RECOGNITION AND REPAIR OF PAH-DNA ADDUCTS

描述：核苷酸切除修复（NER）蛋白的作用是识别和 去除PAH-DNA加合物。 假设PAH-DNA加合物产生 DNA螺旋中的特异性构象变化，其允许被 DNA修复酶 本研究的一个主要目标是系统地 表征DNA修复蛋白与DNA底物的相互作用 含有位点特异性和立体特异性PAH-DNA加合物。 具体 本项目的目的是研究E.杆菌 UvrABC系统和人类损伤识别蛋白XPA与DNA 在确定的DNA中含有几类PAH-DNA加合物的底物 序列上下文。 这些PAH-DNA加合物包括： 苯并[a]芘二醇环氧化物-N2-鸟嘌呤和N6-腺嘌呤， 5-甲基二醇环氧化物-N2-鸟嘌呤，和3） 苯并[c]菲二醇环氧化物-N6-腺嘌呤和N2-鸟嘌呤。 几 生物化学和分子生物学方法将用于表征 这些DNA修复蛋白如何与PAH损伤的DNA相互作用。 域映射 将被用来定义蛋白质基序是重要的损害 识别. 修复蛋白结合的热力学和动力学 不同的位点特异性立体特异性PAH-DNA加合物将 通过几种互补方法（凝胶迁移率变动测定， 和荧光光谱）。 修复酶切口的动力学 还将检查每组立体异构体PAH-DNA加合物。 长 本研究的长期目标是定义特定的结构规则， 通过DNA修复酶有效识别和去除损伤。

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.