The 8-oxo-7,8-dihydrodeoxyguanosine (8oxoG), a major mutagenic DNA lesion, results either from direct oxidation of guanines or misincorporation of 8oxodGTP by DNA polymerases. At present, little is known about the mechanisms preventing the mutagenic action of 8oxodGTP in Saccharomyces cerevisiae. Herein, we report for the first time the identification of an alternative repair pathway for 8oxoG residues initiated by S. cerevisiae AP endonuclease Apn1, which is endowed with a robust progressive 335 exonuclease activity towards duplex DNA. We show that yeast cell extracts, as well as purified Apn1, excise misincorporated 8oxoG, providing a damage-cleansing function to DNA synthesis. Consistent with these results, deletion of both OGG1 encoding 8oxoG-DNA glycosylase and APN1 causes nearly 46-fold synergistic increase in the spontaneous mutation rate, and this enhanced mutagenesis is primarily due to G · C to T · A transversions. Expression of the bacterial 8oxodGTP triphosphotase MutT in the apn1⌬ ogg1⌬ mutant reduces the mutagenesis. Taken together, our results indicate that Apn1 is involved in an S. cerevisiae 8-oxoguanine-DNA glycosylase (Ogg1)-independent repair pathway for 8oxoG residues. Interestingly, the human major AP endonuclease, Ape1, also exhibits similar exonuclease activity towards 8oxoG residues, raising the possibility that this enzyme could participate in the prevention of mutations that would otherwise result from the incorporation of 8oxodGTP.Endogenous oxidative DNA lesions are most abundant and inevitable as cells generate reactive oxygen species through aerobic respiration. The occurrence of oxidized bases in DNA can result from either direct oxidation or misincorporation of oxidized deoxyribonucleoside monophosphate by DNA polymerases from the nucleotide pool (23, 24). These oxidized bases are mutagenic and therefore must be removed by efficient DNA repair processes that include base excision repair (BER) and the nucleotide incision repair (NIR) pathways, as well as by a process involving the sanitization of a pool of oxidized deoxyribonucleotidetriphosphates (dNTPs) (14, 43). In Escherichia coli, three enzymes, formamidopyrimidine-DNA glycosylase (Fpg), adenine-DNA glycosylase (MutY), and a nucleotidase (MutT; 8oxodGTP triphosphatase), play important roles in counteracting the mutagenic effect of the oxidized residue 8-oxo-7,8-dihydrodeoxyguanosine (8oxoG), which forms stable base pairing with adenine (A · 8oxoG) (33). While Fpg excises 8oxoG and MutY excises adenine from the A · 8oxoG mispair in DNA, MutT sanitizes the nucleotide precursor pool of 8oxodGTP by hydrolyzing it to 8oxodGMP and pyrophosphate, thereby preventing the incorporation of the oxidized nucleotide into DNA during replication (12, 32). These three enzymes are also conserved in mammalian cells, thus underscoring the importance of preventing an 8oxoG mutagenic effect (1). However, in the budding yeast Saccharomyces cerevisiae, only a functional homologue of Fpg, Ogg1 (S. cerevisiae 8-oxoguanine-DNA glycosylase), has been is...