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（由申请人提供）：在减数分裂中，同源重组促进遗传多样性并确保第一次减数分裂中染色体的正确分离。重组缺陷会引发异常的染色体分离，是自发性流产（约 35% 临床认可的妊娠）、先天性出生缺陷（如唐氏综合症）（约 1/300 活产）和智力障碍的主要原因。该项目的总体目标是定义减数分裂重组机制，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影响。减数分裂诱导的拓扑异构酶 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频率和在基因组中分布的热点处。裂殖酵母粟酒裂殖酵母具有高度同步的减数分裂和明确的热点（加上出色的遗传学、分子生物学和蛋白质生物化学），为解析重组机制提供了强大的系统。在之前（第一个）资助期，我们定义了Rec12的结构和功能；我们表征了一个包含 Rec12 的大型多亚基减数分裂重组复合体 (MRC)；我们进一步定义了调节重组的途径机制。在第二个资助期，我们将重点关注将 Rec12 发起的重组引导至热点的机制。一种新的观点认为，组蛋白的翻译后修饰 (PTM) 在调节不同物种的重组热点方面发挥着关键作用。然而，有超过一百种不同的组蛋白 PTM，其中很少有人被询问其在重组中可能的作用。我们开发并验证了一种称为微型染色体亲和纯化质谱法 (MiniCAP-MS) 的方法，该方法使我们能够富集和表征染色质离散片段的成分。我们将利用这项革命性技术以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。我们将应用该技术来匹配热点和基础控制等位基因，以鉴定热点特异性结合蛋白和组蛋白 PTM。结合遗传、分子和 ChIP-seq 方法来确定功能意义。我们还开发并验证了一种将热点激活蛋白连接到染色体上的方法。除了确认各个成分（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。