Genetic analysis of early events in mammalian homologous recombination in vivo

在真核生物中，同源重组(HR)具有双重作用。一方面，它在减数分裂过程中导致同源染色体之间的交叉形成，有效地洗牌了双亲的遗传物质，导致了新的等位基因和遗传性状组合的产生。另一方面，在有丝分裂的细胞中，HR参与DNA损伤的修复，特别是DNA双链断裂(DSB)，从而忠实地促进基因组的完整性。几种人类癌症和基因组不稳定疾病增加了对癌症的易感性，这些疾病与HR缺陷有关。在真核生物中，DNA DSB修复的中心步骤涉及RAD51蛋白的多个单体与3‘端单链DNA的结合。得到的RAD51核蛋白细丝通过同源性搜索、DNA链侵入和交换等协调步骤，促进处理过的断裂DNA和同源修复模板之间形成联合分子。在联合分子形成之后是新的DNA合成，它取代了通过DSB形成和3‘端切除而丢失的核苷酸。随后的步骤可能包括解开(溶解)包含新合成的DNA的DNA链并连接到处理过的第二端，或者形成稳定连接的Holliday连接中间体，该中间体可以进一步加工以产生重组产品。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分析方法来关键地评估哺乳动物细胞中HR的机制。作为这项工作的一部分，我们开发了一种测试方法，可以在体内测量通过聚合3‘端形成的新生DNA。在最近的工作中，我们利用这一方法分析了3‘聚合的同源性要求，并分析了RAD51和BRCA2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，开发出了一种 非常灵敏的生化遗传学研究方法 发生在哺乳动物同源基因进化过程中的早期事件 体内重组，我们正在请求NSERC对此的支持 研究工作。 如提案中详细描述的，本申请具有5个具体目标，集中于3‘聚合过程的生化遗传分析，即， 1.RAD52-上位组蛋白和重组介体在HR早期事件中的功能分析 2.染色质免疫沉淀分析检测RAD51：DNA在3‘端侵袭端的相互作用。 3.对3‘-聚合过程进行物理分析 4.研究短链DNA聚合酶在3‘聚合中的作用 5.染色体构象捕获(CCC)分析。

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.