Temperature Dependence and Thermodynamics of Klenow Polymerase Binding to Primed-Template DNA

Abstract

DNA binding of Klenow polymerase has been characterized with respect to temperature to delineate the thermodynamic driving forces involved in the interaction of this polymerase with primed-template DNA. The temperature dependence of the binding affinity exhibits distinct curvature, with tightest binding at 25-30 degrees C. Nonlinear temperature dependence indicates Klenow binds different primed-template constructs with large heat capacity (DeltaCp) values (-870 to -1220 cal/mole K) and thus exhibits large temperature dependent changes in enthalpy and entropy. Binding is entropy driven at lower temperatures and enthalpy driven at physiological temperatures. Large negative DeltaCp values have been proposed to be a 'signature' of site-specific DNA binding, but type I DNA polymerases do not exhibit significant DNA sequence specificity. We suggest that the binding of Klenow to a specific DNA structure, the primed-template junction, results in a correlated thermodynamic profile that mirrors what is commonly seen for DNA sequence-specific binding proteins. Klenow joins a small number of other DNA-sequence independent DNA binding proteins which exhibit unexpectedly large negative DeltaCp values. Spectroscopic measurements show small conformational rearrangements of both the DNA and Klenow upon binding, and small angle x-ray scattering shows a global induced fit conformational compaction of the protein upon binding. Calculations from both crystal structure and solution structural data indicate that Klenow DNA binding is an exception to the often observed correlation between DeltaCp and changes in accessible surface area. In the case of Klenow, surface area burial can account for only about half of the DeltaCp of binding.

Differential Temperature-Dependent Multimeric Assemblies of Replication and Repair Polymerases on DNA Increase Processivity

“…The resulting temperature dependent thermodynamic binding parameters can provide insight into the specificity of the binding process through determination of the heat capacity change ( ΔC o p ) (29, 30). Although a strongly negative ΔC o p has been shown to be the thermodynamic signature of sequence-specific binding (30), the non-sequence specific binding to primer template DNA by the A-family DNA polymerases from Thermus aquaticus (Taq) (31) and Escherichia coli (Klenow) (32) is also associated with large and negative ΔC o p values (29, 33–35). Even though there is no sequence specificity, the negative ΔC o p is consistent with the high structural complementarity (29) of the DNA polymerase binding to the primer template junction, visualized in a variety of crystal structures (18, 36, 37).…”

Directed Evolution of Nucleotide-Based Libraries Using Lambda Exonuclease

“…This mixture was then treated with KF, which is able to synthesize new complementary DNA strands in the presence of dNTPs (47). KF works by adding bases to the 3 hydroxyl groups of a blunt-ended DNA duplex, working in a 5 to 3 direction (48).…”

An optics-based variable-temperature assay system for characterizing thermodynamics of biomolecular reactions on solid support

“…The theoretical consideration and experimental observation of these concepts have been explored and investigated by Lemieux and others for a number of lectin-glycan reactions and other protein-ligand binding reactions. [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15] In general, the net entropy gain can be regarded a measure of the amount of the bound water molecules released in the complex formation as the displacement of one bound water molecule into the bulk decreases the Gibbs free energy by 1-2 kcal/mol at room temperature.…”

“…As a result, the spontaneous association can be entropy-driven when the overall change in entropy S including those of the solvent is positive and dominates the net negative change in the Gibbs free energy G (= H − T S). [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15] It is clear that experimental studies of biomolecular binding reactions should be performed over the entire physiological temperature range instead of merely at room temperature around 300 K. By measuring equilibrium binding constants as a function of temperature, one can determine changes in Gibbs free energy G, enthalpy H, and entropy S (through Van that accompany the main reaction. In practice these insights are useful in guiding drug design and optimization.…”