ENZYMOLOGY OF EUKARYOTIC DNA MISMATCH REPAIR

对细菌系统的分析表明，链特异性DNA 错配校正通过去除基因组中的 来自新复制的DNA和中止重组的生物合成错误 相关但不等位的DNA序列之间的事件。我们最近做了 重组大肠杆菌在纯体系中的甲基定向错配修复 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也有类似的途径。就像 细菌途径，我们在人类细胞中识别的系统能够 识别并以特定于链的方式处理不同的类别 在需要复制物的反应中的碱基错配 (敏感型)DNA聚合酶。这项提案的主要目标是 是确定链特异性错配修复的分子本质为 它发生在人类细胞中。为此，HeLa中发生的反应 核抽提物将根据错配进行表征 切除修复束的特异性、大小和位置，是否涉及 α和德尔塔DNA聚合酶，以及哺乳动物可能的参与 MutS的同源物，MutS是介导错配识别的蛋白质 细菌系统。这项研究的一个主要目的是隔离和 人体反应所需成分的表征， 希望，就像在大肠杆菌的情况下，我们将能够重建 在一个确定的系统中的反应。由于不可能监视 在活细胞内发生错配修复事件的过程中， 上面概述的核提取研究将提供以下标准 评价反应在纯化系统中的成功重组。 作为解决失配修复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 参与一项协作研究，该研究将比较以下方面 他们在错配纠正方面的熟练程度，一个“野生型”的CHO细胞系和一个 含有跨越编码基因的缺失的突变衍生品 哺乳动物MutS同源基因。错配修复中的缺陷是否应关联 随着哺乳动物MutS的缺失，野生型的自发突变 并对突变品系进行评估。

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.