Nucleotide addition in different families of nucleic acid polymerases is generally thought to occur by a conserved mechanism but the details of that mechanism have remained elusive to experimental and computational probes. Multiple proton transfers throughout a solvent-exposed active site and the complex electronic structure of a metal-coordinated nucleotide triphosphate (NTP) substrate have limited the ability of simulations to aid the interpretation of experiments. To overcome these challenges, we have applied recent advances in hybrid quantum mechanics/molecular mechanics (QM/MM) simulations to test mechanistic hypotheses in DNA polymerase and particularly, how the mechanism is influenced by a third Mg<sup>2+</sup> ion that was recently observed in the active site. We combine simulations of free energy surfaces for nucleotide addition with simulations of pKa for active site moieties to gauge the likelihood of certain mechanisms versus others. The results support a mechanism where proton abstraction from the 3’-OH is concerted with nucleophilic attack on the NTP and a third Mg<sup>2+</sup> is mildly catalytic. We will discuss how additional experiments can further distinguish between mechanistic alternatives.

Effects of metal cations on mechanisms of nucleotide addition in DNA polymerase

2019 Fall National Meeting

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