Novel activities of human uracil DNA N-glycosylase for cytosine-derived products of oxidative DNA damage

Uracil DNA N-glycosylase is a repair enzyme that releases uracil from DNA. A major function of this enzyme is presumably to protect the ge nome from pre-mutagenic uracil resulting from deamination of cytosine in DNA. Here, we report that human uracil DNA N-glycosylase also rec ognizes three uracil derivatives that are generated as major products of cytosine in DNA by hydroxyl radical attack or other oxidative pro cesses. DNA substrates were prepared by gamma-irradiation of DNA in a erated aqueous solution and incubated with human uracil DNA N-glycosy lase, heat-inactivated enzyme or buffer. Ethanol-precipitated DNA and supernatant fractions were then separated. Supernatant fractions aft er derivatization, and pellets after hydrolysis and derivatization we re analyzed by gas chromatography/isotope-dilution mass spectrometry. The results demonstrated that human uracil DNA N-glycosylase excised isodialuric acid, 5-hydroxyuracil and alloxan from DNA with apparent K(m) values of approximately 530, 450 and 660 nM, respectively. The excision of these uracil analogues is consistent with the recently de scribed mechanism for recognition of uracil by human uracil DNA N-gly cosylase [Mol,C.D., Arval,A.S., Slupphaug,G., Kavil,B., Alseth,I., Kr okan,H.E. and Tainer,J.A. (1995) Cell, 80, 869-878]. Nine other pyrim idine- and purine-derived products that were identified in DNA sample s were not substrates for the enzyme. The results indicate that human uracil DNA N-glycosylase may have a function in the repair of oxidat ive DNA damage.