Correlation Between DNA Binding Thermodynamics and Functional Behavior of Pol I DNA Polymerases

Abstract

Primer-template DNA (pt-DNA) binding by DNA polymerases is the first step of the polymerization cycle. We have previously characterized the thermodynamics of pt-DNA binding with respect to temperature for the “large fragments” of DNA polymerase I, Klentaq and Klenow, from Thermus aquaticus and Escherichia coli, respectively. Results with both polymerases showed that DNA binding is enthalpy-driven near their respective physiological temperatures. Here, nucleotide incorporation activity was measured with respect to temperature to examine how the thermodynamics of initial pt-DNA binding relates to the enzymatic activities of Klentaq and Klenow. For both polymerases it is observed that a negative enthalpy of initial binding (ΔH) is required for nucleotide incorporation activity, and that a negative entropy of binding (TΔS) inhibits the catalytic activity. Nucleotide incorporation activity was also examined with respect to KCl concentration. As reported previously, thermodynamic linkage plots for pt-DNA binding with respect to KCl concentration (∂ln1/Kd versus∂ln[KCl]) exhibit negative slopes for both polymerases and indicate net ion releases of ∼3 and ∼5 ions upon pt-DNA binding by Klentaq and Klenow, respectively. Interestingly, linkage plots for the steady-state rate of incorporation activity with respect to KCl concentration (∂ln1/ssRateversus∂ln[KCl]) exhibit the same slopes as the linkage plots of pt-DNA binding. This result suggests that salt dependence of initial pt-DNA binding dictates the salt dependence of the overall incorporation activity. It is striking that for both salt and temperature dependences, the detailed thermodynamics of DNA binding so directly correlate with overall functional behavior.This work is supported by National Science Foundation.