DNA alkylation is almost unavoidable due to the abundant presence of alkylating agents in the environment and within cells. Alkylation damage poses considerable threats to human health, particularly the minor groove O2-alkyl thymidine lesions, which are known to be resistant to DNA repair and persistent in mammalian tissues. However, it has not been systematically explored how the size and branching of the alkyl group attached to the O2 of thymidine affect DNA replication. Hence, we synthesized oligodeoxyribonucleotides (ODNs) containing a site-specifically incorporated O2-AlkyldT, ligated these ODNs into single-stranded M13 vectors, and assessed how these lesions compromise the efficiency and fidelity of DNA replication in Escherichia coli cells. Our results revealed that these O2-alkyldT lesions are highly blocking, with the highest bypass efficiency being only around 30% in SOS-induced E. coli cells. In addition, the bypass efficiency decreased with the size of alkyl group. For most O2-alkyldT lesions, all three types of base substitutions, i.e., T?A, T?C and T?G mutations, were observed. However, a TG?CA mutation was uniquely observed for O2-iPrdT in all E. coli strains. Our results also revealed that Pol V played a crucial role in the translesion synthesis of these O2-alkyldT lesions in E. coli cells, especially in terms of T?A and T?G mutations; and pol IV is indispensable for the T?G mutation.

In vivo replication studies of <i>O</i><sup>2</sup>-alkyl thymidine adducts

248th National Meeting (2014)

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In vivo replication studies of O2-alkyl thymidine adducts

In vivo replication studies of O²-alkyl thymidine adducts