Genetic analysis of early events in mammalian homologous recombination in vivo

在真核生物中，同源重组具有双重作用。一方面，它在减数分裂期间导致同源染色体之间的交叉形成，实际上是对双亲的遗传物质进行洗牌，导致等位基因和遗传性状的新组合的产生。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），从而忠实地促进基因组完整性。导致癌症易感性增加的几种人类癌症和基因组不稳定疾病与HR缺陷有关。在真核生物中，DNA DSB修复的核心步骤是将RAD51蛋白的多个单体结合到通过溶核切除产生的3 '端单链DNA上。由此产生的RAD51核蛋白丝通过同源性搜索、DNA链入侵和交换等精心安排的步骤，促进断裂DNA与同源修复模板之间的连接分子的形成。关节分子形成之后是新的DNA合成，取代通过DSB形成和3 '端切除丢失的核苷酸。随后的步骤可能包括解绕（溶解）含有新合成DNA的DNA链并连接到加工后的第二端，或者形成稳定连接的Holliday连接中间体，可以进一步加工以产生重组产物。不幸的是，我们对HR过程的大部分知识来自微生物系统的研究。我们的实验室已经开发出敏感的检测方法，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，我们开发了一种检测方法，可以测量体内通过聚合3 '端形成的新生DNA。在最近的工作中，我们利用该方法分析了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，开发了非常敏感的检测方法，用于研究哺乳动物同源重组过程中发生的一年生事件的生化遗传学

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 avery sensitive assay for studying the biochemical genetics of veryearly events that occur during the process of mammalian homologousrecombination