Abstract
The tobacco-derived nitrosamines 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N'-nitrosonornicotine (NNN) are known human carcinogens. Following metabolic activation, NNK and NNN can induce a number of DNA lesions, including several 4-(3-pyridyl)-4-oxobut-1-yl (POB) adducts. However, it remains unclear to what extent these lesions affect the efficiency and accuracy of DNA replication and how their replicative bypass is influenced by translesion synthesis (TLS) DNA polymerases. In this study, we investigated the effects of three stable POB DNA adducts (O2-POB-dT, O4-POB-dT, and O6-POB-dG) on the efficiency and fidelity of DNA replication in HEK293T human cells. We found that, when situated in a double-stranded plasmid, O2-POB-dT and O4-POB-dT moderately blocked DNA replication and induced exclusively T→A (∼14.9%) and T→C (∼35.2%) mutations, respectively. On the other hand, O6-POB-dG slightly impeded DNA replication, and this lesion elicited primarily the G→A transition (∼75%) together with a low frequency of the G→T transversion (∼3%). By conducting replication studies in isogenic cells in which specific TLS DNA polymerases (Pols) were deleted by CRISPR-Cas9 genome editing, we observed that multiple TLS Pols, especially Pol η and Pol ζ, are involved in bypassing these lesions. Our findings reveal the cytotoxic and mutagenic properties of specific POB DNA adducts and unravel the roles of several TLS polymerases in the replicative bypass of these adducts in human cells. Together, these results provide important new knowledge about the biological consequences of POB adducts.
Highlights
Tobacco smoking leads to more than 30% of all cancer deaths in developed countries, and ϳ90% of male lung cancer deaths and 75– 80% of female lung cancer deaths in the United States can be attributed to tobacco smoking [1, 2]
Both NNK and NNN can be metabolically activated by the cytochrome P450 family of enzymes, and the resulting metabolites can subsequently react with DNA to yield 4-(3-pyridyl)4-oxobutyl (POB) adducts along with other DNA lesions (Fig. 1) [5, 6]
The competitor vector was co-transfected at fixed molar ratios with the lesion-harboring or control plasmids into HEK293T cells or isogenic cells, with Pol , Pol , Pol , Pol , or both Pol and Pol being depleted by the CRISPR-Cas9 genomic editing method (Fig. S4 shows the sequencing results for confirming the dual knockout of Pol and Pol )
Summary
Tobacco smoking leads to more than 30% of all cancer deaths in developed countries, and ϳ90% of male lung cancer deaths and 75– 80% of female lung cancer deaths in the United States can be attributed to tobacco smoking [1, 2]. The carcinogenic effects of NNN and NNK are thought to be attributed to their metabolic activation, the subsequent formation of DNA adducts, and the ensuing induction of mutations during DNA replication [4] Both NNK and NNN can be metabolically activated by the cytochrome P450 family of enzymes, and the resulting metabolites can subsequently react with DNA to yield 4-(3-pyridyl)4-oxobutyl (POB) adducts along with other DNA lesions (Fig. 1) [5, 6]. Weerasooriya et al [6] found that both the bypass efficiency and the mutagenicity of O2-POB-dT in HEK293T cells were polymerase; TLS, translesion synthesis; SSPCR-CRAB, strand-specific PCRcompetitive replication and adduct bypass; ODN, oligodeoxyribonucleotide; RBE, relative bypass efficiency; MF, mutation frequency; HEK, human embryonic kidney; AGT, O6-alkylguanine-DNA alkyltransferase; SAP, shrimp alkaline phosphatase
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