Genetic analysis of early events in mammalian homologous recombination in vivo

哺乳动物体内同源重组早期事件的遗传分析

• 批准号: RGPIN-2016-03633
• 负责人: Baker, Mark
• 金额: $ 2.26万
• 依托单位: University of Guelph
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2018
• 资助国家: 加拿大
• 起止时间: 2018-01-01 至 2019-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=644503
• 关键词: Genetic; analysis; early; events; mammalian

In eukaryotic organisms, homologous recombination (HR) serves a dual role. On one hand, it results in crossover formation between homologous chromosomes during meiosis, in effect, shuffling the genetic material of both parents and leading to the generation of new combinations of alleles and genetic traits. On the other hand, in mitotically-dividing cells, HR serves in the repair of DNA damage, especially DNA double strand breaks (DSB) and thus, faithfully promotes genome integrity. Several human cancers and genome-destabilizing diseases that confer increased susceptibility to cancer are linked to defects in HR. In eukaryotes, the central step in the repair of a DNA DSB involves the binding of multiple monomers of the RAD51 protein to 3' ending single stranded DNA created by nucleolytic resection. The resulting RAD51 nucleoprotein filament promotes formation of a joint molecule between the processed broken DNA and the homologous repair template by the orchestrated steps of homology searching, DNA strand invasion and exchange. Joint molecule formation is followed by new DNA synthesis, which replaces nucleotides lost through DSB formation and 3' end resection. Subsequent steps may involve unwinding (dissolution) of the DNA strand containing the newly synthesized DNA and ligation to the processed second end, or the formation of a stably-joined Holliday junction intermediate that can be processed further to yield recombinant products. Unfortunately, most of our knowledge of the HR process comes from studies in microbial systems. Our laboratory has developed sensitive assays to critically assess the mechanisms of HR in mammalian cells. As part of this effort, we developed an assay that measures the nascent DNA formed by polymerizing 3' ends in vivo. In recent work, we exploited this assay in the analysis of the homology requirements for 3' polymerization and the analysis of the role of Rad51 and Brca2 in the process. Thus, our laboratory is in the very unique position of having developed a*very sensitive assay for studying the biochemical genetics of very*early events that occur during the process of mammalian homologous*recombination in vivo and we are requesting NSERC support for this*research work. ***As detailed in the proposal, this application has 5 specific aims focused on the biochemical genetic analysis of the 3' polymerization process, namely, ***1. Functional analysis of Rad52-epistasis group proteins and recombination mediators in the early events of HR. ***2. Detecting Rad51:DNA interactions at a 3' invading end by chromatin immunoprecipitation (CHIP) analysis. ***3. To conduct a physical analysis of the 3' polymerization process***4. Investigate the role of short tract DNA polymerases involved in 3' polymerization***5. Chromosome conformation capture (CCC) analysis. *****

在真核生物中，同源重组（HR）起着双重作用。一方面，它导致减数分裂期间同源染色体之间的交叉形成，实际上，改组双亲的遗传物质，并导致产生等位基因和遗传性状的新组合。另一方面，在有丝分裂细胞中，HR用于修复DNA损伤，特别是DNA双链断裂（DSB），因此忠实地促进基因组完整性。在真核生物中，DNA DSB修复的中心步骤涉及RAD 51蛋白的多个单体与溶核切除产生的3'末端单链DNA的结合。所得的RAD 51核蛋白丝通过同源性搜索、DNA链侵入和交换的协调步骤促进加工的断裂DNA和同源修复模板之间的接合分子的形成。接合分子形成之后是新的DNA合成，其替换通过DSB形成和3'端切除而丢失的核苷酸。随后的步骤可能涉及含有新合成的DNA的DNA链的解旋（溶解）和连接到加工的第二末端，或形成稳定连接的霍利迪连接中间体，其可以进一步加工以产生重组产物。不幸的是，我们对HR过程的大部分知识都来自于对微生物系统的研究。我们的实验室已经开发出灵敏的检测方法，以严格评估哺乳动物细胞中HR的机制。作为这项工作的一部分，我们开发了一种测定方法，该方法测量通过在体内聚合3'末端形成的新生DNA。在最近的工作中，我们利用该测定分析3'聚合的同源性要求和分析Rad 51和Brca 2在该过程中的作用。因此，我们的实验室处于非常独特的地位，已经开发了一种非常敏感的检测方法，用于研究哺乳动物体内同源重组过程中发生的非常早期事件的生化遗传学，我们正在请求NSERC支持这项研究工作。* 如提案中所详述，本申请具有5个具体目标，集中于3'聚合过程的生物化学遗传分析，即 *1. Rad 52-上位组蛋白和重组介质在HR早期事件中的功能分析。*2.通过染色质免疫沉淀（CHIP）分析检测Rad 51：DNA在3'侵入末端的相互作用。*3。进行3'聚合过程的物理分析 *4.研究参与3'聚合的短链DNA聚合酶的作用 *5。染色体构象捕获（CCC）分析。*****