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

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. The primary DNA-binding subsite of the enzyme, independent of the DNA conformation, is located on the C-terminal domain. Association of the bound DNA with the catalytic N-terminal domain finalizes the engagement of the total DNA-binding site of the enzyme and induces a large topological change in the structure of the bound ssDNA. The free energy of binding includes a conformational transition of the protein. Large positive enthalpy changes accompanying the ASFV pol X-ssDNA association indicate that conformational changes of the complex are induced by the engagement of the N-terminal domain. The enthalpy changes are offset by large entropy changes accompanying the DNA binding to the C-terminal domain and the total DNA-binding site, predominantly resulting from the release of water molecules.

Graphical abstractDownload full-size imageResearch highlights► The primary DNA-binding site of ASFV pol X is located on the C-terminal domain. ► The total free energy of binding includes communication between the C-terminal and catalytic domains. ► The pol X - ssDNA association is an entropy-driven process. ► Engagement of the N-terminal domain is accompanied by a large positive enthalpy change. ► Entropy change results from the water release accompanying the binding.