High Sensitivity of Human Translesion DNA Synthesis Polymerase κ to Variation in O⁶-Carboxymethylguanine Structures

Carboxymethylation of DNA, including the formation of the DNA adduct O⁶-carboxymethylguanine ( O⁶-CMG), is associated with lifestyle factors, such as diet. It can impede replicative polymerases (Pols) and lead to replication fork stalling, or an alternative means for replication to proceed by translesion DNA synthesis (TLS). TLS requires specialized DNA Pols characterized by open and preformed active sites capable of preferential bypass of alkylated DNA adducts but that have high error rates, leading to mutations. Human TLS Pols can bypass O⁶-CMG with varying degrees of accuracy, but it is not known how the chemical structure of the O⁶-CMG adduct influences polymerase proficiency or fidelity. To better understand how adduct structure determines dNTP selection at lesion sites, we prepared DNA templates with a series of O⁶-CMG structural analogs and compared the primer extension patterns of Y- and X-family Pols in response to these modifications. The results indicate that the structure of the DNA adduct had a striking effect on dNTP selection by Pol κ and that an increased steric size influences the fidelity of Pol η, whereas Pol ι and β function were only marginally affected. To test the hypothesis that specific hydrogen bonding interactions between the templating base and the incoming dNTP are a basis of this selection, we modeled the structural analogs with incoming dNTP in the Pol κ active site. These data indicate that the base pairing geometry and stabilization by a dense hydrogen bonding network are important molecular features for dNTP incorporation, providing a basis for understanding error-free bypass of O⁶-CMG by Pol κ.