The Energetic Difference between Synthesis of Correct and Incorrect Base Pairs Accounts for Highly Accurate DNA Replication

Abstract

To better understand the energetics of accurate DNA replication, we directly measured ΔGO for the incorporation of a nucleotide into elongating dsDNA in solution (ΔGOincorporation). Direct measurements of the energetic difference between synthesis of correct and incorrect base pairs found it to be much larger than previously believed (average ΔΔGOincorporation = 5.2±1.34 kcal mol−). Importantly, these direct measurements indicate that ΔΔGOincorporation alone can account for the energy required for highly accurate DNA replication. Evolutionarily, these results indicate that the earliest polymerases did not have to evolve sophisticated mechanisms to replicate nucleic acids, they may have only had to take advantage of the inherently more favorable ΔGO for polymerization of correct nucleotides. These results also provide a basis for understanding how polymerases replicate DNA (or RNA) with high fidelity.

Kinetic selection vs. free energy of DNA base pairing in control of polymerase fidelity

“…Olson et al (12), using Vent pol with 12 p/t DNA configurations, found for each example that a leveling off of right and wrong incorporations occurred in the presence of high PP i concentration. The differences in the apparent K eq for each of the steady states reflected sufficiently large free energy differences to account for pol fidelity (average ΔΔG°i nc = 5.2 ± 1.34 kcal/mol), thus obviating the need for pol active-site intervention to amplify the very small free energy differences between right and wrong base pairs deduced from thermal melting studies (ΔΔG°∼ 0.3 kcal/mol).…”

“…Apparent values of K eq were determined and used to calculate ΔG°v alues for R and W. Free energy differences (ΔΔG°i nc ) for incorporation of R and W were found to range from 3.5 kcal/mol to 7 kcal/mol and were thus sufficient to account for pol fidelity with no need for active-site amplification, but instead apparently reflecting intrinsic properties of p/t DNAs, dNTPs, and PP i , unperturbed by presence of polymerase. It was inferred, although not directly measured, that the leveling off in incorporation profiles was caused by sufficient pyrophosphorolysis to equilibrate forward and reverse reactions for both R and W substrates (12). Although the reverse pyrophosphorolysis reaction was reported to occur for correctly paired termini, it did not enter into the analysis and was not measured for mispaired termini.…”

“…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 ΔG°= −RT ln K eq , indicating ΔΔG°of 3.5-7 kcal/mol, sufficient to account for pol accuracy without need of kinetic selection.…”

“…Because these were far too small to account for fidelity, it was then proposed that steric constraints imposed by the pol active site could "amplify" the small free energy differences between R and W base pairing to attain ΔΔG°i nc ∼ 5.5-7 kcal/mol (7-9). In the absence of significant reverse reaction (pyrophosphorolysis) at low inorganic pyrophosphate (PP i ) levels in vivo or in a test tube, nucleotide incorporation reactions apparently proceed far from equilibrium (10)(11)(12).…”

“…A need for energetic amplification in the pol active site was recently challenged in a thoughtful and important paper (12) that attempted to operate DNA polymerases at equilibrium. Vent pol was used to elongate primer/template (p/t) DNA by incorporating either R or W nucleotides in the presence of PP i .…”