Interactions of the DNA Polymerase X of African Swine Fever Virus with Double-stranded DNA. Functional Structure of the Complex

Research output: Contribution to journalArticle

17 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)75-95
Number of pages21
JournalJournal of Molecular Biology
Volume373
Issue number1
DOIs
StatePublished - Oct 12 2007

Fingerprint

African Swine Fever Virus
DNA
Fluorescence
Energy Transfer
Ions
Ion Exchange
Ultracentrifugation
Enzymes
Tryptophan
Base Pairing
Anions
Proteins
Nucleotides
Binding Sites

Keywords

  • DNA polymerases
  • DNA repair
  • DNA replication
  • protein-DNA interactions
  • quantitative fluorescence titrations

ASJC Scopus subject areas

  • Virology

Cite this

@article{464695a74019468ea1001aecaf55fa0e,
title = "Interactions of the DNA Polymerase X of African Swine Fever Virus with Double-stranded DNA. Functional Structure of the Complex",
abstract = "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.",
keywords = "DNA polymerases, DNA repair, DNA replication, protein-DNA interactions, quantitative fluorescence titrations",
author = "Jezewska, {Maria J.} and Pawel Bujalowski and Wlodzimierz Bujalowski",
year = "2007",
month = "10",
day = "12",
doi = "10.1016/j.jmb.2007.06.054",
language = "English (US)",
volume = "373",
pages = "75--95",
journal = "Journal of Molecular Biology",
issn = "0022-2836",
publisher = "Academic Press Inc.",
number = "1",

}

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, Pawel

AU - Bujalowski, Wlodzimierz

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

UR - http://www.scopus.com/inward/record.url?scp=34548653578&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=34548653578&partnerID=8YFLogxK

U2 - 10.1016/j.jmb.2007.06.054

DO - 10.1016/j.jmb.2007.06.054

M3 - Article

VL - 373

SP - 75

EP - 95

JO - Journal of Molecular Biology

JF - Journal of Molecular Biology

SN - 0022-2836

IS - 1

ER -