Repair and translesion synthesis of O6-alkylguanine DNA lesions in human cells

O-6-alkyl-2 '-deoxyguanosine (O-6-alkyl-dG) lesions are among the most mutagenic and prevalent alkylated DNA lesions that are associated with cancer initiation and progression. In this study, using a shuttle vector-based strand-specific PCR-competitive replication and adduct bypass assay in conjunction with tandem MS for product identification, we systematically assessed the repair and replicative bypass of a series of O-6-alkyl-dG lesions, with the alkyl group being a Me, Et, nPr, iPr, nBu, iBu, or sBu, in several human cell lines. We found that the extent of replication-blocking effects of these lesions is influenced by the size of the alkyl groups situated on the O-6 position of the guanine base. We also noted involvement of distinct DNA repair pathways and translesion synthesis polymerases (Pols) in ameliorating the replication blockage effects elicited by the straight- and branched-chain O-6-alkyl-dG lesions. We observed that O-6-methylguanine DNA methyltransferase is effective in removing the smaller alkyl groups from the O-6 position of guanine, whereas repair of the branched-chain lesions relied on nucleotide excision repair. Moreover, these lesions were highly mutagenic during cellular replication and exclusively directed G -> A mutations; Pol eta and Pol zeta participated in error-prone bypass of the straight-chain lesions, whereas Pol kappa preferentially incorporated the correct dCMP opposite the branched-chain lesions. Together, these results uncover key cellular proteins involved in repair and translesion synthesis of O-6-alkyl-dG lesions and provide a better understanding of the roles of these types of lesions in the etiology of human cancer.