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 geno me from pre-mutagenic uracil resulting from deamination of cytosine in DNA, Here, we report that human uracil DNA N-glycosylase also recogni zes three uracil derivatives that are generated as major products of c ytosine in DNA by hydroxyl radical attack or other oxidative processes , DNA substrates were prepared by gamma-irradiation of DNA in aerated aqueous solution and incubated with human uracil DNA N-glycosylase, he at-inactivated enzyme or buffer. Ethanol-precipitated DNA and supernat ant fractions were then separated, Supernatant fractions after derivat ization, and pellets after hydrolysis and derivatization were 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 similar to 530, 450 and 660 nM, respectively, The excision of these u racil analogues is consistent with the recently described mechanism fo r recognition of uracil by human uracil DNA N-glycosylase [Mol,C.D., A rval,A,S., Slupphaug,G., Kavil,B., Alseth,l., Krokan,H.E. and Tainer,J .A. (1995) Cell, 80, 869-878], Nine other pyrimidine and purine-derive d products that were identified in DNA samples were not substrates for the enzyme, The results indicate that human uracil DNA N-glycosylase may have a function in the repair of oxidative DNA damage.