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 3′→5′ 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 apn1Δ ogg1Δ 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.
ACKNOWLEDGMENTS
We thank Jacques Laval and Pat Foster for discussions, Dmitry Zharkov for the E. coli Nei protein, and Grigory Dianov for sharing results prior to publication.
This work was supported by grants from the European Community (RISC-RAD FI6R-CT-2003-508842), the Association pour la Recherche sur le Cancer, and Electricité de France Contrat Radioprotection (to M.S.), and the Natural Sciences and Engineering Council of Canada (to D.R.). A.A.I. was supported by a postdoctoral fellowships from the Association pour la Recherche sur le Cancer. D.R. is the recipient of a senior scholarship from the Fonds de la Recherche en Sante du Quebec.