天性缺陷，如唐氏综合症（〜1/300 Live Births）和智力残疾。该项目的总体果阿是定义减数分裂重组的机制，这对减数分裂性非整倍体的病因具有链接映射和基因组进化动力学的影响。通过诱导的，拓扑异构酶II样蛋白REC12（SPO11）催化启动重组的DNA双链断裂（DSB）的形成。有趣的是，重组更优选地聚集在调节其基因组频率和分布的热点上。裂变酵母菌酸果实POMBE具有高度同步的减数分裂和明确定义的热点（结合出色的遗传学，分子生物学和蛋白质生物化学）为解剖重组机制提供了有力的系统。在上一个（第一个）资金期间，我们定义了REC12的结构和功能；我们表征了包含REC12的大型多亚基减数分裂复合复合物（MRC）。我们进一步定义了调节重组的途径机制。在第二个融资期间，我们将重点关注将REC12引发重组的机制。一个新兴的观点是，组蛋白的翻译后修饰（PTM）在控制潜水员物种的重组热点方面具有关键作用。但是，有一百多个不同的组蛋白PTM，甚至很少有人被审问以在重组中发挥作用。我们开发并验证了一种用质谱（Minicap-MS）的称为小染色体亲和力纯化的方法，该方法使我们能够丰富和表征染色质的离散片段的构成。我们将使用这种革命性技术以公正的方式识别调节热点激活的组蛋白PTM和蛋白质。我们将将这项技术应用于匹配的热点和基本控制等位基因，以识别热点特异性结合蛋白和组蛋白PTM。遗传，分子和CHIP-seq方法的结合已适当，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绳到染色体的方法。除了确认单个成分的顺式作用特异性（例如，组蛋白修饰酶）外，该系统还将用于上学分析，以阐明在调节减数分裂重组的染色质重塑途径内的功能顺序。