DNA, the key component of genetic material, is constantly under attack by exogenous environmental factors, as well as natural cellular metabolites, resulting in an array of covalent DNA modifications. Alkylation represents a common type of DNA modification, where the size and position of alkyl group may vary depending on the chemical nature of the alkylating agents. These DNA modifications may perturb the flow of genetic information by impeding DNA replication and transcription and eliciting mutations during the processes. To maintain genome stability, cells are equipped with DNA repair and damage tolerance mechanism in all domains of life. Translesion synthesis (TLS) is one damage tolerance mechanism where specialized DNA polymerase can synthesize across the damage or extend from it when replicative DNA polymerase is blocked. While the role of TLS polymerases in replication is well documented, it’s largely unknown whether TLS polymerase would assist transcriptional bypass. Recently, growing evidence revealed the versatile roles of TLS polymerases aside from canonical TLS. For example, human DNA polymerase h has reverse transcriptase activity in cellular environment. In this study, we employed our recently developed competitive transcription and adduct bypass assay (CTAB), where non-replicative double-stranded plasmids harboring site-specifically inserted N2-alkyl-dG lesions are used, to assess their transcriptional bypass efficiency and mutation frequency in HEK293T human embryonic kidney cells. We also conducted the transcription experiments in the isogenic cells where individual translesion synthesis DNA polymerases were depleted by the CRISPR-Cas9 genome editing method. Thus far, our results showed that, N2-alkyl-dG lesions are moderately blocking to transcription, and deficiency of Pol k and Pol i, but not Pol z, further exacerbate the effect. In addition, simultaneous ablation of Pol k and Pol i resulted in diminished transcriptional bypass efficiency than depletion either polymerase alone, indicating that Pol k and Pol i function in separate pathways. Together, our results provide new insights into transcriptional perturbation of N2-alkyl-dG lesions, and the biological functions of TLS polymerases.

Human translesion synthesis DNA polymerases function in transcriptional bypass and repair of N2-alkyl-2'-deoxyguanosine lesions

2019 Fall National Meeting

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