title: Translesion synthesis of O4-alkylthymidine lesions in human cells.
abstract: Environmental exposure, endogenous metabolism and cancer chemotherapy can give rise to alkylation of DNA, and the resulting alkylated thymidine (alkyldT) lesions were found to be poorly repaired and persistent in mammalian tissues. Unrepaired DNA lesions may compromise genomic integrity by inhibiting DNA replication and inducing mutations in these processes. In this study, we explored how eight O4-alkyldT lesions, with the alkyl group being a Me, Et, nPr, iPr, nBu, iBu, (R)-sBu and (S)-sBu, are recognized by DNA replication machinery in HEK293T human embryonic kidney cells. We found that the O4-alkyldT lesions are moderately blocking to DNA replication, with the bypass efficiencies ranging from 20 to 33% in HEK293T cells, and these lesions induced substantial frequencies T→C transition mutation. We also conducted the replication experiments in the isogenic cells where individual translesion synthesis (TLS) DNA polymerases were depleted by the CRISPR/Cas9 genome editing method. Our results showed that deficiency in Pol η or Pol ζ, but not Pol κ or Pol ι, led to pronounced drops in bypass efficiencies for all the O4-alkyldT lesions except O4-MedT. In addition, depletion of Pol ζ resulted in significant decreases in T→C mutation frequencies for all the O4-alkyldT lesions except O4-MedT and O4-nBudT. Thus, our study provided important new knowledge about the cytotoxic and mutagenic properties of the O4-alkyldT lesions and defined the roles of TLS polymerases in bypassing these lesions in human cells.
authors.affiliation: Department of Chemistry, University of California, Riverside, CA 92521-0403, USA.
authors.forename: Jun
authors.initials: J
authors.lastname: Wu
authors.affiliation: Department of Chemistry, University of California, Riverside, CA 92521-0403, USA.
authors.forename: Lin
authors.initials: L
authors.lastname: Li
authors.affiliation: Environmental Toxicology Graduate Program, University of California, Riverside, CA 92521-0403, USA.
authors.forename: Pengcheng
authors.initials: P
authors.lastname: Wang
authors.affiliation: Department of Chemistry, University of California, Riverside, CA 92521-0403, USA.
authors.forename: Changjun
authors.initials: C
authors.lastname: You
authors.affiliation: Environmental Toxicology Graduate Program, University of California, Riverside, CA 92521-0403, USA.
authors.forename: Nicole L
authors.initials: NL
authors.lastname: Williams
authors.affiliation: Department of Chemistry, University of California, Riverside, CA 92521-0403, USA Yinsheng.Wang@ucr.edu.|Environmental Toxicology Graduate Program, University of California, Riverside, CA 92521-0403, USA.
authors.forename: Yinsheng
authors.initials: Y
authors.lastname: Wang
mesh_terms: D000478:Alkylation; D064113:CRISPR-Cas Systems; D004249:DNA Damage; D004260:DNA Repair; D004261:DNA Replication; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D000072669:Gene Editing; D057809:HEK293 Cells; D006801:Humans; D009154:Mutation; D013936:Thymidine / Q000737:chemistry / Q000378:metabolism*
chemical_list: D004259:DNA-Directed DNA Polymerase; D013936:Thymidine