The first step in the DNA base excision repair pathway is the hydrolytic cleavage of the glycosidic bond of a damaged or mismatched base by a damage-specific glycosylase. The long-term goal of this research is to obtain a fundamental understanding of how these enzymes specifically recognize and excise damaged bases in a sea of normal base pairs. DNA glycosylases are of health related interest because of their role in policing the genome for premutagnenic lesions, and have been implicated in modulating the efficacy of several widely used chemotherapeutic agents that modify DNA, such as 5-fluorouracil and various alkylating agents. The specific aims of this proposal are to (i) Identify and quantify the forces exerted on the DNA substrate that lead to extrahelical flipping of the damaged base into the enzyme active site. Uracil DNA glycosylase (UDG) will be used as a paradigm system to evaluate the role of induced helix strain and the "pinch-push-pull" model for base flipping using NMR and fluorescence spectroscopy, rapid kinetic measurements, enzyme mutagenesis and engineered DNA analogs. (ii) Test the role of ground state destabilization in the UDG reaction. The recent crystal structure of UDG bound to a C-glycoside substrate analog indicates that the enzyme bends the glycosidic bond by approximately 41 degree possibly to lower the activation barrier. The principal investigator will investigate alternative explanations for this intriguing mechanism using NMR, mutagenesis and Raman spectroscopy approaches. (iii) Determine the features of the active site environment of UDG that allow the formation of a remarkable oxacarbenium ion-uracil anion intermediate by measuring kinetic isotope effects with active site mutants and modified substrates expected to destabilize this intermediate. (iv) Discover the mechanism for recognition and removal of cationic purine bases from DNA by solving the NMR solution structure of 3-methyladenine DNA glycosylase I (TAG) bound to 3-MeA in combination with complementary biophysical studies. Together, these studies will fill significant gaps in our understanding of DNA repair mechanisms, and in addition, will serve to address several fundamental issues concerning the nature of enzymic catalysis. It is anticipated that the detailed knowledge of the chemistry and energetics of these reactions will contribute to the development of novel small molecules to modulate these enzyme activities in vivo. Such inhibitors or activators may find use as anti-viral or anti-cancer agents.