Ionizing radiation produces a wide spectrum of damages to the base and sugar moieties of DNA. Enzymes such as endonucleases III, IV, VIII, and IX and exonuclease III from Escherichia coli have been shown to recognize radiation-induced DNA lesions in vitro. The long range goal of this research is to elucidate the enzymatic mechanism(s) by which radiation repair enzymes recognize and remove lesions from damaged DNA. Both the substrate specificities and the kinetics of recognition of DNA substrates containing unique radiolysis products will be investigated for E. coli endonucleases III, IV, VIII, and IX and exonuclease III. Novel substrates will be prepared by an enzymatic method utilizing T4 RNA ligase. The hypothesis that ring opening of the deoxyribose moiety is a necessary step in catalysis by glycosylases/endonucleases that recognize radiation damages will be tested by determining the effect of chemical reduction of the imine or aldehyde bond formed during catalysis, as well as the solvent isotope effect on the rate of catalysis. The active site of the E. coli radiation repair enzyme will be probed by isolating the enzyme-damage (DNA) complex. Lastly, the effect of chemical modification of active site amino acid residues will be studied in order lo identify essential amino acids involved in catalysis. The chemical basis of catalysis elucidated in these studies should aid in the development of specific inhibitors of radiation repair enzymes that could be used in conjunction with radiotherapy for cancer treatment.
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