Free radical damage to DNA, which has been associated with carcinogenesis, the normal aging process, and neurodegeneration, produces a variety of lesions. Many of these damages are various abasic lesions (sites of base loss). These include 1 -oxidized, 4 -oxidized or fragmented deoxyribose residues, known products of agents such as H2O2, ionizing radiation and bleomycin. Such damages have cytotoxic and mutagenic potential that might underlie the age and disease effects cited above, but these oxidized deoxyribose lesions have been difficult to measure specifically, sensitively and quantitatively by existing methods. This project proposes to develop new methods for such analysis, with an emphasis on the use of DNA repair enzymes to liberate specific damages for analysis by advanced techniques of mass spectrometry and capillary electrophoresis. These methods will be used to explore the formation and repair of individual oxidized abasic sites in vivo. Abasic (AP) endonucleases have been implicated in the repair of these damages, but this specificity has not been demonstrated in vivo. Key proposed studies include analyzing chromosomal DNA to determine the specific role of E. coli exonuclease III in repairing H2O2-induced DNA damage, and of E. coli endonuclease IV and yeast Apnl in the repair of oxidized abasic sites. The ability of human Ape endonuclease to correct specific repair defects in mutant strains of E. coli and yeast will be assessed by the novel methods, as will mutated forms of endonuclease IV and Apnl. Not only will this work be important for future work toward understanding the role of free-radical damage in aging, cancer, and neurodegeneration, but it will also provide quantum improvements in the methodology for molecular analysis of an important class of oxidative DNA damage.
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