Cytotoxic and mutagenic properties of O4-alkylthymidine lesions in Escherichia coli cells.

Abstract

Due to the abundant presence of alkylating agents in living cells and the environment, DNA alkylation is generally unavoidable. Among the alkylated DNA lesions, O4-alkylthymidine (O4-alkyldT) are known to be highly mutagenic and persistent in mammalian tissues. Not much is known about how the structures of the alkyl group affect the repair and replicative bypass of the O4-alkyldT lesions, or how the latter process is modulated by translesion synthesis polymerases. Herein, we synthesized oligodeoxyribonucleotides harboring eight site-specifically inserted O4-alkyldT lesions and examined their impact on DNA replication in Escherichia coli cells. We showed that the replication past all the O4-alkyldT lesions except (S)- and (R)-sBudT was highly efficient, and these lesions directed very high frequencies of dGMP misincorporation in E. coli cells. While SOS-induced DNA polymerases play redundant roles in bypassing most of the O4-alkyldT lesions, the bypass of (S)- and (R)-sBudT necessitated Pol V. Moreover, Ada was not involved in the repair of any O4-alkyldT lesions, Ogt was able to repair O4-MedT and, to a lesser extent, O4-EtdT and O4-nPrdT, but not other O4-alkyldT lesions. Together, our study provided important new knowledge about the repair of the O4-alkyldT lesions and their recognition by the E. coli replication machinery.