TY - JOUR
T1 - Interactions of the DNA Polymerase X of African Swine Fever Virus with Double-stranded DNA. Functional Structure of the Complex
AU - Jezewska, Maria J.
AU - Bujalowski, Paul J.
AU - Bujalowski, Wlodzimierz
N1 - Funding Information:
This work was supported by NIH grant GM-58565 to W. B. The authors thank Mrs Gloria Bellard for reading the manuscript.
PY - 2007/10/12
Y1 - 2007/10/12
N2 - Interactions of the polymerase X of African swine fever virus with the double-stranded DNA (dsDNA) have been studied with fluorescent dsDNA oligomers, using quantitative fluorescence titrations, analytical ultracentrifugation, and fluorescence energy transfer techniques. Studies with unmodified dsDNAs were performed, using competition titration method. ASV pol X binds the dsDNA with a site-size of n = 10(±2) base-pairs, which is significantly shorter than the total site-size of 16(±2) nucleotides of the enzyme-ssDNA complex. The small site size indicates that the enzyme binds the dsDNA exclusively using the proper DNA-binding subsite. Fluorescence energy transfer studies between the tryptophan residue W92 and the acceptor, located at the 5′ or 3′ end of the dsDNA, suggest strongly that the proper DNA-binding subsite is located on the non-catalytic C-terminal domain. Moreover, intrinsic interactions with the dsDNA 10-mer or 20-mer are accompanied by the same net number of ions released, independent of the length of the DNA, indicating the same length of the DNA engaged in the complex. The dsDNA intrinsic affinity is about two orders of magnitude higher than the ssDNA affinity, indicating that the proper DNA-binding subsite is, in fact, the specific dsDNA-binding site. Surprisingly, ASFV pol X binds the dsDNA with significant positive cooperativity, which results from protein-protein interactions. Cooperative interactions are accompanied by the net ion release, with anions participating in the ion-exchange process. The significance of these results for ASFV pol X activity in the recognition of damaged DNA is discussed.
AB - Interactions of the polymerase X of African swine fever virus with the double-stranded DNA (dsDNA) have been studied with fluorescent dsDNA oligomers, using quantitative fluorescence titrations, analytical ultracentrifugation, and fluorescence energy transfer techniques. Studies with unmodified dsDNAs were performed, using competition titration method. ASV pol X binds the dsDNA with a site-size of n = 10(±2) base-pairs, which is significantly shorter than the total site-size of 16(±2) nucleotides of the enzyme-ssDNA complex. The small site size indicates that the enzyme binds the dsDNA exclusively using the proper DNA-binding subsite. Fluorescence energy transfer studies between the tryptophan residue W92 and the acceptor, located at the 5′ or 3′ end of the dsDNA, suggest strongly that the proper DNA-binding subsite is located on the non-catalytic C-terminal domain. Moreover, intrinsic interactions with the dsDNA 10-mer or 20-mer are accompanied by the same net number of ions released, independent of the length of the DNA, indicating the same length of the DNA engaged in the complex. The dsDNA intrinsic affinity is about two orders of magnitude higher than the ssDNA affinity, indicating that the proper DNA-binding subsite is, in fact, the specific dsDNA-binding site. Surprisingly, ASFV pol X binds the dsDNA with significant positive cooperativity, which results from protein-protein interactions. Cooperative interactions are accompanied by the net ion release, with anions participating in the ion-exchange process. The significance of these results for ASFV pol X activity in the recognition of damaged DNA is discussed.
KW - DNA polymerases
KW - DNA repair
KW - DNA replication
KW - protein-DNA interactions
KW - quantitative fluorescence titrations
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U2 - 10.1016/j.jmb.2007.06.054
DO - 10.1016/j.jmb.2007.06.054
M3 - Article
C2 - 17765921
AN - SCOPUS:34548653578
SN - 0022-2836
VL - 373
SP - 75
EP - 95
JO - Journal of Molecular Biology
JF - Journal of Molecular Biology
IS - 1
ER -