Genotoxic effects of the major alkylation damage N7-methylguanine and methyl formamidopyrimidine

Various alkylating agents are known to preferentially modify guanine in DNA, resulting in the formation of N7-alkylguanine (N7-alkylG) and the imidazole ring opened alkyl-formamidopyrimidine (alkyl-FapyG) lesions. Evaluating the mutagenic effects of N7-alkylG has been challenging due to the instability of the positively charged N7-alkylG. To address this issue, we developed a 2′-fluorine-mediated transition-state destabilization approach, which stabilizes N7-alkylG and prevents spontaneous depurination. We also developed a postsynthetic conversion of 2′-F-N7-alkylG DNA into 2′-F-alkyl-FapyG DNA. Using these methods, we incorporated site-specific N7-methylG and methyl-FapyG into pSP189 plasmid and determined their mutagenic properties in bacterial cells using the supF-based colony screening assay. The mutation frequency of N7-methylG was found to be less than 0.5%. Our crystal structure analysis revealed that N7-methylation did not significantly alter base pairing properties, as evidenced by a correct base pairing between 2′-F-N7-methylG and dCTP in Dpo4 polymerase catalytic site. In contrast, the mutation frequency of methyl-FapyG was 6.3%, highlighting the mutagenic nature of this secondary lesion. Interestingly, all mutations arising from methyl-FapyG in the 5′-GGT(methyl-FapyG)G-3′ context were single nucleotide deletions at the 5′-G of the lesion. Overall, our results demonstrate that 2′-fluorination technology is a useful tool for studying the chemically labile N7-alkylG and alkyl-FapyG lesions.