ENZYMOLOGY OF EUKARYOTIC DNA MISMATCH REPAIR

人类细胞拥有一种链特异性错配修复系统，这是一种 判断细菌甲基导向途径的同源性 在特异性和机制上具有相似性。像细菌系统一样， 人类链特异性修复有助于遗传稳定，因为 几种类型的高度可变的人类细胞在反应中是不足的。 这样的突变体细胞包括某些对 DNA烷基化试剂的细胞毒性作用，定义为第二类 由遗传不稳定的RER+(易于复制错误)的肿瘤细胞 发生在遗传性非息肉病结肠的个体身上 癌症(HNPCC)。在本应用程序中，我们建议沿着两个主要方向进行工作 第一个将扩展我们对诱变子细胞系的研究 就像上面提到的那些。与博士的实验室合作。 Bert Vogelstein，我们将扩大对RER+肿瘤细胞系的分析， 这项研究的目的是评估 错配缺陷与RER+表型和TO的关系 基于体外互补的修复缺陷品系分类。这个 突变细胞工作的其他方面将涉及对RER+肿瘤的测试 耐受烷基化剂杀戮的线，并在 与Henry Friedman博士合作进行错配修复测试 已产生耐受性的临床来源的肿瘤细胞的缺陷 化疗性DNA甲基化药物。第二条主线是 这项工作将与诱变细胞工作并行进行， 将涉及人类修复系统的分裂，最终 目标是在纯化系统中重建反应，以及 阐明其分子机制。使用生化方法和 RER+核抽提物和烷基化产物的互补性 耐受突变细胞，我们已经鉴定出六个(可能是七个)不同的 反应所需的活动。我们打算寻求隔离 这六个组成部分和任何其他可能需要的活动 在研究过程中确定的，最初侧重于 突变细胞系所缺乏的成分。我们这项工作的目的是 将获得每种活性的近乎均一的制剂。在……里面 除了它们在解决机制问题方面的效用外， 提纯成分的可用性应有助于识别 与HNPCC相关的新的遗传基因座。此外，建议的 机械分析可能产生的信息将被证明在 这种遗传性疾病的生化诊断技术的发展。

Human cells possess a strand-specific mismatch repair system that is a homolog of the bacterial methyl-directed pathway as judged by similarities in specificity and mechanism. Like the bacterial system, human strand-specific repair contributes to genetic stability since several type of hypermutable human cell are deficient in the reaction. Such mutator cells include certain mutants that are tolerant to the cytotoxic action of DNA alkylating agents, with a second class defined by genetically unstable, RER+ (replication error prone) tumor cells such as those that occur in individuals with heritable nonpolyposis colon cancer (HNPCC). In this application we propose work along two major lines, the first of which will extend our study of mutator cell lines like those mentioned above. In collaboration with the laboratory of Dr. Bert Vogelstein, we will expand our analysis of RER+ tumor cell lines, with the objectives of this study being to assess generality of the association of mismatch deficiency with the RER+ phenotype and to classify repair-defective lines based on in vitro complementation. The other facet of the mutator cell work will involve test of RER+ tumor lines for tolerance to killing by alkylating agents, and in a collaborative study with Dr. Henry Friedman, test for mismatch repair defects in clinically derived tumor cells that have developed tolerance to chemotherapeutic DNA methylating agents. The second major line of work, which will be conducted in parallel with the mutator cell work, will involve fractionation of the human repair system, with the ultimate goals being reconstitution of the reaction in a purified system and elucidation of its molecular mechanism. Using biochemical methods and complementation of nuclear extracts derived from RER+ and alkylation- tolerant mutant cells, we have identified six (possibly seven) distinct activities required for the reaction. We intend to pursue isolation of these six components and any other required activities that may be identified during the course of the study, focusing initially on those components that are lacking in mutant cell lines. Our aim in this work will be to obtain near homogeneous preparations of each activity. In addition to their utility in addressing questions of mechanism, availability of purified components should facilitate identification of novel genetic loci associated with HNPCC. Moreover, the proposed mechanistic analysis may yield information that will prove useful in the development of biochemical diagnostics for this inherited disease.