Preferential DNA damage and poor repair determine ras gene mutational hotspot in human cancer

Background: Mutations in ras genes are commonly found in human cancers and in animal models. Although mutations at codons 12, 13, and 61 of H-, N- and K-ras genes can activate their oncogenic function, mutations at codon 12 of K-ras are the most common mutations found among the three ras genes in human cancers. To investigate whether codon 12 of human K-ras is especially susceptible to carcinogens and/or whether carcinogen-DNA adducts at this codon are repaired less efficiently, we examined tobacco smoke carcinogen-induced DNA damage in normal human bronchial epithelial and fibroblast cells. Methods: We used the UvrABC nuclease incision method in combination with ligation-mediated polymerase chain reaction to map the distribution of DNA adducts induced by benzo[a]pyrene diol epoxide (BPDE) and other bulky carcinogens within exons 1 and 2 in H-ras, N-ras, and K-ras. We also analyzed BPDE-DNA adduct repair efficiency in these three genes using the same method. Results: Codons 12 and 14 of the K-ras gene were hotspots for carcinogen-DNA adduct formation, with little and no adduct formation at codons 13 and 61, respectively. The BPDE-DNA adducts formed at codon 14 were repaired almost twice as quickly as those formed at codon 12. There was some BPDE-DNA adduct formation at codons 12 of H-ras and N-ras, but this codon was not a hotspot. Furthermore, no substantial difference in repair rates between codon 12 and the other codons analyzed (codons 3 and 18) was observed in either the H-ras or N-ras genes. [Conclusion: These findings link the human cancer mutational hotspot at codon 12 of K-ras to preferential DNA damage and poor repair.