ENZYMOLOGY OF EUKARYOTIC DNA MISMATCH REPAIR

人类细胞具有链特异性错配修复系统，该系统是细菌和酵母途径的同源，根据MutS和MutL样活性在特异性、机制和参与方面的相似性来判断。正如在微生物系统中一样，人类错配修复突变赋予了高度变异性。人类修复系统中的基因缺陷是遗传性非息肉病性结肠癌的原因，与各种组织中出现的一组散发性肿瘤有关，也使人对某些化疗药物产生耐药性，包括DNA甲基化和顺铂。因此，阐明人类错配修复反应的分子性质对于人类恶性肿瘤的起源和治疗都是相关的。我们和其他人已经确定了六种参与人类修复反应的活性：错配识别活动MutS-α(MSH2.MSH6异二聚体)和MutS-beta(MSH2.MSH3异二聚体)，MutL-Alpha(MLH1.PMS2异二聚体)，增殖细胞核抗原，RPA和DNA聚合酶增量。我们要求延长这项工作的主要目标之一将是鉴定和分离修复系统的其他组件，重点是参与反应切除步骤的解旋酶和/或核酸外切酶活性。这项分离工作还将包括分离和鉴定几种新的MutL样活性，这些活性已从HeLa细胞和某些MLH1缺陷的肿瘤和耐药细胞系的提取物中鉴定出来。这项工作的第二个目标将是进一步阐明错配修复反应中所需活性的分子作用。初步研究将强调起始复合体的组装，包括蛋白质-蛋白质相互作用，以及ATP在起始复合体形成和进化中的作用。如果切除活动在项目过程中可用，分子研究将扩展到包括这些活动。

Human cells possess a strand-specific mismatch repair system that is a homolog of the bacterial and yeast pathways as judged by similarities in specificity, mechanism, and involvement of MutS and MutL-like activities. As in microbial systems, human mismatch repair mutations confer hypermutability. Genetic defects in the human repair system are the cause of hereditary nonpolyposis colon cancer, have been implicated in a subset of sporadic tumors that arise in a variety of tissues, and also confer resistance to certain chemotherapeutic agents, including DNA methylators and cisplatin. Clarification of the molecular nature of the human mismatch repair reaction is therefore relevant to both the origin and treatment of human malignancies. We and others have identified six activities that are involved in the human repair reaction: the mismatch recognition activities MutS-alpha (MSH2.MSH6 heterodimer) and MutS-beta (MSH2.MSH3 heterodimer), MutL-alpha (MLH1.PMS2 heterodimer), PCNA, RPA, and DNA polymerase delta. One of our major goals for the requested extension of this work will be the identification and isolation of other components of the repair system, with emphasis on helicase and/or exonuclease activities involved in the excision step of the reaction. This fractionation work will also include the isolation and characterization of several novel MutL-like activities that have been identified in extracts derived from HeLa cells and certain MLH1- deficient tumor and drug-resistant cell lines. The second aim of the work will be to further clarify the molecular roles of the required activities in the mismatch repair reaction. Initial studies will emphasize the assembly of the initiation complex, including protein- protein interactions, and the role of ATP in initiation complex formation and evolution. Should excision activities become available during the course of the project, the molecular studies will be extended to include these activities.