N7-methyl-2’-deoxyguanosine (m7dG) and N3-methy-2’-deoxyladenosine (m3dA) can be generated in DNA from SN2–type methylation reactions. Owing to the presence of a positive charge located at the methylated nitrogen atom, these modified nucleosides can undergo spontaneous N-glycosidic bond cleavage, which leads to depurination and formation of abasic site. It remains unclear how these two chemically unstable lesions perturb DNA replication and how they are repaired in cells. Here, we applied both transition-state destabilization and carbon replacement strategies with the use of 2'-fluro-m7dG, 7-deaza-m7dG, 2'-fluro-m3dA, 3-deaza-m3dA as stable analogues. Oligodeoxyribonucleotides (ODNs) containing a site-specifically inserted analogue have been synthesized and characterized. Those ODNs will be incorporated into single-stranded M13 plasmids and introduced into E. colicells to investigate their biological consequences regarding how they impede DNA replication and induce mutations. We will also define the identities of translesion synthesis DNA polymerases involved in their replicative bypass and the DNA glycosylases involved in their repair.

Replication studies of chemically stable analogs of N7-methylguanine and N3-methyladenine in Escherichia coli cells

2019 Fall National Meeting

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