ENZYMOLOGY OF EUKARYOTIC DNA MISMATCH REPAIR

真核 DNA 错配修复的酶学

• 批准号: 2634687
• 负责人: PAUL LAWRENCE MODRICH
• 金额: $ 22.87万
• 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 1998-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2634687
• 关键词: DNA binding protein; DNA directed DNA polymerase; DNA repair; HeLa cells; alkylating agents; colorectal neoplasms; drug tolerance; enzyme activity; enzyme mechanism; enzyme reconstitution; gene mutation; genetic disorder; helicase; high performance liquid chromatography; human tissue; laboratory rabbit; neoplasm /cancer genetics; neoplastic cell; pancreas neoplasms; phenotype; phosphodiesterase I; phosphoester ligase; protein purification; spleen exonuclease; tissue /cell culture

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.

人类细胞具有链特异性错配修复系统， 细菌甲基导向途径的同源物， 特异性和机制的相似性。就像细菌系统一样， 人类链特异性修复有助于遗传稳定性， 几种类型的超变人类细胞在该反应中是缺陷的。 这样的增变细胞包括某些对所述突变体耐受的突变体。 DNA烷化剂的细胞毒性作用，定义了第二类 通过遗传不稳定的RER+（复制错误倾向）肿瘤细胞， 与遗传性非息肉病性结肠炎患者相同 癌症（HNPCC）。在本申请中，我们提出沿着沿着两个主要方向工作 线，其中第一个将扩展我们的研究增变细胞系 就像上面提到的。与博士的实验室合作。 Bert Vogelstein，我们将扩大我们对RER+肿瘤细胞系的分析， 本研究的目的是评估 错配缺陷与RER+表型的相关性， 基于体外互补对修复缺陷系进行分类。的 突变细胞工作的其他方面将涉及RER+肿瘤的测试 对烷化剂杀灭的耐受性线， 与亨利弗里德曼博士的合作研究，错配修复试验 临床来源的肿瘤细胞的缺陷， 到化疗DNA甲基化剂。第二条主线 工作，这将与增变细胞工作平行进行， 将涉及人类修复系统的分离，最终 目标是在纯化的系统中重建反应， 阐明其分子机制。使用生物化学方法， 来自RER+和烷基化-的核提取物的互补 耐受突变细胞，我们已经确定了六个（可能是七个）不同的 反应所需的活动。 我们打算孤立 这六个组成部分和任何其他必要的活动， 在研究过程中确定的，最初侧重于那些 突变细胞系中缺乏的成分。我们在这项工作中的目标 将获得每种活性的近似均匀的制剂。在 除了在解决机制问题方面的作用外， 纯化组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，拟议的 机械分析可能产生的信息，将证明是有用的， 开发这种遗传性疾病的生化诊断方法。