Alkylating agents are commonly found in the environment, and previous studies showed that DNA ethylation might play an important role in cigarette smoke-induced cancer formation. Particularly, O4-, N3-, and O2-ethylthymidine (O4-, N3-, and O2-EtdT) could be detected in DNA from smokers. However, the regio-specific effects of the ethylated thymidine lesions on DNA replication have never been explored. We synthesized oligodeoxyribonucleotides (ODNs) containing a site-specifically incorporated O4-, N3, and O2-EtdT, 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. In keeping with previous studies showing that O4-EtdT is a promutagenic lesion, our results demonstrated that, among these three Et-dT lesions, O4-EtdT could be bypassed with the highest efficiency and the greatest mutation frequency, leading to almost 60% T?C mutation. On the other hand, N3-EtdT gave rise to the highest frequency of �1 deletion at the lesion site. The O2-EtdT, however, has comparable bypass efficiency and T?C mutation frequency as N3-EtdT, while the frequency of -1 deletion is similar as that of O4-EtdT. Furthermore, Pol V was found to play a crucial role in the translesion synthesis of these three lesions in E. coli cells. Together, our studies revealed the common and unique effects of the three regioisomers of Et-dT lesions on DNA replication in E. coli cells.

In vivo replication study of ethylated thymidine adducts

246th National Meeting (2013)

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