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Alternative nucleotide incision repair pathway for oxidative DNA damage

Abstract

The DNA glycosylase pathway1, which requires the sequential action of two enzymes for the incision of DNA2, presents a serious problem for the efficient repair of oxidative DNA damage, because it generates genotoxic intermediates such as abasic sites and/or blocking 3′-end groups that must be eliminated by additional steps before DNA repair synthesis can be initiated. Besides the logistical problems, biological evidence hints at the existence of an alternative repair pathway. Mutants of Escherichia coli3 and mice (ref. 4 and M. Takao et al., personal communication) that are deficient in DNA glycosylases that remove oxidized bases are not sensitive to reactive oxygen species, and the E. coli triple mutant nei, nth, fpg is more radioresistant than the wild-type strain5. Here we show that Nfo-like endonucleases nick DNA on the 5′ side of various oxidatively damaged bases, generating 3′-hydroxyl and 5′-phosphate termini. Nfo-like endonucleases function next to each of the modified bases that we tested, including 5,6-dihydrothymine, 5,6-dihydrouracil, 5-hydroxyuracil and 2,6-diamino-4-hydroxy-5-N-methylformamidopyrimidine residues. The 3′-hydroxyl terminus provides the proper end for DNA repair synthesis; the dangling damaged nucleotide on the 5′ side is then a good substrate for human flap-structure endonuclease6 and for DNA polymerase I of E. coli.

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Figure 1: Incision activity of various AP endonucleases towards oxidative DNA lesions.
Figure 2: Nfo protein is the endonuclease that recognizes DHPy.
Figure 3: Cleavage of oligonucleotides containing modified bases by whole-cell extracts from S. cerevisiae and HeLa cells.
Figure 4: Exonucleolytic activities of E. coli polymerase I and human FEN-1 proteins at the nicked duplex DNA containing 5′-dangling nucleotide.

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Acknowledgements

We thank J. Laval for stimulating discussions and financial support; G. Baldacci for human PCNA; C. V. Privezentzev for human HAP-1 and FEN-1; A. Maksimenko and C. Delaporte for human cells; D. Ramotar for yeast strains; and P. R. Strauss, A. Kuzminov and D. Zharkov for critically reading the manuscript. This work was supported by the European Community (M.K.S.), Association pour la Recherche sur le Cancer (J.L.), and fellowships from Centre français pour l'accueil et les échanges internationaux (EGIDE) to A.A.I.

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Correspondence to Murat K. Saparbaev.

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Ischenko, A., Saparbaev, M. Alternative nucleotide incision repair pathway for oxidative DNA damage. Nature 415, 183–187 (2002). https://doi.org/10.1038/415183a

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