Oxidative damage to DNA is constant and inevitable. Reactive oxygen species are produced by normal metabolic processes and ionizing radiation. There is an increasing body of evidence that indicates oxidative damage to DNA plays a critical role in the degenerative processes leading to aging and cancer. The study of oxidation DNA damage and repair is, therfore, of great importance in understanding these processes. The goal of this proposal is to characterize a human DNA repair enzyme, redoxy-endonuclease, that recognizes oxdidative and radiation-induce DNA damage. A second goal of this proposal is to determine the role of this enzyme in the maintenance of DNA stability in a cellular environment containing numerous damaging agents.
The specific aims are directed toward obtaining an understanding of the enzymology, genetics, regulation, and subcellular distribution of redoxy-endonuclease. The human redoxy-endonuclease will be purified to homogeneity. The substrate specificity of the enzyme following damage to DNA with oxidizing agents, ionizing radiation, and UV light will be determined. For these studies, end-labeled DNA fragments of defined sequence will be utilized as DNA substrates and the enzyme-generated scission products will be analyanalyzed using DNA sequencing methodologies. The mechanism of action of the enzyme will be deduced through the use of DNA sequencing and HPLC methodologies. A partial amino acid sequence of the enzyme will be obtained by utilization of protein microsequencing technologies. This information will be used to construct synthetic oligodeoxynucleotide gene probes for human redoxy-endonucleases. Such probes and recombinant DNA methodologies will be used to screen human genomic and cDNA libraries to identify and characterize molecular clones of the redoxy-endonuclease gene. The characterization of the redoxy-enonuclease gene will permit study of the regulation of this enzyme in response to DNA damage and also in human cells with different DNA repair backgrounds. The subcellular distribution of the redoxy-endonuclease will be determined by comparing the levels of enzyme activity associated with chromatin and mitochondria.
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