RECOGNITION AND REPAIR OF PAH-DNA ADDUCTS

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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.