Tobacco-Specific Nitrosamine-Derived O²-Alkylthymidines Are Potent Mutagenic Lesions in SOS-Induced Escherichia coli

Abstract

To investigate the biological effects of the O2-alkylthymidines induced by the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), we have replicated a plasmid containing O2-methylthymidine (O2-Me-dT) or O2-[4-(3-pyridyl-4-oxobut-1-yl]thymidine (O2-POB-dT) in Escherichia coli with specific DNA polymerase knockouts. High genotoxicity of the adducts was manifested in the low yield of transformants from the constructs, which was 2–5% in most strains but increased 2–4-fold with SOS. In the SOS-induced wild type E. coli, O2-Me-dT and O2-POB-dT induced 21% and 56% mutations, respectively. For O2-POB-dT, the major type of mutation was T → G followed by T → A, whereas for O2-Me-dT, T → G and T → A occurred in equal frequency. For both lesions, T → C also was detected in low frequency. The T → G mutation was reduced in strains with deficiency in any of the three SOS polymerases. By contrast, T → A was abolished in the pol V– strain, while its frequency in other strains remained unaltered. This suggests that pol V was responsible for the T → A mutations. The potent mutagenicity of these lesions may be related to NNK mutagenesis and carcinogenesis.

DNA Polymerases η and ζ Combine to Bypass O2-[4-(3-Pyridyl)-4-oxobutyl]thymine, a DNA Adduct Formed from Tobacco Carcinogens

“…Perhaps this structure explains the mutagenic incorporation of dGTP opposite O 2 alkyl-dT by pol V in E. coli . 42,73 However, the preference for the incorporation of dT and dA opposite O 2 -alkyl-dG in humans cells is not evident. Structural and functional studies must be undertaken to elucidate the interactions that control the identity of the nucleotide inserted opposite O 2 -alkyl-dTs.…”

“…42,46 The oligodeoxynucleotide containing 6-carboxyfluorescein was purchased from Integrated DNA Technologies (Coralville, Iowa). The sequences of the oligodeoxynucleotides are shown in Chart 1.…”

“…While dA is inserted opposite O 2 -Me-dT and O 2 -POB-dT most often, the major mutagenic process is the incorporation of dC opposite the adducts. 42 More recently, the bypass of O 2 -Me-dT and O 2 -POB-dT were evaluated in mammalian cells. 44 Both dATP and dTTP were incorporated opposite the adducts, and pol η , ξ , and REV1 are the primary polymerases involved.…”

Context Matters: Contribution of Specific DNA Adducts to the Genotoxic Properties of the Tobacco-Specific Nitrosamine NNK

“…68 Consistent with these observation, this adduct is a block to replication in E. coli and HEK293T cells. 51, 52 The absence of NER in the UV5 CHO cell line also resulted in an increase in NNKOAc-induced hprt mutations, specifically TA to AT mutations, providing support for the importance of O 2 -pobdThd in the overall mutagenic activity of pyridyloxobutylating agents. 68 Pol ζ and other Y-family polymerases are thought to play a major role in the error-prone and error-free bypass of this bulky lesion.…”

“…The pyridyloxobutyl DNA adduct, O 2 -pobdThd, is modestly mutagenic in bacterial strains; TA to GC was the major mutation (37%) compared to TA to AT (12%) induced by this adduct. 51 The pyridyloxobutyl analog was more miscoding than the corresponding methyl analog. 51 …”