The Role of Late MMR Proteins in DNA Repair and Cancer

描述(申请人提供)：我们研究的主要目标是分析哺乳动物DNA错配修复系统(MMR)的生物学功能，并确定MMR基因突变如何影响DNA修复和癌症易感性。MMR对于维持哺乳动物基因组的完整性是必不可少的，而MMR基因的突变会导致癌症易感性增加和减数分裂失败。真核MMR是一个复杂的系统，需要多个MutS和MutL蛋白相互作用才能启动修复反应。在错配识别之后，下游事件被激活，导致错合核苷酸(S)被切除，并通过DNA重新合成填补产生的单链缺口。在过去的资助期间，我们对外切酶1(Exol)突变小鼠进行了全面的分析，Exol是目前已知的唯一在真核切除反应中起作用的外切酶。我们确定Exol在Msh2-MSH6依赖的碱基错配修复中起作用，Exol失活导致高度渗透性癌症易感表型。此外，Exol功能的丧失会导致雄性和雌性小鼠不育，这表明Exol在哺乳动物减数分裂中起着至关重要的作用。我们假设，一组定向错义突变的产生将阐明Exol如何发挥抑制癌症的功能，并将允许研究其在减数分裂中的作用。此外，努力确定与Exol相互作用的蛋白质，将有助于阐明MMR晚期的其他关键成分。这项建议的具体目的是：1.模拟在人类癌症患者中发现的人类Exol错义突变以及在小鼠中普遍存在的编码单核苷酸多态(SNPs)，并分析由此产生的癌症易感性表型。2.确定Exol错义突变对MMR和突变避免的影响。我们将在体外分析Exol敲入突变对DNA修复功能的影响，并确定在小鼠组织中产生的体内突变表型。3.研究哺乳动物减数分裂过程中错义突变对Exol生物学功能的影响。我们将进行详细的组织病理学和细胞遗传学研究，以确定Exol错义突变可能扰乱精母细胞和卵母细胞I期前期进展的机制。4.分析一种新的Exol相互作用蛋白对MMR的意义，建立MMR复合体的体内分析系统。我们已经确定RuvBL2解旋酶是一种Exol相互作用酶，并将研究它在MMR中的潜在作用。我们还建议建立一个体内系统，用于分析MMR复合体的形成和鉴定小鼠组织中新的MMR相关蛋白。

DESCRIPTION (provided by applicant): The main goal of our research is to analyze the biological functions of the mammalian DNA mismatch repair system (MMR) and to determine how mutations in MMR genes affect DNA repair and cancer susceptibility. MMR is essential for maintaining the integrity of the mammalian genome and mutations in MMR genes result in increased cancer susceptibility and meiotic failure. Eukaryotic MMR is a complex system that requires the interaction of several MutS and MutL proteins for the initiation of the repair reaction. Subsequent to mismatch recognition, downstream events are activated that lead to the excision of the misincorporated nucleotide(s) and the filling in of the resulting single strand gap by DNA resynthesis. In the past funding period, we performed a comprehensive analysis of Exonuclease 1 (Exol) mutant mice, the only currently identified exonuclease known to function in the eukaryotic excision reaction. We determined that Exol functions in the Msh2-Msh6-dependent repair of base-base mismatches and that Exol inactivation causes a highly penetrant cancer predisposition phenotype. In addition, loss of Exol function caused infertility in male and female mice, indicating an essential role for Exol in mammalian meiosis. We hypothesize that the generation of a set of targeted missense mutations will elucidate how Exol functions to suppress cancer and will allow a study of its role in meiosis. In addition, efforts to identify proteins that interact with Exol, will help elucidate other key components of the late stages of MMR. The specific aims of this proposal are: 1. To model human Exol missense mutations found in human cancer patients as well as prevalent coding single nucleotide polymorphisms (SNPs) in mice and analyze the resulting cancer susceptibility phenotype. 2. To determine the effect of Exol missense mutations on MMR and mutation avoidance. We will analyze the effect of the Exol knock-in mutations on DNA repair functions in vitro and determine the resulting in vivo mutator phenotype in mouse tissues. 3. To determine the effects of missense mutations on the biological functions of Exol in mammalian meiosis. We will perform detailed histopathological and cytogenetic studies to determine the mechanisms by which Exol missense mutations may disrupt prophase I progression in spermatocytes and oocytes. 4. To analyze the significance of a novel Exol interacting protein for MMR and to establish a system for the in vivo analysis of MMR complexes. We have identified RuvBL2 helicase as an Exol interacting enzyme and will study its potential role in MMR. We also propose to establish an in vivo system for the analysis of MMR complex formation and the identification of novel MMR associated proteins in mouse tissue.