Oxidatively generated guanine(C8)-Thymine(N3) intrastrand cross-links in double-stranded DNA are repaired by base excision repair pathways

Oxidatively generated guanine radical cations in DNA can undergo various nucleophilic reactions including the formation of C8-guanine cross-links with adjacent or nearby N3-thymines in DNA in the presence of O₂. The G^∗[C8-N3]T^∗ lesions have been identified in the DNA of human cells exposed to oxidative stress, and are most likely genotoxic if not removed by cellular defense mechanisms. It has been shown that the G^∗[C8-N3]T^∗ lesions are substrates of nucleotide excision repair in human cell extracts. Cleavage at the sites of the lesions was also observed but not further investigated (Ding et al. (2012) Nucleic Acids Res. 40, 2506-2517). Using a panel of eukaryotic and prokaryotic bifunctional DNA glycosylases/lyases (NEIL1, Nei, Fpg, Nth, and NTH1) and apurinic/apyrimidinic (AP) endonucleases (Apn1, APE1, and Nfo), the analysis of cleavage fragments by PAGE and MALDI-TOF/MS show that the G^∗[C8-N3]T^∗ lesions in 17-mer duplexes are incised on either side of G^∗, that none of the recovered cleavage fragments contain G^∗, and that T^∗ is converted to a normal T in the 3′-fragment cleavage products. The abilities of the DNA glycosylases to incise the DNA strand adjacent to G^∗, while this base is initially cross-linked with T^∗, is a surprising observation and an indication of the versatility of these base excision repair proteins.