Interactions of the DNA Polymerase X From African Swine Fever Virus with the ssDNA. Properties of the Total DNA-Binding Site and the Strong DNA-Binding Subsite

Abstract

Interactions of the polymerase X from the African Swine Fever Virus with the ssDNA have been studied, using quantitative fluorescence titration and fluorescence resonance energy transfer techniques. Direct binding experiments were performed with fluorescent etheno-derivative of ssDNA oligomers. Studies of unmodified ssDNA oligomers were carried out using the competition titration method. The primary DNA-binding subsite of the enzyme, which establishes the contact with the nucleic acid independent of the DNA conformation, is located on the C-terminal domain of the enzyme. Association of the bound DNA with the weak DNA-binding subsite on the catalytic N-terminal domain, finalizes the engagement of the total DNA-binding site of the enzyme in interactions with the nucleic acid. Moreover, it induces a large topological change in the structure of the bound ssDNA. The intrinsic free energy of binding of the total DNA-binding site is not a simple sum of contributions from the strong and the weak DNA-binding subsites. Large positive enthalpy changes accompanying the ASFV pol X - ssDNA association indicate that specific conformational changes of the complex are induced by the engagement of the weak DNA-binding subsite on the N-terminal domain. The unfavorable enthalpy changes are offset by large entropy changes accompanying the DNA binding to the strong DNA-binding subsite and the total DNA-binding site, and, predominantly, result from the release of the water molecules.