Mutagenic and Repair Mechanisms of Endogenous DNA Damage

该项目的长期目标是阐明突变、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。其具体目标是(A)建立人类细胞中跨损伤合成通过氧化损伤的dna的途径(S)，使用一种新的实验系统来量化这一过程的效率和保真度(B)在最近发现的无数dna聚合酶中识别那些专门参与复制和修复人类细胞跨损伤合成的酶以及(C)识别参与识别的氨基酸残基。 DNA损伤和底物特异性的测定。这项研究将集中在DNA内源性氧化损伤的主要形式，包括胸腺嘧啶二醇、甲酰胺并嘧啶、外环DNA加合物和8-氧鸟嘌呤。一种新的穿梭载体系统已经开发出来，它将使我们能够探索人类细胞中的跨损伤合成事件。这一数量 该系统衡量进行跨病变合成的损伤的效率、保真度和编码特性，并将用于确定受损DNA碱基的遗传毒性。核糖核酸干扰技术将被用来探索跨损伤合成专门化DNA聚合酶在人类细胞中的作用和功能(S)。通过x射线结晶学获得的dna糖基酶的结构信息，将与从生物信息学和分子模拟方法中获得的见解相结合，以探索dna的作用机制。 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的分子生物学以及DNA糖基酶的几个功能提供了重要的见解，跨损伤合成是DNA聚合酶错误编码的中心事件，DNA糖基酶是启动DNA修复的酶。因此，这项研究形成了项目2、3和4的生物焦点。

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.