The Role of Late MMR Proteins in DNA Repair and Cancer

晚期 MMR 蛋白在 DNA 修复和癌症中的作用

• 批准号: 7323699
• 负责人: WINFRIED EDELMANN
• 金额: $ 36.79万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-01-01 至 2012-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7323699
• 关键词: Address; Affect; Base-Base Mismatch; Biochemical; Biological Assay; Biological Process; Cancer Patient; Cell Extracts; Code; Complex; Cytogenetics; DNA; DNA Repair; Defect; Depth; Enzymes; Event; Excision; Exodeoxyribonuclease I; Exonuclease; Failure; Female; Fertility Study; Funding; Generations; Genes; Genetic; Genetic Polymorphism; Genome Stability; Goals; Human; In Vitro; Infertility; Knock-in Mouse; Knockout Mice; Lead; Malignant Neoplasms; Meiosis; Meiotic Prophase I; Mismatch Repair; Missense Mutation; Modeling; Mus; Mutant Strains Mice; Mutation; Nucleotides; Oocytes; Phenotype; Predisposition; Proteins; Proteomics; Range; Reaction; Research; Role; Series; Single Nucleotide Polymorphism; Spermatocytes; Staging; System; Systems Analysis; Tertiary Protein Structure; Testing; Tissues; Transgenic Mice; base; carcinogenesis; embryonic stem cell; genetic analysis; helicase; in vivo; insight; male; mutant; novel; prevent; repaired; reproductive

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.

描述（由申请人提供）：我们研究的主要目标是分析哺乳动物DNA不匹配修复系统（MMR）的生物学功能，并确定MMR基因中的突变如何影响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和MMR基因中的突变至关重要，从而导致癌症易感性和减数分裂衰竭增加。真核MMR是一个复杂的系统，它需要几种MUT和MUTL蛋白的相互作用来启动修复反应。在不匹配识别之后，激活下游事件，从而导致不合格的核苷酸（S）切除，并通过DNA重新合成所产生的单链间隙填充。在过去的资金期间，我们对核酸外切酶1（外来）突变小鼠进行了全面的分析，这是当前已知的外核酸酶在真核切除反应中起作用。我们确定外醇在基碱不匹配的MSH2-MSH6依赖性修复中起作用，并且外来失活导致高度渗透性的癌症倾向表型。此外，外摩尔功能的丧失导致雄性和雌性小鼠的不育症，这表明外生在哺乳动物减数分裂中起着至关重要的作用。我们假设一组靶向错义突变的产生将阐明海外功能如何抑制癌症，并允许研究其在减数分裂中的作用。此外，识别与Exol相互作用的蛋白质的努力将有助于阐明MMR后期阶段的其他关键组成部分。该提案的具体目的是：1。对人类癌症患者发现的人类外来错过突变进行建模以及小鼠中普遍编码的单核苷酸多态性（SNP），并分析所得的癌症易感性表型。 2。确定外来错义突变对MMR和避免突变的影响。我们将分析Exol敲进突变对体外DNA修复功能的影响，并确定导致小鼠组织中体内突变器表型的影响。 3。确定错义突变对哺乳动物减数分裂中海ol的生物学功能的影响。我们将执行详细的组织病理学和细胞遗传学研究，以确定外摩尔错义突变可能破坏精子细胞和卵母细胞中预言的进展的机制。 4。分析新型外醇相互作用蛋白对于MMR的重要性，并为MMR复合物的体内分析建立系统。我们已经将RUVBL2解旋酶确定为一种外醇相互作用酶，并将研究其在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复合物的形成和小鼠组织中新型MMR相关蛋白的鉴定。