Translesion synthesis of O(4)-alkylthymidine lesions in human cells

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 O(4)-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 O(4)-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 O(4)-alkyldT lesions except O(4)-MedT. In addition, depletion of Pol ζ resulted in significant decreases in T→C mutation frequencies for all the O(4)-alkyldT lesions except O(4)-MedT and O(4)-nBudT. Thus, our study provided important new knowledge about the cytotoxic and mutagenic properties of the O(4)-alkyldT lesions and defined the roles of TLS polymerases in bypassing these lesions in human cells.