Abstract
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 O6-alkyl-2'-deoxyguanosine (O6-alkyl-dG) adducts, i.e. O6-pyridyloxobutyl-dG (O6-POB-dG) and O6-pyridylhydroxybutyl-dG (O6-PHB-dG), derived from tobacco-specific nitrosamines, and O6-carboxymethyl-dG (O6-CM-dG), induced by endogenous N-nitroso compounds. We also investigated two neutral analogs of O6-CM-dG, i.e. O6-aminocarbonylmethyl-dG (O6-ACM-dG) and O6-hydroxyethyl-dG (O6-HOEt-dG). We found that, in Escherichia coli cells, these lesions mildly (O6-POB-dG), moderately (O6-PHB-dG), or strongly (O6-CM-dG, O6-ACM-dG, and O6-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 O6-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 O6-alkyl-dG lesions investigated. DNA polymerase IV (Pol IV) and Pol V, however, were uniquely required for inducing the G → A transition for O6-CM-dG exposure. Together, our study expands our knowledge about the recognition of important NOC-derived O6-alkyl-dG lesions by the E. coli DNA replication machinery.
Highlights
N-Nitroso compounds (NOCs) are common DNA-alkylating agents, are abundantly present in food and tobacco, and can be generated endogenously
It was reported that appreciable levels of O4-POB-dT along with the above-mentioned O2-POB-dT and O6-POB-dG could be formed in cultured mammalian cells upon exposure to 4-(acetoxymethylnitrosamino)-1(3-pyridyl)-1-butanone, a model pyridyloxobutylating agent [21, 22]
A previously described competitive replication and adduct bypass (CRAB) assay (Fig. 3 and Fig. S8) was employed to determine the impacts of these lesions on DNA replication in E. coli cells and to determine how replication across these lesions is influenced by SOS-induced DNA polymerases [39, 40]
Summary
N-Nitroso compounds (NOCs) are common DNA-alkylating agents, are abundantly present in food and tobacco, and can be generated endogenously. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Metabolic activation of NNK and NNN via cytochrome P450 enzymes can produce reactive intermediates that can react with DNA to yield POB and/or PHB adducts along with other DNA lesions, which may induce mutations during DNA replication (5, 8 –15). Another type of extensively investigated promutagenic lesions, carboxymethyl (CM) adducts, can be induced in DNA through metabolic activation of endogenous NOCs, e.g. N-nitroso bile acid conjugate or N-nitrosoglycine (16 –18). O6-CM-dG could be induced in cultured human cells upon treatment with azasertrosonornicotine; CM, carboxymethyl; Pol, polymerase; ESI, electrospray ionization; ODN, oligodeoxyribonucleotide; PNK, polynucleotide kinase
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