ENZYMOLOGY OF EUKARYOTIC DNA MISMATCH REPAIR

真核 DNA 错配修复的酶学

• 批准号: 3304557
• 负责人: PAUL LAWRENCE MODRICH
• 金额: $ 21.62万
• 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/3304557
• 关键词: 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; gene conversion; gene deletion mutation; gene rearrangement; genetic mapping; genetic recombination; high performance liquid chromatography; human tissue; 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， 错配校正通过去除 来自新复制DNA的生物合成错误和中止重组 相关但非等位基因DNA序列之间的事件。 我们最近 重组E.纯系统中大肠杆菌甲基指导的错配修复 由8种蛋白质组成，并且还证明了 在来自人类细胞的核提取物中存在类似的途径。 像 细菌途径，我们在人类细胞中发现的系统能够 以特定于链的方式识别和处理不同的类 碱基错配的反应需要一个复制的 （aphidicolin敏感的）DNA聚合酶。 该提案的主要目标是 是建立链特异性错配修复的分子本质， 它发生在人体细胞中。 为此，HeLa细胞中发生的反应 核提取物的特征在于不匹配 切除修复道的特异性、大小和位置， α和δ DNA聚合酶，以及哺乳动物的可能参与 MutS的同源物，MutS是介导细胞内错配识别的蛋白质， 细菌系统 本研究的一个主要目的是分离和 人体反应所需组分的表征， 希望像E.我们就能重建 在一个特定的系统中的反应。 由于不可能监视 在活细胞内发生的错配修复事件的过程中， 上述核提取物研究将提供以下标准： 评价反应在纯化系统中的成功重构。 作为解决错配修复与 在哺乳动物系统中的熟练程度和遗传稳定性，我们还将 参与一项合作研究， 他们在错配校正方面的熟练程度，“野生型”CHO细胞系和 突变体衍生物，其含有跨越编码所述突变体衍生物的基因的缺失。 哺乳动物MutS同源物。 错配修复中的缺陷是否与 随着哺乳动物MutS缺失，野生型的自发变异 并评估突变株系。