An Enthalpic “On/Off Switch” Linking DNA Binding and Nucleotide Incorporation Activity in 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 Klenow and Klentaq “large fragment domains” of DNA polymerase I, from Thermus aquaticus and Escherichia coli, respectively. DNA binding affinities for both polymerases are quite tight across wide temperature ranges, 5-70 °C for Klentaq and 5-37 °C for Klenow. Both polymerases show significant heat capacity changes upon binding, yielding curved ΔG versus temperature dependences. This results in large changes in ΔH and TΔS with temperature, including a sign change from positive to negative for both enthalpy and entropy as temperature increases. For both polymerases, DNA binding is enthalpy-driven near their respective physiological temperatures.Herein, 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. It is found that both polymerases are enzymatically inactive until the temperature reaches the point where the enthalpy of binding becomes negative (favorable), despite the fact that they both bind DNA quite well at lower temperatures. The data suggest that, for both polymerases, a negative free energy of binding alone is insufficient to drive catalysis, and that a negative enthalpy of initial binding (ΔH) is required for nucleotide incorporation activity. This work is supported by the National Science Foundation.