RECOGNITION AND REPAIR OF PAH-DNA ADDUCTS

描述：核苷酸切除修复(NER)蛋白作用于识别和 去除多环芳烃-DNA加合物。假设多环芳烃-DNA加合物产生 DNA螺旋中的特定构象变化允许通过 DNA修复酶。这项研究的一个主要目标是系统地 DNA修复蛋白与DNA底物相互作用的表征 含有位点特异性和立体特异性的多环芳烃-DNA加合物。具体的 本项目的目的是研究大肠杆菌的相互作用。 UvrABC系统和人类损伤识别蛋白XPA与DNA 特定DNA中含有几类多环芳烃-DNA加合物的底物 序列上下文。这些多环芳烃-DNA加合物包括：1) 苯并[a]芘二醇环氧-N_2-鸟嘌呤和N_6-腺嘌呤，2) 5-甲基黄烯二醇环氧-N-鸟嘌呤，以及3) 苯并[c]菲二醇环氧-N6-腺嘌呤和N-鸟嘌呤。几个 将使用生化和分子生物学方法来表征 这些DNA修复蛋白如何与多环芳烃损伤的DNA相互作用。域映射 将用来定义对损伤很重要的蛋白质模体 承认。修复蛋白结合的热力学和动力学 不同的位置特定的立体特定的PAH-DNA加合物将是 通过几种互补的方法进行研究(凝胶迁移率变化分析， 和荧光光谱学)。修复酶伤口的动力学研究 每组立体异构体PAH-DNA加合物也将被检测。《长河》 本研究的术语目标是定义特定的结构规则，以允许 通过DNA修复酶高效识别和移除损伤。

DESCRIPTION: Nucleotide excision repair (NER) proteins act to recognize and remove PAH-DNA adducts. It is hypothesized that PAH-DNA adducts produce specific conformational changes in the DNA helix which allow recognition by DNA repair enzymes. A major goal of this study is to systematically characterize the interaction of DNA repair proteins with DNA substrates containing site-specific and stereospecific PAH-DNA adducts. The specific objectives of this project are to study the interaction of the E. coli UvrABC system and the human damage recognition protein, XPA, with DNA substrates containing several classes of PAH-DNA adducts in defined DNA sequence contexts. These PAH-DNA adducts include: 1) stereoisomers of benzo[a]pyrene diol epoxide-N2-guanine and N6-adenine, 2) stereoisomers of 5-methylchrysene diol epoxide-N2-guanine, and 3) stereoisomers of benzo[c]phenanthrene diol epoxide-N6-adenine and N2-guanine. Several biochemical and molecular biology approaches will be used to characterize how these DNA repair proteins interact with PAH damaged DNA. Domain mapping will be used to define the protein motifs which are important for damage recognition. The thermodynamics and kinetics of repair protein binding to the different site-specific stereo-specific PAH-DNA adducts will be investigated by several complementary approaches (gel mobility shift assays, and fluorescence spectroscopy). The kinetics of repair enzyme incision for each set of stereoisomer PAH-DNA adducts will also be examined. The long term goal of this study is to define specific structural rules which allow efficient damage recognition and removal by DNA repair enzymes.