Thermodynamics of the binding of Thermus aquaticus DNA polymerase to primed-template DNA
AUTOR(ES)
Datta, Kausiki
FONTE
Oxford University Press
RESUMO
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 non- specific 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.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=206472Documentos Relacionados
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