Sequence and time-dependent deamination of cytosine bases in UVB-induced cyclobutane pyrimidine dimers in vivo.
The mutational specificity of UV-light is characterized by an abundance of C to T transition mutations at dipyrimidines containing cytosine or 5-methylcytosine. A significant percentage of these mutations are CC to TT double transitions. Of the major types of UV-induced DNA lesions, the cis-syn cyclobutane pyrimidine dimers (CPDs) are thought to be the most mutagenic lesions, at least in mammalian cells. It has been proposed that the CPDs become mutagenic perhaps only after cytosine bases within these dimers deaminate to uracil and the resulting U-containing photolesions are correctly bypassed by DNA polymerases. In order to assess the significance of this proposed mutagenic mechanism, we have developed two methods to specifically measure deaminated CPDs in UV-irradiated human cells or DNA. The first method is based on enzymatic photoreversal of CPDs, followed by cleavage of the DNA with uracil DNA glycosylase, an AP lyase activity, and ligation-mediated PCR to map the resulting strand breaks. The second method, which can be used to detect double deamination events (CC to UU), is PCR amplification of photolyase-treated DNA using primers complemetary to the deaminated sequences. We have measured deamination events in the human p53 gene, which contains a large percentage of C to T transitions in skin cancers. The deamination reactions are specific for cytosine within CPDs, are negligible immediately after irradiation, and are time-dependent and DNA sequence context-dependent. Twenty four hours after irradiation of human fibroblasts with UVB light, between 10 and 60% of most CPD signals are converted to the deaminated form, depending on the sequence. Significant deamination occurs at skin cancer mutation sites in the p53 gene. Double deamination also occurs and this reaction can involve dimers containing 5-methylcytosine or cytosine. These double events are expected to occur more frequently in cells with a DNA repair defect because there is more time for deamination in unrepaired lesions. This may explain the relatively high frequency of CC to TT mutations in skin cancers from xeroderma pigmentosum patients. In summary, these novel detection techniques demonstrate that deamination of cytosine in pyrimidine dimers is a significant event that most likely contributes to the mutational specificity of UVB irradiation in human cells.
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Department of Biology, Beckman Research Institute of the City of Hope, Duarte, CA, 91010, USA.
Dammann
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Pfeifer
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D019757:Carbon-Oxygen Lyases / Q000378:metabolism; D003596:Cytosine / Q000378:metabolism*; D004247:DNA / Q000378:metabolism / Q000528:radiation effects*; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D003641:Deamination; D004255:Deoxyribodipyrimidine Photo-Lyase / Q000378:metabolism; D043223:Deoxyribonuclease IV (Phage T4-Induced); D005347:Fibroblasts / Q000528:radiation effects; D016758:Genes, jun; D016158:Genes, p53; D006801:Humans; D016296:Mutagenesis; D011740:Pyrimidine Dimers / Q000378:metabolism*; D012878:Skin Neoplasms / Q000235:genetics; D013997:Time Factors; D014466:Ultraviolet Rays / Q000009:adverse effects*
D011740:Pyrimidine Dimers; D003596:Cytosine; D004247:DNA; D043223:Deoxyribonuclease IV (Phage T4-Induced); D004255:Deoxyribodipyrimidine Photo-Lyase; D019757:Carbon-Oxygen Lyases; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase