Polymerase Local Deformations Revealed by Dynamics Simulations of DNA Polymerase with DNA Mismatches at the Primer Terminus

Nanosecond dynamics simulations for DNA polymerase /DNA complexes with three mismatched base pairs, either GG, CA, or CC (primer/template) at the DNA polymerase active site, are performed to investigate the mechanism of polymerase opening and how the mispairs may affect the DNA extension step; these trajectories are compared to the behavior of a DNA polymerase /DNA complex with the correct base pair GC, and assessed with the aid of targeted molecular dynamics simulations of all systems from the closed to the open enzyme state. DNA polymerase conformational changes (subdomain closing and opening) have been suggested to play a critical role in DNA synthesis fidelity since these changes are associated with the formation of the substrate binding pocket for the nascent base pair. Here we observe different large C-terminal subdomain (thumb) opening motions in the simulations of DNA polymerase with GC versus GG base pairs. Whereas the conformation of DNA polymerase in the former approaches the observed open state in the crystal structures, the enzyme in the latter does not. Interestingly, rotation of Arg258 toward Asp192 which coordinates both active site metals in the closed `active' complex, occurs rapidly in the GG simulation. We have previously suggested that this rotation is a key slow step in the closed to open transition. Targeted molecular dynamics simulations also point to a unique pathway for Arg258 rotation in the GG mispair complex. Further structural analysis in the active site reveals distorted geometries of all these mispair complexes examined. The hierarchy of these distortions (GG > CC > CA) parallels the experimentally deduced inability of DNA polymerase to extend these mispairs. These studies on the DNA polymerase opening provide structural and dynamic insights, helping to explain experimental results regarding DNA extension following misincorporation.

Click to go back to the publication list