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.

细菌系统的分析表明，链特异性DNA 不匹配校正可以通过去除来保护基因组免受突变 来自新复制的DNA并流产重组的生物合成错误 相关但非平行性DNA序列之间的事件。 我们最近有 重构的大肠杆菌甲基指导的不匹配修复在纯系统中 由8种蛋白质组成，也证明了 来自人类细胞的核提取物中的类似途径。 喜欢 细菌途径，我们在人类细胞中发现的系统能够 以特定方式识别和处理不同类别 在需要复制的反应中的基碱碱不含量 （蚜虫敏感）DNA聚合酶。 该提议的主要目标 是确定链特异性不匹配修复的分子性质 它发生在人类细胞中。 为此，反应发生在Hela中 核提取物将相对于不匹配而表征 切除修复区的特异性，大小和位置，参与 α和三角洲DNA聚合酶，以及哺乳动物的可能参与 MUT的同源物，介导的蛋白质的蛋白质介导 细菌系统。 这项研究的主要目的是孤立和 人类反应所需的成分的表征， 希望与大肠杆菌一样，我们将能够重建 定义系统中的反应。 由于无法监视 在活细胞内发生的不匹配维修事件的过程， 上面概述的核提取研究将为 评估纯化系统中反应的成功重建。 作为解决不匹配维修之间关系的初步尝试 在哺乳动物系统中的熟练程度和遗传稳定性，我们还将 参加一项合作研究，该研究将与 他们熟练校正的熟练程度，“野生型” CHO细胞系和A 突变衍生物包含跨基因编码的缺失 哺乳动物Muts同源物。 不匹配维修缺陷是否相关 随着哺乳动物的删除，野生型的自发企业 将评估突变线。