Oxidative DNA damage induced by copper and hydrogen peroxide promotes CG→TT tandem mutations at methylated CpG dinucleotides in nucleotide excision repair-deficient cells

Oxidative DNA damage may play an important role in human disease including cancer. Previously, mutational spectra have been determined using systems that include transition metal ions and hydrogen peroxide (H2O2). G-->T transversions and C-->T transitions were the most common mutations observed including some CC-->TT tandem mutations. C-->T transition mutations at methylated CpG dinucleotides are the most common mutations in human genetic diseases. It has been hypothesized that oxidative stress may increase the frequency of mutations at methylated CpG sequences. Here we have used a CpG-methylated shuttle vector to derive mutational spectra of copper/H2O2-induced DNA damage upon passage of the shuttle vector through human fibroblasts. We find that copper/H2O2 treatment produces higher numbers of CpG transition mutations when the CpGs are methylated but does not create clear C-->T hotspots at these sites. More strikingly, we observed that this treatment produces a substantial frequency of mutations that were mCG-->TT tandem mutations. Six of seven tandem mutations were of this type. mCG-->TT mutations (6/63 = 10% of all mutations) were observed only in nucleotide excision repair-deficient (XP-A) cells but were not found in repair-proficient cells. The data suggest that this novel type of mutation may be produced by vicinal or cross-linked base damage involving 5-methylcytosine and a neighboring guanine, which is repaired by nucleotide excision repair. We suggest that the underlying oxidative lesions could be responsible for the progressive neurodegeneration seen in XP-A individuals.