PCR-based approaches to adduct analysis.

Abstract

Ligation-mediated polymerase chain reaction (LMPCR) is a PCR-based method for the detection of DNA adducts at individual nucleotide positions in mammalian genes. Adduct-specific enzymes, such as T4 endonuclease V, various base excision repair enzymes, UvrABC nuclease, and chemical cleavage techniques can be used to convert the adducts into DNA strand breaks. The positions of these breaks are then detected by LMPCR. This method has been used primarily to map the distribution of UV-induced DNA lesions and adducts of polycyclic aromatic hydrocarbons. The number and diversity of mutations in the p53 mutation database provides indirect evidence that environmental mutagens may be involved in human carcinogenesis. We hypothesize that there is a limited involvement of selection for specific mutations in the central domain of the p53 protein, and that the distribution of DNA damage along the p53 gene caused by environmental carcinogens can be correlated with the mutational spectra, i.e. hotspots and types of mutations, of certain cancers. This concept has been validated by experiments with sunlight and the cigarette smoke component benzo[a]pyrene representing the polycyclic aromatic hydrocarbon class of carcinogens. The damage and repair data obtained for these mutagens can predict certain parameters of the mutational spectra of human non-melanoma skin cancers and lung cancers from smokers. Future studies with suspected mutagens may help to implicate causative agents involved in other cancers, where the exact carcinogen has not yet been identified but an environmental factor is suspected.