DNA polymerase X from African swine fever virus: quantitative analysis of the enzyme-ssDNA interactions and the functional structure of the complex.

Abstract:

Interactions of polymerase X from African swine fever virus with single-stranded DNA (ssDNA) have been studied, using quantitative fluorescence titration and analytical ultracentrifugation techniques. Experiments were performed with a fluorescent etheno-derivative of ssDNA oligomers. Studies of unmodified ssDNA oligomers were carried out using the competition titration method. The total site-size of the pol X-ssDNA complex is 16(+/-1) nucleotide residues. The large total ssDNA-binding site has a complex heterogeneous structure. It contains the proper ssDNA-binding site that encompasses only 7(+/-1) residues. As the length of the ssDNA increases, the enzyme engages an additional binding area in interactions with the DNA, at a distance of approximately 7-8 nucleotides from the proper site, which is located asymmetrically within the polymerase molecule. As a result, the net ion release accompanying the interactions with the DNA, increases from approximately 1 for the proper DNA-binding site to approximately 6 for the total DNA-binding site. Unlike in the case of the mammalian polymerase beta that belongs to the same polymerase X family, the DNA-binding areas within the total DNA-binding site of pol X are not autonomous. Consequently, the enzyme does not form different binding modes with different numbers of occluded nucleotide residues, although the interacting areas are structurally separated. The statistical thermodynamic model that accounts for the engagement of the proper and the total DNA-binding site in interactions with the DNA provides an excellent description of the binding process. Pol X binds the ssDNA without detectable cooperativity and with very modest base specificity.

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