Genomic integrity is constantly challenged by exposure to environmental and endogenous genotoxic agents. Reactive oxygen species (ROS) represent one of the most common types of DNA damaging agents. While ROS mainly induce single-nucleobase lesions, epimeric 2-deoxyribose lesions can also be induced upon hydrogen atom abstraction from the C1, C3, or C4 carbon and the subsequent incorrect chemical repair of the resulting carbon-centered radicals. Herein, we investigated the replicative bypass of the C1- and C3-epimeric lesions of the four 2-deoxynucleosides in HEK293T human embryonic kidney epithelial cells. Our results revealed distinct bypass efficiencies and mutagenic properties of these two types of epimeric lesions. Replicative bypasses of all C1-epimeric lesions except α-dA are mutagenic in HEK293T cells, and their mutagenic properties are further modulated by translesion synthesis (TLS) DNA polymerases. By contrast, none of the four C3-epimeric lesions are mutagenic, and the replicative bypass of these lesions is not compromised upon depletion of polymerase η, ι, κ, or ζ. Together, our results provide important new knowledge about the cytotoxic and mutagenic properties of C1 and C3 epimeric lesions, and reveal the roles of TLS DNA polymerases in bypassing these lesions in human cells.

Exogenous and endogenous chemicals can react with DNA to produce DNA lesions that may block DNA replication. Not much is known about the roles of polymerase (Pol) ν and Pol θ in translesion synthesis (TLS) in cells. Here we examined the functions of these two polymerases in bypassing major-groove O6-alkyl-2-deoxyguanosine (O6-alkyl-dG) and minor-groove N2-alkyl-dG lesions in human cells, where the alkyl groups are ethyl, n-butyl (nBu), and, for O6-alkyl-dG, pyridyloxobutyl. We found that Pol ν and Pol θ promote TLS across major-groove O6-alkyl-dG lesions. O6-alkyl-dG lesions mainly induced G→A mutations that were modulated by the two TLS polymerases and the structures of the alkyl groups. Simultaneous ablation of Pol ν and Pol θ resulted in diminished mutation frequencies for all three O6-alkyl-dG lesions. Depletion of Pol ν alone reduced mutations only for O6-nBu-dG, and sole loss of Pol θ attenuated the mutation rates for O6-nBu-dG and O6-pyridyloxobutyl-dG. Replication across the two N2-alkyl-dG lesions was error-free, and Pol ν and Pol θ were dispensable for their replicative bypass. Together, our results provide critical knowledge about the involvement of Pol ν and Pol θ in bypassing alkylated guanine lesions in human cells.

To cope with unrepaired DNA lesions, cells are equipped with DNA damage tolerance mechanisms, including translesion synthesis (TLS). While TLS polymerases are well documented in facilitating replication across damaged DNA templates, it remains unknown whether TLS polymerases participate in transcriptional bypass of DNA lesions in cells. Herein, we employed the competitive transcription and adduct bypass assay to examine the efficiencies and fidelities of transcription across N2-alkyl-2-deoxyguanosine (N2-alkyl-dG, alkyl = methyl, ethyl, n-propyl, or n-butyl) lesions in HEK293T cells. We found that N2-alkyl-dG lesions strongly blocked transcription and elicited CC → AA tandem mutations in nascent transcripts, where adenosines were misincorporated opposite the lesions and their adjacent 5 nucleoside. Additionally, genetic ablation of Pol η, but not Pol κ, Pol ι, or Pol ζ, conferred marked diminutions in the transcriptional bypass efficiencies of the N2-alkyl-dG lesions, which is exacerbated by codepletion of Rev1 in Pol η-deficient background. We also observed that the repair of N2-nBu-dG was not pronouncedly affected by genetic depletion of Pol η or Rev1. Hence, our results provided insights into transcriptional perturbations induced by N2-alkyl-dG lesions and expanded the biological functions of TLS DNA polymerases.

Endogenous metabolites and exogenous chemicals can induce covalent modifications on DNA, producing DNA lesions. The N2 of guanine was shown to be a common alkylation site in DNA; however, not much is known about the influence of the size of the alkyl group in N2-alkyldG lesions on cellular DNA replication or how translesion synthesis (TLS) polymerases modulate DNA replication past these lesions in human cells. To answer these questions, we employ a robust shuttle vector method to investigate the impact of four N2-alkyldG lesions (i.e., with the alkyl group being a methyl, ethyl, n-propyl, or n-butyl group) on DNA replication in human cells. We find that replication through the N2-alkyldG lesions was highly efficient and accurate in HEK293T cells or isogenic CRISPR-engineered cells with deficiency in polymerase (Pol) ζ or Pol η. Genetic ablation of Pol ι, Pol κ, or Rev1, however, results in decreased bypass efficiencies and elicits substantial frequencies of G → A transition and G → T transversion mutations for these lesions. Moreover, further depletion of Pol ζ in Pol κ- or Pol ι-deficient cells gives rise to elevated rates of G → A and G → T mutations and substantially decreased bypass efficiencies. Cumulatively, we demonstrate that the error-free replication past the N2-alkyldG lesions is facilitated by a specific subset of TLS polymerases, and we find that longer alkyl chains in these lesions induce diminished bypass efficiency and fidelity in DNA replication.

Environmental, endogenous and therapeutic alkylating agents can react with internucleotide phosphate groups in DNA to yield alkyl phosphotriester (PTE) adducts. Alkyl-PTEs are induced at relatively high frequencies and are persistent in mammalian tissues; however, their biological consequences in mammalian cells have not been examined. Herein, we assessed how alkyl-PTEs with different alkyl group sizes and stereochemical configurations (S P and R P diastereomers of Me and nPr) affect the efficiency and fidelity of transcription in mammalian cells. We found that, while the R P diastereomer of Me- and nPr-PTEs constituted moderate and strong blockages to transcription, respectively, the S P diastereomer of the two lesions did not appreciably perturb transcription efficiency. In addition, none of the four alkyl-PTEs induced mutant transcripts. Furthermore, polymerase η assumed an important role in promoting transcription across the S P-Me-PTE, but not any of other three lesions. Loss of other translesion synthesis (TLS) polymerases tested, including Pol κ, Pol ι, Pol ξ and REV1, did not alter the transcription bypass efficiency or mutation frequency for any of the alkyl-PTE lesions. Together, our study provided important new knowledge about the impact of alkyl-PTE lesions on transcription and expanded the substrate pool of Pol η in transcriptional bypass.

Chemically induced DNA adducts can lead to mutations and cancer. Unfortunately, because common analytical methods (e.g., liquid chromatography-mass spectrometry) require adducts to be digested or liberated from DNA before quantification, information about their positions within the DNA sequence is lost. Advances in nanopore sequencing technologies allow individual DNA molecules to be analyzed at single-nucleobase resolution, enabling us to study the dynamic of epigenetic modifications and exposure-induced DNA adducts in their native forms on the DNA strand. We applied and evaluated the commercially available Oxford Nanopore Technology (ONT) sequencing platform for site-specific detection of DNA adducts and for distinguishing individual alkylated DNA adducts. Using ONT and the publicly available ELIGOS software, we analyzed a library of 15 plasmids containing site-specifically inserted O6- or N2-alkyl-2-deoxyguanosine lesions differing in sizes and regiochemistries. Positions of DNA adducts were correctly located, and individual DNA adducts were clearly distinguished from each other.