Comparison between DNA melting thermodynamics and DNA polymerase fidelity

The relation between DNA polymerase fidelity and base pairing stability is investigated by using DNA primer- template duplexes that contain a common 9-base template sequence but have either correct (A·T) or incorrect (G·T, C·T, T·T) base pairs at the primer 3' terminus. Thermal melting and enzyme kinetic measurements are compared for each kind of terminus. Analysis of melting temperatures finds that differences between the free energy changes upon dissociation (ΔΔG°) are only 0.2, 0.3, and 0.4 kcal·mol^-1 (1 cal = 4.18 J) for terminal A·T compared to G·T, C·T, and T·T mispairs, respectively, at 37°C. We show that enthalpy changes are directly correlated with entropy changes for normal and abnormal base pairs in DNA in aqueous solution and that ΔΔG° values are small because of near cancelation of corresponding enthalpy and entropy components. The kinetics of elongating primer termini are measured with purified Drosophila DNA polymerase α. The matched A·T terminus is found to be extended ≃ 200 times faster than a G·T mismatch and 1400 and 2500 times faster than C·T and T·T mismatches, respectively. Enzymatic discrimination against elongating mismatched termini is based mainly on K(m) rather than V(max) differences. From K(m) at 37°C, we find ΔΔG° values of 2.6-3.7 kcal·mol^-1, about an order of magnitude greater than indicated by melting data. A similar measurement of nucleotide insertion kinetics has previously found rates of forming A·T base pairs to be 5000 times greater than G·T mispairs and 20,000 times greater than C·T and T·T mispairs. Here also, K(m) differences are mainly responsible for discrimination and indicate even larger ΔΔG° values (4.3-4.9 kcal·mol^-1). Thus, free energy differences between correct and incorrect base pairs in the active site cleft of polymerase appear to be >10 times as large as in aqueous medium. We explore the idea that a binding cleft that snugly fits correct base pairs and excludes water at the active site may amplify base-pair free energy differences by reducing entropy differences and increasing enthalphy differences sufficiently to account for nucleotide insertion and extension fidelity.