Faculty Bibliography

Radiation and oxidation agents damage the pyrimidine bases of DNA. The resulting modified bases can be recognized by enzymes which effect the removal of base lesions, leaving an apurinic site in the DNA backbone. We have been studying three distinct classes of pyrimidine base damage: a) saturation of the 5,6-ethylenic bond to produce the corresponding glycol, b) hydration of the 5,6 double bond to produce the corresponding 6-hydroxy-5,6-dihydroderivative, and c) oxidation of the methyl group of thymine and 5-methylcytosine to produce the corresponding hydroxymethylated compound

UV irradiation of poly(dG-dC) and poly(dA-dU) in solution produces pyrimidine hydrates which are repaired by bacterial and mammalian DNA glycosylases (Biochemistry, 28: 6164, 1989). E. coli endonuclease III was used to quantitate the formation and stability of these hydrates in poly(dG-dC) and poly(dA-dU). When poly(dG-dC) was irradiated with 100 kJ/m2 of 254 nm light at pH 8.0, 2.2% of Cyt residues were converted to cytosine hydrate (6-hydroxy-5,6-dihydrocytosine) while 0.09% were converted to uracil hydrate (6-hydroxy-5,6-dihydrouracil). To measure the stability of these photoproducts, poly(dG-dC) was incubated in solution for up to 24 hours after UV irradiation. Cytosine hydrate was stable at 4-degrees-C and decayed at 25- degrees-C, 37-degrees-C and 55-degrees-C with half lives of 75, 25 and 6 hours, respectively. Uracil hydrate produced in irradiated poly(dA-dU) was stable at 4-degrees-C and at 25- degrees-C, and decayed with a half life of 6 hours at 37- degrees-C and less than one half-hour at 55-degrees-C. Uracil hydrate and uracil were also shown to be formed in irradiated poly(dG-dC). These experiments demonstrate that far-UV induced cytosine hydrate may persist in DNA for prolonged time periods and also undergoes deamination to uracil hydrate which, in turn, undergoes dehydration to yield uracil. The formation and stability of these photoproducts in DNA may have promoted the evolutionary development of the repair enzyme endonuclease III and analogous DNA glycosylase/endonuclease activities of higher organisms, as well as the development of uracil-DNA glycosylase

Ultraviolet irradiation of poly(dG-dC) and poly(dA-dU) in solution produces pyrimidine hydrates that are repaired by bacterial and mammalian DNA glycosylases [Boorstein et al. (1989) Biochemistry 28, 6164-6170]. Escherichia coli endonuclease III was used to quantitate the formation and stability of these hydrates in the double-stranded alternating copolymers poly(dG-dC) and poly(dA-dU). When poly(dG-dC) was irradiated with 100 kJ/m2 of 254-nm light at pH 8.0, 2.2% of the cytosine residues were converted to cytosine hydrate (6-hydroxy-5,6-dihydrocytosine) while 0.09% were converted to uracil hydrate (6-hydroxy-5,6-dihydrouracil). To measure the stability of these products, poly(dG-dC) was incubated in solution for up to 24 h after UV irradiation. Cytosine hydrate was stable at 4 degrees C and decayed at 25, 37, and 55 degrees C with half-lives of 75, 25, and 6 h. Uracil hydrate produced in irradiated poly(dA-dU) was stable at 4 degrees C and at 25 degrees C and decayed with a half-life of 6 h at 37 degrees C and less than 0.5 h at 55 degrees C. Uracil hydrate and uracil were also formed in irradiated poly(dG-dC). These experiments demonstrate that UV-induced cytosine hydrate may persist in DNA for prolonged time periods and also undergo deamination to uracil hydrate, which in turn undergoes dehydration to yield uracil. The formation and stability of these photoproducts in DNA may have promoted the evolutionary development of the repair enzyme endonuclease III and analogous DNA glycosylase/endonuclease activities of higher organisms, as well as the development of uracil-DNA glycosylase

Escherichia coli endonuclease III and mammalian repair enzymes cleave UV-irradiated DNA at AP sites formed by the removal of cytosine photoproducts by the DNA glycosylase activity of these enzymes. Poly(dG-[3H]dC) was UV irradiated and incubated with purified endonuclease III. 3H-Containing material was released in a fashion consistent with Michaelis-Menten kinetics. This 3H material was determined to be cytosine by chromatography in two independent systems and microderivatization. 3H-Containing material was not released from nonirradiated copolymer. When poly(dA-[3H]dU) was UV irradiated, endonuclease III released 3H-containing material that coeluted with uracil hydrate (6-hydroxy-5,6-dihydrouracil). Similar results are obtained by using extracts of HeLa cells. There results indicate that the modified cytosine residue recognized by endonuclease III and the mammalian enzyme is cytosine hydrate (6-hydroxy-5,6-dihydrocytosine). Once released from DNA through DNA-glycosylase action, the compound eliminates water, reverting to cytosine. This is consistent with the known instability of cytosine hydrate. The repairability of cytosine hydrate in DNA suggests that it is stable in DNA and potentially genotoxic