Interactions of the DNA polymerase X from African swine fever virus with gapped DNA substrates. Quantitative analysis of functional structures of the formed complexes

Biochemistry. 2007 Nov 13;46(45):12909-24. doi: 10.1021/bi700677j. Epub 2007 Oct 17.


Energetics and specificity of interactions between the African swine fever virus polymerase X and gapped DNA substrates have been studied, using the quantitative fluorescence titration technique. Stoichiometries of pol X complexes, with the DNA substrates, are higher than suggested by NMR studies. This can be understood in the context of the functionally heterogeneous organization of the total DNA-binding site of pol X, which is composed of two DNA-binding subsites. The enzyme forms two different complexes with the gapped DNAs, differing dramatically in affinities. In the high-affinity complex, pol X engages the total DNA-binding site, forming the gap complex, while in the low-affinity the enzyme binds to the dsDNA parts of the gapped DNA, using only one of the DNA-binding subsites. As a result, the net number of ions released in the gap complex formation is significantly larger than in the binding of the dsDNA part. In the presence of Mg+2, pol X shows a strong preference for the ssDNA gaps having one and two nucleotides. Recognition of the short gaps already occurs in the ground state of the enzyme-DNA complex. Surprisingly, the specific structure necessary to recognize the short gaps is induced by magnesium binding to the enzyme. In the absence of Mg+2, pol X looses its selectivity for short ssDNA gaps. Pol X binds gapped DNAs with considerable cooperative interactions, which increase with the decreasing gap size. The functional implications of these findings for ASFV pol X activities are discussed.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • African Swine Fever Virus / enzymology*
  • Bromides / pharmacology
  • DNA, Single-Stranded / metabolism*
  • DNA-Directed DNA Polymerase / chemistry
  • DNA-Directed DNA Polymerase / metabolism*
  • Magnesium / pharmacology
  • Models, Chemical
  • Sodium Compounds / pharmacology
  • Spectrometry, Fluorescence
  • Thermodynamics


  • Bromides
  • DNA, Single-Stranded
  • Sodium Compounds
  • DNA polymerase X
  • DNA-Directed DNA Polymerase
  • Magnesium
  • sodium bromide