Dataset Information

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

ABSTRACT: 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.

SUBMITTER: Cao H

PROVIDER: S-EPMC1976268 | biostudies-literature | 2007

REPOSITORIES: biostudies-literature

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

Cao Huachuan H Wang Yinsheng Y

Nucleic acids research 20070710 14

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 re ...[more]

PMID: 17626047

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Project description:BackgroundDetermining the role of DNA methylation in various biological processes is dependent on the accurate representation of often highly complex patterns. Accurate representation is dependent on unbiased PCR amplification post bisulfite modification, regardless of methylation status of any given epiallele. This is highly dependent on primer design. Particular difficulties are raised by the analysis of CpG-rich regions, which are the usual regions of interest. Here, it is often difficult or impossible to avoid placing primers in CpG-free regions, particularly if one wants to target a specific part of a CpG-rich region. This can cause biased amplification of methylated sequences if the C is placed at those positions or to unmethylated sequences if a T is placed at those positions.MethodsWe examined the effect of various base substitutions at the cytosine position of primer CpGs on the representational amplification of templates and also examined the role of the annealing temperature during PCR. These were evaluated using methylation-sensitive high-resolution melting and Pyrosequencing.ResultsFor a mixture of fully methylated and unmethylated templates, amplification using the C-, C/T (Y-) and inosine-containing primers was biased towards amplification of methylated DNA. The bias towards methylated sequences increased with annealing temperature. Amplification using primers with an A/C/G/T (N) degeneracy at the cytosine positions was not biased at the lowest temperature used but became increasingly biased towards methylated DNA with increased annealing temperature. Using primers matching neither C nor T was in the main unbiased but at the cost of poor PCR amplification efficiency. Primers with abasic sites were also unbiased but could only amplify DNA for one out of the two assays tested. However, with heterogeneous methylation, it appeared that both the primer type and stringency used have a minimal influence on PCR bias.ConclusionsThis is the first comprehensive analysis of base substitutions at CpG sites in primers and their effect on PCR bias for the analysis of DNA methylation. Our findings are relevant to the appropriate design of a wide range of assays, including amplicon-based next-generation sequencing approaches that need to measure DNA methylation.

Project description:BackgroundMammalian CpG islands (CGIs) normally escape DNA methylation in all adult tissues and developmental stages. However, in our previous study we unexpectedly identified many methylated CGIs in human peripheral blood leukocytes. Methylated CpG dinucleotides convert to TpG dinucleotides through deaminization of their cytosine bases more frequently than hypomethylated CpG dinucleotides. Therefore, we wondered how methylated CGIs in germline or non-germline cells maintain their CpG-rich sequences. It is known that events such as germline hypomethylation, CpG selection, biased gene conversion (BGC), and frequent CpG fixation can contribute to the maintenance of CpG-rich sequences in methylated CGIs in germline or non-germline cells. However, it has not been investigated which of the processes maintain CpG-rich sequences of methylated CGIs in each genomic position.ResultsIn this study, we comprehensively examined the contribution of the processes described above to the maintenance of CpG-rich sequences in methylated CGIs in germline and non-germline cells which were classified by genomic positions. Approximately 60-80% of CGIs with high methylation in H1 cell line (H1-HM) in all the genomic positions showed a low average CpG→TpG/CpA substitution rate. In contrast, fewer than half the numbers of CGIs with H1-HM in all the genomic positions showed a low average CpG→TpG/CpA substitution rate and low levels of methylation in sperm cells (SPM-LM). Furthermore, a small fraction of CGIs with a low average CpG→TpG/CpA substitution rate and high levels of methylation in sperm cells (SPM-HM) showed CpG selection. On the other hand, independent of the positions in genes, most CGIs with SPM-HM showed a slightly higher average TpG/CpA→CpG substitution rate compared with those with SPM-LM.ConclusionsRelatively high numbers (approximately 60-80%) of CGIs with H1-HM in all the genomic positions preserve their CpG-rich sequences by a low CpG→TpG/CpA substitution rate caused mainly by their SPM-LM, and for those with SPM-HM partly by CpG selection and TpG/CpA→CpG fixation. BGC has little contribution to the maintenance of CpG-rich sequences of CGIs with SPM-HM which were classified by genomic positions.

Dataset Information

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

Publications

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

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