Environmental Effects on Guanine-Thymine Mispair Tautomerization Explored with Quantum Mechanical/Molecular Mechanical Free Energy Simulations

DNA bases can adopt energetically
unfavorable tautomeric forms that enable the formation of Watson–Crick-like
(WC-like) mispairs, which have been proposed to give rise to spontaneous
mutations in DNA and misincorporation errors in DNA replication and
translation. Previous NMR and computational studies have indicated
that the population of WC-like guanine-thymine (G‑T) mispairs
depends on the environment, such as the local nucleic acid sequence
and solvation. To investigate these environmental effects, herein
G‑T mispair tautomerization processes are studied computationally
in aqueous solution, in A-form and B-form DNA duplexes, and within
the active site of a DNA polymerase λ variant. The wobble G‑T
(wG‑T), WC-like G‑T*, and WC-like G*-T forms are considered,
where * indicates the enol tautomer of the base. The minimum free
energy paths for the tautomerization from the wG‑T to the WC-like
G‑T* and from the WC-like G‑T* to the WC-like G*-T are
computed with mixed quantum mechanical/molecular mechanical (QM/MM)
free energy simulations. The reaction free energies and free energy
barriers are found to be significantly influenced by the environment.
The wG‑T→​G‑T* tautomerization is predicted
to be endoergic in aqueous solution and the DNA duplexes but slightly
exoergic in the polymerase, with Arg517 and Asn513 providing electrostatic
stabilization of G‑T*. The G‑T*→​G*-T
tautomerization is also predicted to be slightly more thermodynamically
favorable in the polymerase relative to these DNA duplexes. These
simulations are consistent with an experimentally driven kinetic misincorporation
model suggesting that G‑T mispair tautomerization occurs in
the ajar polymerase conformation or concertedly with the transition
from the ajar to the closed polymerase conformation. Furthermore,
the order of the associated two proton transfer reactions is predicted
to be different in the polymerase than in aqueous solution and the
DNA duplexes. These studies highlight the impact of the environment
on the thermodynamics, kinetics, and fundamental mechanisms of G‑T
mispair tautomerization, which plays a role in a wide range of biochemically
important processes.