DNA replication studies of N-nitroso compound-induced O 6-alkyl-2'-deoxyguanosine lesions in Escherichia coli.

N-Nitroso compounds (NOCs) are common DNA-alkylating agents, are abundantly present in food and tobacco, and can also be generated endogenously. Metabolic activation of some NOCs can give rise to carboxymethylation and pyridyloxobutylation/pyridylhydroxybutylation of DNA, which are known to be carcinogenic and can lead to gastrointestinal and lung cancer, respectively. Herein, using the competitive replication and adduct bypass (CRAB) assay, along with MS- and NMR-based approaches, we assessed the cytotoxic and mutagenic properties of three O 6-alkyl-2'-deoxyguanosine (O 6-alkyl-dG) adducts, i.e. O 6-pyridyloxobutyl-dG (O 6-POB-dG) and O 6-pyridylhydroxybutyl-dG (O 6-PHB-dG), derived from tobacco-specific nitrosamines, and O 6-carboxymethyl-dG (O 6-CM-dG), induced by endogenous N-nitroso compounds. We also investigated two neutral analogs of O 6-CM-dG, i.e. O 6-aminocarbonylmethyl-dG (O 6-ACM-dG) and O 6-hydroxyethyl-dG (O 6-HOEt-dG). We found that, in Escherichia coli cells, these lesions mildly (O 6-POB-dG), moderately (O 6-PHB-dG), or strongly (O 6-CM-dG, O 6-ACM-dG, and O 6-HOEt-dG) impede DNA replication. The strong blockage effects of the last three lesions were attributable to the presence of hydrogen-bonding donor(s) located on the alkyl functionality of these lesions. Except for O 6-POB-dG, which also induced a low frequency of G → T transversions, all other lesions exclusively stimulated G → A transitions. SOS-induced DNA polymerases played redundant roles in bypassing all the O 6-alkyl-dG lesions investigated. DNA polymerase IV (Pol IV) and Pol V, however, were uniquely required for inducing the G → A transition for O 6-CM-dG exposure. Together, our study expands our knowledge about the recognition of important NOC-derived O 6-alkyl-dG lesions by the E. coli DNA replication machinery.