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

Abstract

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.