Essential in both procaryotes and eucaryotes, DNA repair metalloenzymes act to correct mutagenic DNA damage otherwise responsible for cancer, aging, and cell death. To understand the molecular basis for the unique structural chemistry required for the sequence independent surveillance and repair of mutagenic DNA damage, we are successfully pursuing integrated structural and biochemical studies for three major classes of important DNA repair enzymes. Herein, we propose to continue research to comprehensively characterize the structure and function of: 1). apurinic/apyrimidinic (AP) lyases, which cleave 3' to AP sites, have associated N-glycosylase activities, and function by a beta-elimination mechanism (endonuclease III), 2) AP endonucleases, which cleave 5' to AP sites, require a metal ion, and function by a hydrolytic mechanism (exonuclease III and endonuclease IV), and 3) DNA glycosylases, which remove damaged bases from DNA by N-glycosyl bond cleavage creating an abasic site (uracil DNA-glycosylase). Design and construction of DNA substrate analogues, footprinting and chemical protection assays to characterize enzyme-DNA interactions, together with the expression, purification, biochemical, and kinetic characterization of wild-type and mutant enzymes by the Cunningham laboratory (SUNY) will be closely coupled to ongoing crystallographic and computational studies on wild-type, mutant, and DNA-complexed enzymes in the Tainer laboratory (Scripps). We propose a pragmatic, recursive, and integrated approach to understanding the molecular mechanisms of DNA excision-repair enzymes, such that the results from biochemistry, molecular biology, and x-ray crystallography serve to test current understanding and guide future experiments. Structural and biochemical studies include comparative characterizations of human and E. coli AP endonucleases and uracil DNA-glycosylases and of E. coli endonuclease III with a related archaebacterial thermophilic enzyme. Based upon parallel structure-function studies, we will establish common features and variations within and among these three classes of DNA repair enzymes resulting from their folds and binding motifs as well as their active site residues and metal ions ([4fe-4S] cluster, Mg, and Mn and Zn). This research on DNA repair enzymes will probe structural determinants for their biological stability and activity together with their unique chemically specific surveillance and catalytic recognition processes required for the sequence independent discrimination between damaged and undamaged DNA. This knowledge contributes specifically to understanding the action of DNA repair enzymes in the prevention of cancer-causing mutagenesis resulting from oxidative damage, xenobiotic toxins, and other causes, and contributes more generally to defining protein-DNA recognition processes fundamental to cell growth and differentiation. The long term goal is to apply our results to the development of medical and scientific tools, including DNA repair inhibitors as drugs for the enhancement of radiation and chemotherapies, and redesigned excision-repair enzymes useful for genetic engineering. The prominent active site grooves in the endonuclease III and AP endonuclease structures bode well for the structure-based design of DNA repair inhibitors with widespread potential for cancer therapeutics due to important defects in coupling repair to cell cycle progression in cancer cells compared to normal cells.
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