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.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Radiation Study Section (RAD)
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University of Vermont & St Agric College
Schools of Medicine
United States
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Hashimoto, M; Donald, C D; Yannone, S M et al. (2001) A possible role of Ku in mediating sequential repair of closely opposed lesions. J Biol Chem 276:12827-31
Venkhataraman, R; Donald, C D; Roy, R et al. (2001) Enzymatic processing of DNA containing tandem dihydrouracil by endonucleases III and VIII. Nucleic Acids Res 29:407-14
Hashimoto, M; Greenberg, M M; Kow, Y W et al. (2001) The 2-deoxyribonolactone lesion produced in DNA by neocarzinostatin and other damaging agents forms cross-links with the base-excision repair enzyme endonuclease III. J Am Chem Soc 123:3161-2
Rabow, L; Venkataraman, R; Kow, Y W (2001) Mechanism of action of Escherichia coli formamidopyrimidine N-glycosylase: role of K155 in substrate binding and product release. Prog Nucleic Acid Res Mol Biol 68:223-34
He, B; Qing, H; Kow, Y W (2000) Deoxyxanthosine in DNA is repaired by Escherichia coli endonuclease V. Mutat Res 459:109-14
Liu, J; He, B; Qing, H et al. (2000) A deoxyinosine specific endonuclease from hyperthermophile, Archaeoglobus fulgidus: a homolog of Escherichia coli endonuclease V. Mutat Res 461:169-77
Yao, M; Kow, Y W (1997) Further characterization of Escherichia coli endonuclease V. Mechanism of recognition for deoxyinosine, deoxyuridine, and base mismatches in DNA. J Biol Chem 272:30774-9
Willemoes, M; Hove-Jensen, B (1997) Binding of divalent magnesium by Escherichia coli phosphoribosyl diphosphate synthetase. Biochemistry 36:5078-83
Rabow, L E; Kow, Y W (1997) Mechanism of action of base release by Escherichia coli Fpg protein: role of lysine 155 in catalysis. Biochemistry 36:5084-96
Yao, M; Kow, Y W (1996) Cleavage of insertion/deletion mismatches, flap and pseudo-Y DNA structures by deoxyinosine 3'-endonuclease from Escherichia coli. J Biol Chem 271:30672-6

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