Cytotoxic and mutagenic properties of regioisomeric O2-, N3- and O4-ethylthymidines in bacterial cells

Exposure to environmental agents and endogenous metabolism can both give rise to DNA alkylation. Thymine is known to be alkylated at O 2, N3 and O 4 positions; however, it remains poorly explored how the regioisomeric alkylated thymidine lesions compromise the flow of genetic information by perturbing DNA replication in cells. Herein, we assessed the differential recognition of the regioisomeric O 2-, N3- and O 4-ethylthymidine (O 2-, N3- and O 4-EtdT) by the DNA replication machinery of Escherichia coli cells. We found that O 4-EtdT did not inhibit appreciably DNA replication, whereas O 2- and N3-EtdT were strongly blocking to DNA replication. In addition, O 4-EtdT induced a very high frequency of T→C mutation, whereas nucleotide incorporation opposite O 2- and N3-EtdT was promiscuous. Replication experiments with the use of polymerase-deficient cells revealed that Pol V constituted the major polymerase for the mutagenic bypass of all three EtdT lesions, though Pol IV also contributed to the T→G mutation induced by O 2- and N3-EtdT. The distinct cytotoxic and mutagenic properties of the three regioisomeric lesions could be attributed to their unique chemical properties.