Quantification of oxidative single-base and intrastrand cross-link lesions in unmethylated and CpG-methylated DNA induced by Fenton-type reagents

Methylation of cytosine at CpG sites in mammalian cells plays an important role in the epigenetic regulation of gene expression. Here, we assessed the formation of single-nucleobase lesions and intrastrand cross-link lesions (i.e. G[8-5]C, C[5-8]G, mC[5m-8]G, and G[8-5m]mC, where 'mC' represents 5-methylcytosine) in unmethylated and the corresponding CpG-methylated synthetic double-stranded DNA upon treatment with Fenton-type reagents [i.e. H2O2, ascorbate together with Cu(II) or Fe(II)]. Our results showed that the yields of oxidative single-nucleobase lesions were considerably higher than those of the intrastrand cross-link lesions. Although no significant differences were found for the yields of single-base lesions induced from cytosine and mC, the G[8-5m]mC cross-link was induced approximately 10 times more efficiently than the G[8-5]C cross-link. In addition, the mC[5m-8]G was induced at a level that was approximately 15 times less than G[8-5m]mC, whereas the corresponding C[5-8]G intrastrand cross-link lesion was not detectable. Moreover, Cu(II) is approximately 10-fold as effective as Fe(II) in inducing oxidative DNA lesions. These results suggest that oxidative intrastrand cross-link lesions formed at methylated-CpG sites may account for the previously reported mCG-->TT tandem double mutations induced by Fenton-type reagents.