Abstract
O6-alkyl-2'-deoxyguanosine (O6-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 O6-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 O6 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 O6-alkyl-dG lesions. We observed that O6-methylguanine DNA methyltransferase is effective in removing the smaller alkyl groups from the O6 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 η and Pol ζ participated in error-prone bypass of the straight-chain lesions, whereas Pol κ preferentially incorporated the correct dCMP opposite the branched-chain lesions. Together, these results uncover key cellular proteins involved in repair and translesion synthesis of O6-alkyl-dG lesions and provide a better understanding of the roles of these types of lesions in the etiology of human cancer.
Highlights
O6-alkyl-2-deoxyguanosine (O6-alkyl-dG) lesions are among the most mutagenic and prevalent alkylated DNA lesions that are associated with cancer initiation and progression
We employed a previously published shuttle vector– based SSPCR-CRAB assay [35] (Fig. S1) along with the restriction digestion and post-labeling method (Fig. 2A), to examine the core repair and lesion bypass machineries that counteract the adverse biological effects conferred by O6-alkyl-dG lesions and to unveil how the size and shape of an alkyl group influence the repair and replicative bypass of these lesions in human cells
We examined the roles of MGMT and nucleotide excision repair (NER) in repairing these lesions, assessed the replication blockage and mutagenic effects of these lesions, and investigated how replication across these lesions is modulated by translesion synthesis (TLS) DNA polymerases
Summary
O6-alkyl-2-deoxyguanosine (O6-alkyl-dG) lesions are among the most mutagenic and prevalent alkylated DNA lesions that are associated with cancer initiation and progression. We observed that O6-methylguanine DNA methyltransferase is effective in removing the smaller alkyl groups from the O6 position of guanine, whereas repair of the branched-chain lesions relied on nucleotide excision repair These lesions were highly mutagenic during cellular replication and exclusively directed G3A mutations; Pol and Pol participated in error-prone bypass of the straight-chain lesions, whereas Pol preferentially incorporated the correct dCMP opposite the branched-chain lesions. Several previous studies characterized the biological consequences of O6-alkyl-dG lesions in Escherichia coli and mammalian cells (24 –26) Both O6-methyl-dG (O6-Me-dG) and O6-ethyl-dG (O6-Et-dG) may exert cytotoxic effects via mismatch repair-induced double-strand breaks [26], they do not significantly block DNA synthesis [27, 28]. We examined the roles of DNA repair pathways and TLS polymerases in modulating the replicative bypass of O6-alkyl-dG lesions
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