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. 2002 Aug 15;30(16):3566-73.
doi: 10.1093/nar/gkf478.

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

Dong-Hyun Lee  1 , Timothy R O'Connor, Gerd P Pfeifer
Affiliations

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

Dong-Hyun Lee et al. Nucleic Acids Res. .

Abstract

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.

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Figures

Figure 1
Figure 1
Mutation spectra induced by 5 µM CuCl2 in the absence of H2O2 in unmethylated and CpG-methylated supF shuttle vectors from repair-deficient (XP-A) cells (A) and repair-proficient fibroblasts (B). The mutations introduced into the unmethylated plasmid are shown above the sequence, and the mutations introduced into the methylated plasmid are below the sequence. X, single-base deletions (X flanked by dashes, a nucleotide was deleted within a string of two or more identical nucleotides); * indicates the occurrence of two mutations in the same plasmid. CpG sequences are emphasized by white text on a black box.
Figure 1
Figure 1
Mutation spectra induced by 5 µM CuCl2 in the absence of H2O2 in unmethylated and CpG-methylated supF shuttle vectors from repair-deficient (XP-A) cells (A) and repair-proficient fibroblasts (B). The mutations introduced into the unmethylated plasmid are shown above the sequence, and the mutations introduced into the methylated plasmid are below the sequence. X, single-base deletions (X flanked by dashes, a nucleotide was deleted within a string of two or more identical nucleotides); * indicates the occurrence of two mutations in the same plasmid. CpG sequences are emphasized by white text on a black box.
Figure 2
Figure 2
(A) Mutation spectra induced by Cu(II)/H2O2 in unmethylated and CpG-methylated supF shuttle vectors from nucleotide excision repair-deficient cells. The mutations introduced into the unmethylated plasmid are shown above the sequence, and the mutations introduced into the methylated plasmid are below the sequence. X, single-base deletions; +A, addition of 1 nt. Tandem mutations are underlined. CpG sequences are shown by white text on a black background. (B) Mutation spectra induced by Cu(II)/H2O2 in unmethylated and CpG-methylated supF shuttle vectors from repair-proficient cells. X, single-base deletions; +G, addition of 1 nt. Tandem mutations are underlined. * and # indicate the occurrence of two mutations in the same plasmid.
Figure 2
Figure 2
(A) Mutation spectra induced by Cu(II)/H2O2 in unmethylated and CpG-methylated supF shuttle vectors from nucleotide excision repair-deficient cells. The mutations introduced into the unmethylated plasmid are shown above the sequence, and the mutations introduced into the methylated plasmid are below the sequence. X, single-base deletions; +A, addition of 1 nt. Tandem mutations are underlined. CpG sequences are shown by white text on a black background. (B) Mutation spectra induced by Cu(II)/H2O2 in unmethylated and CpG-methylated supF shuttle vectors from repair-proficient cells. X, single-base deletions; +G, addition of 1 nt. Tandem mutations are underlined. * and # indicate the occurrence of two mutations in the same plasmid.
Figure 3
Figure 3
CG→TT and CG→AA tandem mutations in CpG-methylated supF shuttle vectors treated with copper/H2O2. Sequence scans of two wild-type and mutant plasmids are shown.
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