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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM037216-05
Application #
3292391
Study Section
Radiation Study Section (RAD)
Project Start
1988-07-01
Project End
1992-06-30
Budget Start
1990-07-01
Budget End
1991-06-30
Support Year
5
Fiscal Year
1990
Total Cost
Indirect Cost
Name
University of Vermont & St Agric College
Department
Type
Schools of Medicine
DUNS #
066811191
City
Burlington
State
VT
Country
United States
Zip Code
05405
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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
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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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