Mismatch Repair and the Fidelity Enforcement of Homologous Recombination

描述（由申请人提供）：通过同源依赖性重组进行的双链DNA断裂修复的高保真度源于使用DNA的第二同源链作为修复断裂链的模板。当重组机制使用不正确的模板修复DNA断裂时，发生非法重组，这几乎肯定会导致突变，包括易位和缺失。遗传筛选以鉴定S.酿酒酵母揭示了错配识别酶，RecQ家族解旋酶，内切核酸酶和外切核酸酶的作用。在人类中，错配识别蛋白和几种RecQ家族解旋酶的突变导致癌症的高发病率;这些蛋白质在同源重组的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。虽然这些研究已经导致了几种模型的建议，这些遗传学研究不能确定这些蛋白质在重组途径中何时何地起作用，或者它们采用什么特定机制来防止非法重组中间体的进展。我将用纯化的人类蛋白质和模拟重组中间体的DNA底物，从生物化学的角度研究这个过程。有了这些试剂，我将使用传统的集成方法以及单分子方法来确定何时以及如何错配识别复合物识别潜在的非法重组中间体，以及研究这些复合物的结合如何影响绑定中间体的命运。这些研究将阐明DNA重组的一个重要但知之甚少的方面。在参与同源重组的基因突变和癌症之间存在着明确的联系。从拟议的实验结果将提供有关同源重组的组件如何合作，以确保这种DNA修复过程的高保真革命性的新信息。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个体易患癌症，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.