Mismatch Repair and the Fidelity Enforcement of Homologous Recombination

描述(由申请人提供)：通过同源依赖重组的双链DNA断裂修复的高保真源于使用第二条同源DNA链作为模板来修复断裂的链。当重组机制使用不正确的模板修复DNA断裂时，就会发生非法重组，这几乎肯定会导致包括易位和缺失在内的突变。鉴定酿酒酵母中非法重组抑制子的基因筛查揭示了错配识别酶、RecQ家族解旋酶以及核酸内切和核酸外切酶的作用。在人类中，错配识别蛋白和几个RecQ家族解旋酶的突变会导致癌症的高发病率；这些蛋白质在同源重组保真度增强中的作用可能是这些突变携带者癌症易感性增加的原因之一。虽然这些研究导致了几个模型的提出，但这些遗传学研究无法确定这些蛋白质在重组途径中何时何地发挥作用，或者它们使用什么特定机制(S)来阻止非法重组中间体的进展。我将使用纯化的人类蛋白质和模仿重组中间产物的DNA底物，从生化角度研究这一过程。有了这些试剂，我将使用传统的系综方法以及单分子方法来确定错配识别复合体何时以及如何识别潜在的非法重组中间体，并研究这些复合体的结合如何影响结合中间体的命运。这些研究将阐明DNA重组的一个重要但鲜为人知的方面。参与同源重组的基因突变与癌症之间存在着明确的联系。拟议的实验结果将为同源重组的组成部分如何合作以确保DNA修复过程的高保真提供革命性的新信息。了解这些蛋白质的功能，以及这些基因的突变如何使受影响的人容易患上癌症，有可能为这些患者提供新的治疗策略。

DESCRIPTION (provided by applicant): The high fidelity of double-strand DMA break repair by homology-dependent recombination arises from the use of a second, homologous strand of DMA as a template to repair the broken strand. Illegitimate recombination occurs when the recombination machinery repairs the DNA break using an incorrect template, which will almost certainly result in mutations including translocations and deletions. Genetic screens to identify suppressors of illegitimate recombination in S. cerevisiae revealed roles for the mismatch recognition enzymes, a RecQ family helicase, and endo- and exonucleases. In humans, mutations in the mismatch recognition proteins and several of the RecQ family helicases results in a high incidence of cancer; the role of these proteins in the fidelity enforcement of homologous recombination may account for some of the increased cancer susceptibility of the carriers of these mutations. While these studies have lead to the proposal of several models, these genetic studies cannot determine when and where in the recombination pathway these proteins act, or what specific mechanism(s) they employ to prevent the progression of illegitimate recombination intermediates. I will investigate this process biochemically, using purified human proteins and DNA substrates mimicking recombination intermediates. With these reagents, I will use traditional ensemble methods as well as single molecule approaches to determine when and how the mismatch recognition complexes identify potential illegitimate recombination intermediates, as well as investigate how the binding of these complexes influences the fate of the bound intermediates. These studies will shed light upon an important, yet poorly understood aspect of DNA recombination. There is a well established link between mutations in the genes involved in homologous recombination and cancer. The results from the proposed experiments will provide revolutionary new information about how the components of homologous recombination cooperate to ensure the high fidelity of this DNA repair process. Understanding the function of these proteins, and how mutations in these genes predispose affected individuals to cancer, has the potential to provide new therapeutic strategies for these patients.