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

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Abstract

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.

Original languageEnglish (US)
Pages (from-to)12909-12924
Number of pages16
JournalBiochemistry
Volume46
Issue number45
DOIs
StatePublished - Nov 13 2007

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African Swine Fever Virus
Viruses
DNA
Substrates
Chemical analysis
Enzymes
Binding Sites
DNA polymerase X
Titration
Stoichiometry
Magnesium
Ground state
Nucleotides
Fluorescence
Nuclear magnetic resonance
Ions

ASJC Scopus subject areas

  • Biochemistry

Cite this

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title = "Interactions of the DNA polymerase X from African swine fever virus with gapped DNA substrates. Quantitative analysis of functional structures of the formed complexes",
abstract = "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.",
author = "Jezewska, {Maria J.} and Pawel Bujalowski and Wlodzimierz Bujalowski",
year = "2007",
month = "11",
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T1 - Interactions of the DNA polymerase X from African swine fever virus with gapped DNA substrates. Quantitative analysis of functional structures of the formed complexes

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PY - 2007/11/13

Y1 - 2007/11/13

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