What is the free energy source enabling high-fidelity DNA polymerases (pols) to favor incorporation of correct over incorrect base pairs by 10(3)- to 10(4)-fold, corresponding to free energy differences of Delta Delta G(inc) similar to 5.5-7 kcal/mol? Standard Delta Delta G degrees values (similar to 0.3 kcal/mol) calculated from melting temperature measurements comparing matched vs. mismatched base pairs at duplex DNA termini are far too low to explain pol accuracy. Earlier analyses suggested that pol active-site steric constraints can amplify DNA free energy differences at the transition state (kinetic selection). A recent paper Olson et al. (2013) J Am Chem Soc 135: 1205-1208 used Vent pol to catalyze incorporations in the presence of inorganic pyrophosphate intended to equilibrate forward (polymerization) and backward (pyrophosphorolysis) reactions. A steady-state leveling off of incorporation profiles at long reaction times was interpreted as reaching equilibrium between polymerization and pyrophosphorolysis, yielding apparent Delta G degrees = -RT In K-eq, indicating Delta Delta G degrees of 3.5-7 kcal/mol, sufficient to account for pol accuracy without need of kinetic selection. Here we perform experiments to measure and account for pyrophosphorolysis explicitly. We show that forward and reverse reactions attain steady states far from equilibrium for wrong incorporations such as G opposite T. Therefore, Delta Delta G degrees(inc) values obtained from such steady-state evaluations of K-eq are not dependent on DNA properties alone, but depend largely on constraints imposed on right and wrong substrates in the polymerase active site.
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