Our genetic integrity is assured by the precise repair of DNA damage caused by chemical mutagens, ionizing radiation, and the spontaneous hydrolytic decay of DNA bases. Defects in DNA repair have been linked to a growing repair of inherited diseases of humans. The efficient repair of DNA damage in neoplastic cells poses an obstacle to the effective chemotherapy of cancer with alkylating agents. We are studying the structures of several DNA base excision repair proteins by x-ray crystallographic methods in order to understand how these enzymes located damaged bases in DNA and cleave the N-glycosylic bond, releasing the damaged base from DNA. Several DNA N-glycosylases from E. coli and humans that recognize alkylation-damaged DNA have been crystallized in complexes with specific DNA substrates and inhibitors. Crystal structures of these complexes are being determined by multiple isomorphous replacement and multiple wavelength anomalous diffraction experiments. These repair enzymes have different specificities for the types of alkylated bases that they excise from DNA. Broadly specific enzymes like the E. coli AlkA protein efficiently remove many types of alkylated purines with little regard for the shapes of positions of adducts on the purine base. We previously determined a 1.8 A crystal structure of unliganded AlkA and discovered an active site pocket containing many aromatic residues. This electron-rich environment may serve as a binding site for electron- deficient, alkylated bases that are """"""""flipped out"""""""" of the DNA helix prior to cleavage of the N-glycosyl bond. This hypothesis will be addressed by crystal structures of AlkA complexed to an inhibitory DNA containing a modified a basic site and of an inactive AlkA mutant complexed to a alkylated purine in DNA. The human alkyladenine DNA glycosylase (AAG) is a functional analog of AlkA that bears no sequence resemblance to AlkA. The crystal structure of an AAG-DNA complex is being determined. Conserved features of the active sites of AAG and AlkA are likely to reflect a common strategy for locating damages bases, exposing them to the enzyme active site, and catalyzing the scission of the N-glycosyl bond. In contrast to AlkA and AAG, the E. coli Tag glycosylase is highly selective for the removal of 3-methyladenine from DNA. Crystallographic studies of Tag will address the structural basis of Tag's strict substrate preference and perhaps reveal a different catalytic strategy. Functional studies of AlkA, AAG, and Tag glycosylases are being performed to identify residues with important roles in DNA binding, base-flipping, and enzymatic catalysis.
Showing the most recent 10 out of 19 publications
