The dark repair of damaged DNA can involve a based excision pathway in which glycosylases specific for damaged bases hydrolyze the N-glycosidic bond between the base and the sugar and endonucleases remove the resulting deoxyribose-5-phosphate moiety. Particular attention will be given to the mechanisms of the reactions catalyzed by glycosylases which remove damaged pyrimidine bases, with particular emphasis being placed on the reaction catalyzed by uracil-DNA glycosylase. Model studies will be performed on various deoxynucleosides to determine whether saturation of the 5,6-double bond of the pyrimidine (by nucleophilic addition in the case of uracil) allows an acid-catalyzed mechanisms in which an imonium ion is formed between the base and sugar and the sugar ring is opened; these studies will rely on kinetic isotope effects, elemental substitution in the sugar ring, and 17O NMR spectroscopy. Once the model systems are understood, the mechanisms of the reaction catalyzed by uracil-DNA glycosylase will be examined. The mechanisms of strand cleavage at aldehydic abasic sites catalyzed by both class I and class II AP endonucleases will also be studied. Exonuclease III and endonuclease IV hydrolytically cleave the 5'- phosphodiester bond involving an aldehydic abasic site, and stereochemical studies will be performed to establish mechanistic relationships between these enzymes. Endonucleases III and UV endonuclease V catalyze both a damaged base-DNA glycosylase reaction and cleavage of the 3'-phosphodiester bond involving an aldehydic abasic site by a beta-elimination reaction. Studies of the chemical reactivity of aldehydic abasic sites will be performed so that the beta-elimination reactions catalyzed by these enzymes can be better understood. The relationships between the enzymatic activities and mechanistic details of both activities will be studied by measuring kinetic isotope effects and studying the processing of substrate analogs.
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