A major stable oxidation product of DNA cytosine is uracil glycol (Ug). Because of the potential of Ug to be a strong premutagenic lesion, it is important to assess whether it is a blocking lesion to DNA polymerase as is its structural counterpart, thymine glycol (Tg), and to evaluate its pairing properties. Here, a series of oligonucleotides containing Ug or Tg were prepared and used as templates for a model enzyme, Escherichia coli DNA polymerase I Klenow fragment (exo؊). During translesion DNA synthesis, Ug was bypassed more efficiently than Tg in all sequence contexts examined. Furthermore, only dAMP was incorporated opposite template Ug and Tg and the kinetic parameters of incorporation showed that dAMP was inserted opposite Ug more efficiently than opposite Tg. Ug opposite G and A was also recognized and removed in vitro by the E. coli DNA repair glycosylases, endonuclease III (endo III), endonuclease VIII (endo VIII), and formamidopyrimidine DNA glycosylase. The steady state kinetic parameters indicated that Ug was a better substrate for endo III and formamidopyrimidine DNA glycosylase than Tg; for endonuclease VIII, however, Tg was a better substrate.Free radical-induced DNA damage is a substantial contributor to the spontaneous mutational burden and has been implicated in a variety of disease processes including cancer. Accumulation of free radical-induced DNA damage has also been associated with aging (1-3). A significant number of free radical-induced mutations produced by oxidizing agents and ionizing radiation are C 3 T transitions (4 -8), thus oxidized cytosine residues appear to play an important role in oxidative mutagenesis. Hydroxyl radicals, the principal damaging species produced by oxidizing agents, interact with cytosine residues principally by addition to the 5,6-double bond (9, 10). A major oxidation product of DNA cytosine is cytosine glycol which is unstable and deaminates to uracil glycol (Ug) 1 (11).Cytosine glycol can also dehydrate to form 5-hydroxycytosine (5-OHC) (11-13), while 5-hydroxyuracil (5-OHU) arises from sequential deamination and dehydration (11). Depending on the oxidizing agent used, uracil glycol and 5-hydroxycytosine are formed at comparable levels in DNA and furthermore, the background level of these lesions is high in DNA extracted from untreated cells (11).Oxidized pyrimidine lesions are repaired by a highly conserved process called base excision repair (for reviews, see Refs. 14 and 15). The damaged pyrimidines are recognized by a class of enzymes called DNA glycosylases. The principal activities in Escherichia coli that recognize oxidized pyrimidines are endonuclease III (endo III) and endonuclease VIII (endo VIII) (see Refs. 14 and 15, and references therein); formamidopyrimidine DNA glycosylase (Fpg) has also been shown to recognize oxidized pyrimidines in vitro (16). Upon recognition of an oxidized pyrimidine by a DNA glycosylase, the N-glycosylic bond is cleaved releasing the free base. This is followed by cleavage of the phosphodiester backbone by an associ...

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