Exploring the Role of the Third Active Site Metal Ion in DNA Polymerase η with QM/MM Free Energy Simulations

The enzyme human DNA polymerase η (Pol η) is critical for bypassing lesions during DNA replication. In addition to the two Mg²⁺ ions aligning the active site, experiments suggest that a third Mg²⁺ ion could play an essential catalytic role. Herein the role of this third metal ion is investigated with quantum mechanical/molecular mechanical (QM/MM) free energy simulations of the phosphoryl transfer reaction and a proposed self-activating proton transfer from the incoming nucleotide to the pyrophosphate leaving group. The simulations with only two metal ions in the active site support a sequential mechanism, with phosphoryl transfer followed by relatively fast proton transfer. The simulations with three metal ions in the active site suggest that the third metal ion may play a catalytic role through electrostatic interactions with the leaving group. These electrostatic interactions stabilize the product, making the phosphoryl transfer reaction more thermodynamically favorable with a lower free energy barrier relative to the activated state corresponding to the deprotonated 3′OH nucleophile, and also inhibit the subsequent proton transfer. The possibility that Mg²⁺-bound hydroxide acts as the base deprotonating the 3′OH nucleophile is also explored.