Replicative Bypass Studies of α-Anomeric Lesions of 2′-Deoxyribonucleosides in Vitro

Abstract

Genomic integrity is constantly challenged by a variety of endogenous and exogenous DNA damaging agents, which can lead to the formation of 104-105 DNA lesions per cell per day. Reactive oxygen species (ROS) represent a major type of DNA damaging agent. Specifically, a hydroxyl radical can attack the C1' position of 2-deoxyribose, and the ensuing carbon-centered radical, if improperly repaired, can cause the inversion of stereochemical configuration at the C1' to give α-anomeric lesions. In this study, we assessed the replicative bypass of α-dA, α-dT, α-dC, and α-dG in template DNA by conducting primer extension assays with the use of purified translesion synthesis DNA polymerases. Our results revealed that human polymerase (Pol) η, but not human Pol κ, Pol ι, or yeast Pol ζ, was capable of bypassing all of the α-dN lesions and extending the primer to generate full-length replication products. Data from steady-state kinetic measurements showed that Pol η was the most efficient in inserting the correct nucleotides opposite the modified nucleosides, with the relative efficiencies of nucleotide incorporation following the order of α-dA > α-dG > α-dT > α-dC. Additionally, human Pol η was found to misincorporate dTMP opposite α-dT and dCMP opposite α-dC at frequencies of 66% and 24%, respectively, whereas α-dA and α-dG were weakly miscoding. These findings provided important knowledge about the effects these α-dN lesions have on the fidelity and efficiency of DNA replication mediated by human Pol η.