RECOGNITION AND REPAIR OF UV DAMAGE TO HUMAN DNA

患有色素性干皮病的遗传病患者 (XP) 缺乏 DNA 的核苷酸切除修复 (NER)，因此有很多 接触后致癌概率高于普通人群 到紫外线照射。 NER 是一种主要的细胞生物防御系统，可消除 由于紫外线照射和诱导形成大块损伤而导致 DNA 损伤 环境遗传毒性化学物质和致癌物质。虽然，已经 人们普遍认为DNA损伤识别在NER中起着核心作用， DNA损伤处理的分子和热力学细节在很大程度上仍然存在 不清楚，并且尚未使用严格的生化方法进行系统研究 接近。此外，损伤识别蛋白 XPA、RPA、 和XPC-HR23B一直存在争议。本研究的长期目标是 了解紫外线诱导或相关的分子和生化细节 人类 NER 蛋白对 DNA 损伤的识别和修复及其潜力 这些关系对损伤诱导突变的影响 致癌作用。获得 DNA 损伤的系统且更精确的视图 通过人类 NER 的识别和修复，将解决以下问题： 损伤识别的层次是什么以及损伤是如何动态变化的 以逐步识别机制进行处理？什么结构和化学性质 DNA 螺旋的改变由特定修复蛋白识别 识别步骤？识别的分子结构是什么 中间体？哪些蛋白质结构域对于蛋白质-DNA 和 识别中的蛋白质-蛋白质接触。具体来说，该项目旨在 确定 XPC-HR23B、XPA 相互作用的热力学和动力学， 和含有特定位点紫外光损伤的 DNA 底物的 RPA 和 使用严格的生化方法制备苯并[a]芘二醇环氧化物 (BPDE) DNA 加合物 方法：识别和分析参与的重要蛋白质基序 损伤识别：表征修复中间体的损伤特征 识别紫外线诱导的光损伤和 BPDE-DNA 加合物；并确定 损伤的结构和化学修饰的机制 已识别并修复。

Individuals who suffer from the genetic disease xeroderma pigmentosum (XP) lack nucleotide excision repair (NER) of DNA, and thus have much higher carcinogenic probability than the average population after exposure to UV irradiation. NER is a major cellular biological defense system to remove DNA damage due to the formation of bulky lesions induced by UV irradiation and environmental genotoxic chemicals and carcinogens. Although, it has been generally accepted that DNA damage recognition plays a central role in NER, the molecular and thermodynamic details of DNA damage processing remain largely unclear, and have not been systematically studied using biochemically rigorous approaches. In addition, the roles of the damage recognition proteins XPA, RPA, and XPC-HR23B have been controversial. The long-term objective of this study is to understand the molecular and biochemical details of UV-induced or related DNA damage recognition and repair by human NER proteins, and the potential effects of these relationships on damage-induced mutagenesis and carcinogenesis. To gain a systematic and more precise view of DNA damage recognition and repair by human NER, the following questions will be addressed: What is the hierarchy of damage recognition and how is damage dynamically processed in a stepwise recognition mechanism? What structural and chemical alterations in the DNA helix are identified by repair proteins at specific recognition steps? What is the molecular architecture of recognition intermediates? And what protein domains are important for protein-DNA and protein-protein contacts in the recognition. Specifically, this project aims to determine the thermodynamics and kinetics of the interaction of XPC-HR23B, XPA, and RPA with DNA substrates containing site-specific UV photolesions and benzo[a]pyrene diol epoxide (BPDE) DNA adducts using rigorous biochemical approaches: identify and analyze the important protein motifs involved in damage recognition: characterize the repair intermediates for damage recognition of UV-induced photolesions and BPDE-DNA adducts; and determine the mechanism in which the structural and chemical modifications of damage are recognized and repaired.