) This application deals with the structural biology of DNA 0-alkylation damage. As part of the applicant's long-term objectives of understanding the molecular recognition of damaged DNA, he proposes to determine the solution structure of DNA duplexes containing alkyl lesions and base analogs currently used in chemotherapy. He will establish the three-dimensional structure of the repair protein: 06-methylguanine methyltransferase (Ogt), free and after reaction with enzyme inhibitors, and of the damaged DNA/protein complex. Alkyl transferase activity has been correlated with increased sensitivity to mutations induced by methylating agents. Tumor cell lines expressing high levels of alkyl transferase activity are resistant to the action of therapeutic methylating and chloroethylating agents. In addition, alkyl transferase inhibitors used in combination with methylating chemotherapeutic agents overcome the resistance of some tumor cell lines to the drugs. The information resulting from this project will increase our knowledge of chemical mutagenesis and DNA repair and facilitate the rational design of new alkyl transferase inhibitors, which can be used in cancer therapy. Using phosphoramidite chemistry, he will synthesize large quantities of oligodeoxynucleotide duplexes containing the base analog S6-thio-2'-deoxyguanosine and S6-methyl-6-thio-2'-deoxyguanosine paired opposite to deoxycytidine and thymidine. Their three-dimensional structure will be established by NMR spectroscopy and computational methods. He will use recombinant DNA technology to obtain large quantities of highly pure Ogt protein in natural abundance as well as enriched with 15N and 13C. His solution structure will be determined by using multinuclear -- multidimensional NMR spectroscopy and computational methods. Similar methods will be used to determine the structure of protein after reaction with the enzyme inhibitors 06-methylguanine and 06-benzylguanine.
