Thermodynamics of the binding of Thermus aquaticus DNA polymerase to primed-template DNA
DNA binding of the Type 1 DNA polymerase from Thermus aquaticus (Taq polymerase) and its Klentaq large fragment domain have been studied as a function of temperature. Equilibrium binding assays were performed from 5 to 70°C using a fluorescence anisotropy assay and from 10 to 60°C using isothermal titration calorimetry. In contrast to the usual behavior of thermophilic proteins at low temperatures, Taq and Klentaq bind DNA with high affinity at temperatures down to 5°C. The affinity is maximal at 40-50°C. The ΔH and ΔS of binding are highly temperature dependent, and the ΔCp of binding is -0.7 to -0.8 kcal/mol K, for both Taq and Klentaq, with good agreement between van't Hoff and calorimetric values. Such a thermodynamic profile, however, is generally associated with sequence-specific DNA binding and not nonspecific binding. Circular dichroism spectra show conformational rearrangements of both the DNA and the protein upon binding. The high ΔCp of Taq/Klentaq DNA binding may be correlated with structure-specific binding in analogy to sequence-specific binding, or may be a general characteristic of proteins that primarily bind non-specifically to DNA. The low temperature DNA binding of Taq/Klentaq is suggested to be a general characteristic of thermophilic DNA binding proteins.
Publication Source (Journal or Book title)
Nucleic Acids Research
Datta, K., & LiCata, V. (2003). Thermodynamics of the binding of Thermus aquaticus DNA polymerase to primed-template DNA. Nucleic Acids Research, 31 (19), 5590-5597. https://doi.org/10.1093/nar/gkg774