ENZYMOLOGY OF EUKARYOTIC DNA MISMATCH REPAIR

真核 DNA 错配修复的酶学

• 批准号: 3304559
• 负责人: PAUL LAWRENCE MODRICH
• 金额: $ 21.29万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-01-01 至 1994-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3304559
• 关键词: CHO cells; DNA binding protein; DNA damage; DNA directed DNA polymerase; DNA footprinting; DNA methylation; DNA repair; DNA replication; HeLa cells; complementary DNA; dihydrofolate reductase; enzyme mechanism; gel mobility shift assay; gene conversion; gene deletion mutation; gene rearrangement; genetic mapping; genetic recombination; high performance liquid chromatography; human tissue; nucleic acid sequence; protein purification; radiotracer; restriction mapping; transfection

Analysis of bacterial systems has demonstrated that strand-specific DNA mismatch correction protects the genome against mutation by removing biosynthetic errors from newly replicated DNA and by aborting recombination events between related, but non-allelic DNA sequences. We have recently reconstituted E. coli methyl-directed mismatch repair in a pure system comprised of 8 proteins, and have also demonstrated the existence of a similar pathway in nuclear extracts derived from human cells. Like the bacterial pathway, the system we have identified in human cells is able to recognize and to process in a strand-specific manner the different classes of base-base mispairs in a reaction that requires a replicative (aphidicolin sensitive) DNA polymerase. The primary goal of this proposal is to establish the molecular nature of strand-specific mismatch repair as it occurs in human cells. To this end, the reaction occurring in HeLa nuclear extracts will be characterized with respect to mismatch specificity, size and location of excision repair tracts, involvement of alpha and delta DNA polymerase, and possible involvement of the mammalian homologue of MutS, the protein that mediates mismatch recognition in bacterial systems. A major aim of this study is the isolation and characterization of the components required for the human reaction, with the hope that, as in the case of E. coli, we will be able to reconstruct the reaction in a defined system. Since it is not possible to monitor the course of a mismatch repair event occurring within a living cell, the nuclear extract studies outlined above will provide the criteria for evaluating successful reconstitution of the reaction in a purified system. As an initial attempt to address the relationship between mismatch repair proficiency and genetic stability in mammalian systems, we will also participate in a collaborative study that will compare, with respect to their proficiency in mismatch correction, a "wild type" CHO cell line and a mutant derivative that contains a deletion spanning the gene encoding the mammalian MutS homologue. Should a defect in mismatch repair be associated with the mammalian MutS deletion, the spontaneous mutabilities of wild type and mutant lines will be assessed.

对细菌系统的分析表明，链特异性 DNA 错配校正通过删除来保护基因组免受突变 新复制的 DNA 和重组中止导致的生物合成错误 相关但非等位基因 DNA 序列之间的事件。 我们最近有 在纯系统中重组大肠杆菌甲基定向错配修复 由8种蛋白质组成，并且还证明了存在 来自人类细胞的核提取物中存在类似的途径。 就像 细菌途径，我们在人体细胞中发现的系统能够 识别并以特定于链的方式处理不同的类别 需要复制的反应中的碱基错配 （阿菲迪霉素敏感）DNA 聚合酶。 本提案的主要目标 是为了建立链特异性错配修复的分子性质 它发生在人体细胞中。 为此，HeLa 中发生的反应 核提取物将根据错配进行表征 切除修复束的特异性、大小和位置、参与 α 和 δ DNA 聚合酶，以及哺乳动物的可能参与 MutS 的同源物，介导错配识别的蛋白质 细菌系统。 本研究的一个主要目的是隔离和 人类反应所需成分的表征， 希望我们能够像大肠杆菌一样重建 特定系统中的反应。 由于无法监控 活细胞内发生错配修复事件的过程， 上述核提取物研究将为 评估纯化系统中反应的成功重建。 作为解决错配修复之间关系的初步尝试 哺乳动物系统的熟练程度和遗传稳定性，我们还将 参加一项合作研究，该研究将比较 他们在错配校正方面的熟练程度，“野生型”CHO细胞系和 包含跨越编码基因的缺失的突变衍生物 哺乳动物 MutS 同源物。 是否与错配修复缺陷相关 随着哺乳动物 MutS 缺失，野生型的自发突变 和突变系将被评估。