Interactions of the DNA polymerase X from African swine fever virus with gapped DNA substrates. Quantitative analysis of functional structures of the formed complexes.
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.
Jezewska
Maria J
MJ
Department of Biochemistry and Molecular Biology, The Sealy Center for Structural Biology, Sealy Center for Cancer Cell Biology, The University of Texas Medical Branch at Galveston, 301 University Boulevard, Galveston, Texas 77555-1053, USA.
Bujalowski
Paul J
PJ
Bujalowski
Wlodzimierz
W
D000358:African Swine Fever Virus / Q000201:enzymology*; D001965:Bromides / Q000494:pharmacology; D004277:DNA, Single-Stranded / Q000378:metabolism*; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry / Q000378:metabolism*; D008274:Magnesium / Q000494:pharmacology; D008956:Models, Chemical; D017670:Sodium Compounds / Q000494:pharmacology; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics
D001965:Bromides; D004277:DNA, Single-Stranded; D017670:Sodium Compounds; C109534:DNA polymerase X; D004259:DNA-Directed DNA Polymerase; D008274:Magnesium; C027938:sodium bromide