Novel Activities of Human Uracil DNA N-Glycosylase for Cytosine-Derived Products of Oxidative DNA Damage

Abstract

Uracil DNA N-glycosylase is a repair enzyme that releases uracil from DNA. A major function of this enzyme is presumably to protect the genome from pre-mutagenic uracil resulting from deamination of cytosine in DNA. Here, we report that human uracil DNA N-glycosylase also recognizes three uracil derivatives that are generated as major products of cytosine 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, heat-inactivated enzyme or buffer. Ethanol-precipitated DNA and supernatant fractions were then separated. Supernatant fractions after derivatization, 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 approximately 530, 450 and 660 nM, respectively. The excision of these uracil analogues is consistent with the recently described mechanism for recognition of uracil by human uracil DNA N-glycosylase [Mol,C.D., Arval,A.S., Slupphaug,G., Kavil,B., Alseth,I., Krokan,H.E. and Tainer,J.A. (1995) Cell, 80, 869-878]. Nine other pyrimidine- and purine-derived 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.

DNA glycosylases in the base excision repair of DNA

“…There is, however, sufficient space to allow small substituents such as hydroxy or carbonyl groups at the 5-or 6-positions. Thus certain uracil analogues generated from cytosine by γ-irradiation are recognized by UDG, but are excised at considerably lower rates than uracil [27,29]. This steric shielding has been verified by sitespecific mutagenesis of Tyr"%( of human UDG to Ala [82], which results in a mutant that excises thymine as well as uracil from DNA ( Figure 6).…”

“…These include 5-fluorouracil in DNA found after treatment with 5-fluorouracil [26], as well as isodialuric acid, 5-hydroxyuracil and alloxan, which are all formed from cytosine in DNA after exposure to γ-irradiation or oxidative stress [27][28][29]. Uracil at the 3h-end of a DNA chain is not removed by human [30] or E. coli [31] UDGs, whereas uracil at the 5h-end is removed provided that it is phosphorylated.…”

Identification and Characterization of a Novel Human DNA Glycosylase for Repair of Cytosine-derived Lesions

“…Surprisingly, while 5-OHU is the preferred substrate of NEIL2, three other human enzymes, namely NEIL1, NTH1, and uracil-DNA glycosylase, are also able to excise this lesion from DNA (10,23,24). This raises the possibility that 5-OHU, an important mutagenic lesion generated in significant amount in the genome, requires several back-up enzymes for its repair.…”

Oxidative DNA damage repair in mammalian cells: A new perspective

“…In other studies, the activity and expression of NTH1 and OGG1 were found to be significantly altered during the acute (16-18 weeks) and early chronic (24 weeks) phases of hepatitis in the Long Evans Cinnamon (LEC) rat, an animal model for Wilson's disease (WD) which is said to cause a "genetically induced" oxidative condition and higher risk for liver cancer [89]. Several studies indicated that SMUG1 and UNG1 are also capable of excising oxidized derivatives of pyrimidines and siRNA-mediated downregulation of these genes in mouse embryo fibroblasts significantly increased radio sensitivity [91,92]. In view of the presence of multiple DNA glycosylases further studies are required before we could evaluate their contribution to normal cellular functions.…”