Extensive free-energy simulations identify water as the base in nucleotide addition by DNA polymerase

Transphosphorylation of nucleotide triphosphates is the central reaction in DNA replication by DNA polymerase as well as many other biological processes. Despite its importance, the microscopic chemical mechanism of transphosphorylation of nucleotide triphosphates is, in most cases, unknown. Here we use extensive simulations of DNA polymerase η to test mechanistic hypotheses. We systematically survey the reactive space by calculating 2D free-energy surfaces for 10 different plausible mechanisms that have been proposed. We supplement these free-energy surfaces with calculations of pK_a for a number of potentially acidic protons in different states relevant to the catalytic cycle. We find that among all of the conditions that we test, the smallest activation barrier occurs for a reaction where a Mg²⁺-coordinated water deprotonates the nucleophilic 3'-OH, and this deprotonation is concerted with the phosphoryl transfer. The presence of a third Mg²⁺ in the active site lowers the activation barrier for the water-as-base mechanism, as does protonation of the pyrophosphate leaving group, which is consistent with general acid catalysis. The results demonstrate the value of simulations, when used in conjunction with experimental data, to help establish a microscopic chemical mechanism in a complex environment.