Title
Temperature dependence and thermodynamics of Klenow polymerase binding to primed-template DNA
Document Type
Article
Publication Date
3-1-2006
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.
Publication Source (Journal or Book title)
Biophysical journal
First Page
1739
Last Page
51
Recommended Citation
Datta, K., Wowor, A. J., Richard, A. J., & LiCata, V. J. (2006). Temperature dependence and thermodynamics of Klenow polymerase binding to primed-template DNA. Biophysical journal, 90 (5), 1739-51. https://doi.org/10.1529/biophysj.105.071837