Ultraviolet-induced thymine hydrates in DNA are excised by bacterial and human DNA glycosylase activities.
Ultraviolet irradiation of DNA results in various pyrimidine modifications. We studied the excision of an ultraviolet thymine photoproduct by Escherichia coli endonuclease III and by a preparation of human WI-38 cells. These enzymes cleave UV-irradiated DNA at apyrimidinic sites formed by glycosylic removal of the photoproduct. Poly(dA-[3H]dT).poly(dA-[3H]dT) was UV irradiated and incubated with purified E. coli endonuclease III. 3H-Containing material was released in a manner consistent with Michaelis-Menten kinetics. This 3H-labeled material was determined to be a mixture of thymine hydrates (6-hydroxy-5,6-dihydrothymine), separable from unmodified thymine by chromatography in three independent systems. Both cis-thymine hydrate and trans-thymine hydrate were chemically and photochemically synthesized. These coeluted with the enzyme-released 3H-containing material. No thymine glycol was released from the UV-irradiated polymer. Similar results were obtained with extracts of WI-38 cells as the enzyme source. The release of thymine hydrates by both glycosylase activities was directly proportional to the amount of enzyme and the irradiation dose to the DNA substrate. These results demonstrate the modified thymine residues recognized and excised by endonuclease III and the human enzyme to be a mixture of cis-thymine hydrate and trans-thymine hydrate. The reparability of these thymine hydrates suggests that they are stable in DNA and therefore potentially genotoxic.
Department of Pathology, Temple University School of Medicine, Philadelphia, Pennsylvania 19140.
T
T
Ganguly
K M
KM
Weems
N J
NJ
Duker
D001426:Bacterial Proteins / Q000378:metabolism*; D002478:Cells, Cultured; D004249:DNA Damage; D045647:DNA Glycosylases; D004260:DNA Repair; D043224:Deoxyribonuclease (Pyrimidine Dimer); D004706:Endodeoxyribonucleases / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D005347:Fibroblasts / Q000201:enzymology*; D006801:Humans; D009699:N-Glycosyl Hydrolases / Q000378:metabolism*; D011067:Poly dA-dT / Q000378:metabolism / Q000528:radiation effects; D013941:Thymine / Q000031:analogs & derivatives* / Q000378:metabolism; D014466:Ultraviolet Rays
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; C066110:6-hydroxy-5,6-dihydrothymine; D011067:Poly dA-dT; C029389:thymine glycol; D004706:Endodeoxyribonucleases; D043224:Deoxyribonuclease (Pyrimidine Dimer); C106403:NTH protein, E coli; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; D013941:Thymine