The 3′→5′ Exonuclease of Apn1 Provides an Alternative Pathway To Repair 7,8-Dihydro-8-Oxodeoxyguanosine in Saccharomyces cerevisiae

Abstract

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...

Evidence for Extrinsic Exonucleolytic Proofreading

“…37 The 3' exonuclease of the yeast apurinic endonuclease Apn1 has recently been implicated in the repair of 8-oxo-guanine in vivo. 38 Consistent with the possibility of proofreading by apurinic endonuclease during snBER (Fig. 1D) is the observation that the fidelity of a snBER reaction involving pol β and the apurinic endonuclease APE is 3-to 8-fold higher than is the fidelity of gap-filling by pol β alone.…”

Intrinsic apurinic/apyrimidinic (AP) endonuclease activity enables Bacillus subtilis DNA polymerase X to recognize, incise, and further repair abasic sites

“…S2A and SI Discussion). In addition, besides its AP endonuclease, EndoIV also possesses a 3′-5′-exonuclease activity governed by the same active site (25,(27)(28)(29). Altogether, the results lead us to propose for the PHP domain of bacterial/archaeal PolXs a three metal ions mechanism similar to EndoIV for recognition of damaged DNA, binding, and incision, suggesting a convergent evolution to give rise to a 3′-OH end required to prime further DNA repair synthesis.…”

Uncoupling of the base excision and nucleotide incision repair pathways reveals their respective biological roles

“…It was thought that these AP endonucleases mainly function in the BER pathway by protecting cells from abasic sites and single-strand breaks with 3Ј-blocking groups induced either by DNA-damaging agents or by DNA glycosylases (13,27). Recently, we have demonstrated that AP endonuclease-initiated NIR works in concert with the BER pathway to cleanse genomic DNA of potentially mutagenic and lethal lesions (3,28). In the present study, a genetic dissection of the BER and NIR functions of endonuclease IV reveals that the drug sensitivity of E. coli correlates with the specific lack of NIR activity, thus strengthening our previous conclusions.…”