There is a well established link between inefficient DNA repair of ultraviolet (UV) light induced damage and carcinogenesis in man, as demonstrated through the study of the human disease, xeroderma pigmentosum and epidemiological studies of nonmelanoma skin cancer. Enhanced UV exposure and persistence of dipyrimidine photoproducts have been correlated with oncogene activation and tumor suppressor inactivation . Thus, the ability to maintain DNA integrity is rooted in accurate DNA repair systems that catalyze the restoration of damaged DNAs to their native state as well as high fidelity DNA replication. The variety of ubiquitous DNA repair pathways include recombinational, nucleotide, and base excision repair. The initial steps of the base excision pathway consist of the location, recognition, and release of damaged bases by DNA glycosylases. These enzymes sometimes have a concomitant AP lyase activity. One of these enzymes, T4 endonuclease V, is a cyclobutane pyrimidine dimer DNA glycosylase-AP lyase. Previous studies have revealed the fundamental mechanisms by which this enzyme initiates repair at this UV-induced lesion, and include the determination of its active site, the sequences that are responsible for DNA binding and the chemical basis for catalysis. In order for endonuclease V to continue to serve as the prototype for mechanistic studies of this class of enzymes, the following specific aims are proposed: 1) Develop fine structure kinetic analyses of endonuclease V-catalyzed reactions. order to determine the progression of steps that ultimately lead to enzyme-mediated catalysis, a full kinetic heme will be developed using rapid quench-flow technologies and novel DNA substrates. 2) Determine the molecular architecture of endonuclease V bound to cyclobutane dimer.containing DNA. It is hypothesized that endonuclease V undergoes a conformational change on dimer-specific binding. This hypothesis will be tested by biophysically characterizing DNA-protein completes before and after the glycosylase step. 3) Describe the architecture of endonuclease V-nontarget DNA interactions and determine the mechanism of target site location. A variety of genetic and biophysical methods will be used to investigate hypotheses concerning the associations between nontarget DNA and endonuclease V. 4) Determine the analytic mechanisms for other pyrimidine dimer-specific DNA glycosylases-AP lyases. It is hypothesized that all DNA glycosylases-AP lyases function through a common chemistry. This will be tested by elucidating the catalytic mechanism of action for other dimer-specific DNA glycosylase-AP lyases. These studies may the foundation for developing unifying principles for initiating the restoration of damaged DNA by DNA glycosylases. In the long term, this will involve the following determinations: 1) what are the mechanisms by se proteins locate sites of specific damage within vast excesses of undamaged DNA; 2) what are the principles for specific substrate recognition and what are the structural motifs within these proteins that achieve differential binding; 3) what are the active site residues within these enzymes and how do these residues suggest catalytic mechanisms; 4 what is the chemical basis for glycosyl bond cleavage?
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