The base excision repair (BER) pathway is responsible for the removal of most forms of endogenous DNA base damage and many types of base alterations due to replication errors or from exogenous sources. Within the last decade, significant advances have been made in understanding the chemical mechanisms by which the enzymes that initiate BER, the DNA glycosylases and glycosylase/AP lyases, catalyze the release of inappropriate bases. Additionally, the X-ray crystal structures of several DNA glycosylases have been elucidated and in some cases, catalytically-compromised mutants have been co-crystallized with substrate DNAs. Unexpectedly, these co-crystal complexes have revealed that the mechanism of specific base recognition involves an additional step of base (nucleotide) flipping. As a collaborative investigation between the Lloyd and Tainer laboratories, structure/function studies on MutY, an adenine- specific DNA glycosylase have been initiated. MutY is composed of a N-terminal 26kDa domain which retains catalytic activity and a C-terminal l3kDa domain. This enzyme catalyzes the release of adenine from DNA containing mismatches A 8-oxoG, A G and A C. In order to gain insight into the mechanism of specific base recognition and to resolve a long-standing controversy on the catalytic mechanism of MutY, the structure of the catalytically competent domain of MutY and several mutant proteins have been solved at 1.1Angstrom units resolution in the presence of several inhibitor molecules. In this application, the exquisite details of these structures and the relative positions of their catalytic inhibitors have led to hypotheses for: 1) the specific amino acid side chains lining a groove on the surface of the enzyme that binds (and bends) DNA into the active site; 2) specific residues and mechanisms by which both specific base recognition is achieved within the active site and the adenine is flipped to an extrahelical position within a 20Angstrom units deep binding pocket; and 3) residues that are responsible for the separate and distinct chemical reactions that catalyze glycosyl and phosphodiester bond scission. Additional studies are proposed to solve the structure and ascertain the biological significance of the additional 13kDa C-terminal domain of MutY.
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