Mutagenic and Repair Mechanisms of Endogenous DNA Damage

内源性DNA损伤的诱变与修复机制

• 批准号: 6990324
• 负责人: Arthur Patrick Grollman
• 金额: $ 15.13万
• 依托单位: STATE UNIVERSITY NEW YORK STONY BROOK
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-06-21 至 2009-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6990324
• 关键词: DNA damage; DNA directed DNA polymerase; DNA repair; N glycosidase; RNA interference; X ray crystallography; adduct; aminoacid; bioinformatics; cell line; computer simulation; enzyme substrate; gene mutation; gene targeting; molecular dynamics; neoplasm /cancer genetics; nucleic acid biosynthesis; nucleic acid structure; oxidative stress; site directed mutagenesis

The long term goal of this project is to elucidate molecular mechanisms of mutagenesis, translesion DNA synthesis (TLS) and base excision repair, correlating these biological findings with the three-dimensional structure and thermodynamic properties of oxidatively damaged DNA. The specific aims are (a) to establish the pathway(s) of translesion synthesis past oxidatively damaged DNA in human cells, using a novel experimental system to quantify the efficiency and fidelity of this process (b) to identify, among the myriad of recently discovered DNA polymerases, those enzymes specifically engaged in replicative and repair translesion syntheses in human cells and (c) to identify amino acid residues that participate in recognition of DNA damage and in determining substrate specificity. This research will focus on major forms of oxidative damage found endogenously in DNA, including thymine glycol, formamidopyrimidines, exocyclic DNA adducts and 8-oxoguanine. A novel shuttle vector system has been developed that will allow us to explore translesion synthesis events in human cells. This quantitative system measures the efficiency, fidelity and coding properties of lesions undergoing translesion synthesis and will be used to establish the genotoxicity of damaged DNA bases. RNA interference technology will be used to explore the role and function(s) of translesion synthesis-specialized DNA polymerases in human cells. Structural information on DNA glycosylases, obtained by x-ray crystallography, will be combined with insights gained from bioinformatics and molecular modeling methods to explore mechanisms of DNA damage recognition during base excision repair. These studies provide significant insights into the molecular biology of translesion synthesis, the central event in miscoding by DNA polymerases, and into several functions of DNA glycosylases, enzymes that initiate DNA repair. As such, this research forms the biologic focus for projects 2, 3 and 4.

该项目的长期目标是阐明诱变、跨损伤DNA合成（TLS）和碱基切除修复的分子机制，将这些生物学发现与氧化损伤DNA的三维结构和热力学性质相关联。具体目的是（a）建立人细胞中氧化损伤DNA的跨损伤合成途径，使用新的实验系统来量化该过程的效率和保真度（B），以在最近发现的无数DNA聚合酶中鉴定，这些酶特异性参与人细胞中的复制和修复跨损伤合成，以及（c）以鉴定参与识别的氨基酸残基， DNA损伤和确定底物特异性。这项研究将集中在主要形式的氧化损伤发现内源性DNA，包括胸腺嘧啶乙二醇，甲酰氨基嘧啶，环外DNA加合物和8-氧代鸟嘌呤。一种新的穿梭载体系统已经开发出来，这将使我们能够探索在人类细胞中的translesion合成事件。这种定量 该系统测量了进行跨损伤合成的损伤的效率、保真度和编码特性，并将用于确定受损DNA碱基的遗传毒性。RNA干扰技术将用于探索跨损伤合成特异性DNA聚合酶在人类细胞中的作用和功能。通过X射线晶体学获得的DNA糖基化酶的结构信息将与生物信息学和分子建模方法的见解相结合，以探索DNA糖基化酶的机制。 在碱基切除修复过程中的损伤识别。这些研究为跨损伤合成（DNA聚合酶错误编码的核心事件）的分子生物学以及DNA糖基化酶（启动DNA修复的酶）的多种功能提供了重要见解。因此，这项研究形成了项目2、3和4的生物学重点。