Thermodynamic characterization of DNA binding by type I DNA polymerases from Thermus aquaticus and Escherichia coli

DNA binding of the DNA polymerase I from Thermus aquaticus (Taq and Klentaq fragment) and E. coli (Klenow fragment) have been studied as a function of [salt] and temperature in order to understand their DNA binding thermodynamics. Binding of the two different species of polymerases occurs with sub-micromolar affinities in very different salt concentration ranges. Thus, at similar [KCl] the binding of Klenow is ~ 3kcal/mol (150X) tighter than the binding of Taq/Klentaq. Linkage analysis of the [KCl] dependence of DNA binding at 25°C reveals a net release of 2-3 ions for Taq/Klentaq and 4-5 ions for Klenow. DNA binding of Taq at 60°C only slightly decreases the linked ion release. Linkage analysis of [MgCl2] dependence of DNA binding indicates that formation of protein-DNA complex in both the polymerases is linked to the release of approximately one Mg+2 ion. The temperature dependencies of DNA binding were studied at 5-70°C for Taq/Klentaq, and 5-37°C for Klenow. The temperature dependencies of ΔG of binding by Taq/Klentaq and Klenow show strong curvature due to the presence of negative ΔCp of binding, which was confirmed using isothermal titration calorimetry. DNA binding by both species of polymerase show enthalpy-entropy compensation, with binding being enthalpy driven at their respective physiological temperatures. It is notable that Taq/Klentaq binds DNA as low as 5°C, even though it has almost no catalytic activity at room temperature. Circular dichroism and small angle x-ray scattering measurements show small observable conformational rearrangements upon complex formation for both polymerases. Large negative ΔCp are typically associated with sequence specific DNA binding. Sequence specificity for the single-stranded template overhang was examined by altering the sequence to poly-A, poly-T and poly-C respectively. The affinity difference from weakest to tightest is only about one order of magnitude. Preliminary studies of the binding of Klenow and Klentaq to different DNA structures show that the two polymerases have different DNA structure preferences. We propose that the relatively large negative ΔCp for Klenow and Klentaq DNA binding might be due to DNA structure specificity or a general characteristic of primarily non-sequence specific DNA binding proteins that bind with high affinity.