19F NMR studies of vaccinia type IB topoisomerase. Conformational dynamics of the bound DNA substrate

Keehwan Kwon, Yu Lin Jiang, Fenhong Song, James Stivers

Research output: Contribution to journalArticle

Abstract

The site-specific DNA cleavage and religation activities of the vaccinia virus type IB topoisomerase at (C/T)CCTT+1X-1 sites in duplex DNA have allowed detailed investigations of the chemical and conformational steps on the reaction pathway of this enzyme (see accompanying article (Kwon, K., and Stivers, J. T. (2002) J. Biol. Chem. 277, 345-352)). To extend these studies to the DNA substrate, we have performed 19F NMR experiments using substrates in which the +1 T has been replaced with the NMR-sensitive thymidine base analogue 5-fluoro-2′-deoxyuridine (5-F-dUrd). Substitution of 5-F-dUrd has little effect on the binding affinity of topoisomerase I for DNA, results in small changes in the cleavage and religation rate constants, and produces a net 3-fold decrease in the cleavage equilibrium constant as compared with the CCCTT consensus DNA. One-dimensional 19F NMR experiments show that the +1 5-F-dUrd is in a dynamic equilibrium between a stacked and unstacked state in both the noncovalent complex and the covalent phosphotyrosine complex. These NMR observations are supported by the selective sensitivity of the +1 T and +1 5-F-dUrd to KMnO4 oxidation. A role for localized DNA distortion in the topoisomerase I mechanism is suggested.

Original languageEnglish (US)
Pages (from-to)353-358
Number of pages6
JournalJournal of Biological Chemistry
Volume277
Issue number1
DOIs
StatePublished - Jan 4 2002

Fingerprint

Vaccinia
Nuclear magnetic resonance
Type I DNA Topoisomerase
DNA
Substrates
DNA Cleavage
Phosphotyrosine
Vaccinia virus
Thymidine
Equilibrium constants
Viruses
Rate constants
Substitution reactions
Experiments
5-fluoro-2'-deoxyuridine
Enzymes
Oxidation

ASJC Scopus subject areas

  • Biochemistry

Cite this

19F NMR studies of vaccinia type IB topoisomerase. Conformational dynamics of the bound DNA substrate. / Kwon, Keehwan; Jiang, Yu Lin; Song, Fenhong; Stivers, James.

In: Journal of Biological Chemistry, Vol. 277, No. 1, 04.01.2002, p. 353-358.

Research output: Contribution to journalArticle

@article{998c1d184c4d47cebcbf01324d7408da,
title = "19F NMR studies of vaccinia type IB topoisomerase. Conformational dynamics of the bound DNA substrate",
abstract = "The site-specific DNA cleavage and religation activities of the vaccinia virus type IB topoisomerase at (C/T)CCTT+1X-1 sites in duplex DNA have allowed detailed investigations of the chemical and conformational steps on the reaction pathway of this enzyme (see accompanying article (Kwon, K., and Stivers, J. T. (2002) J. Biol. Chem. 277, 345-352)). To extend these studies to the DNA substrate, we have performed 19F NMR experiments using substrates in which the +1 T has been replaced with the NMR-sensitive thymidine base analogue 5-fluoro-2′-deoxyuridine (5-F-dUrd). Substitution of 5-F-dUrd has little effect on the binding affinity of topoisomerase I for DNA, results in small changes in the cleavage and religation rate constants, and produces a net 3-fold decrease in the cleavage equilibrium constant as compared with the CCCTT consensus DNA. One-dimensional 19F NMR experiments show that the +1 5-F-dUrd is in a dynamic equilibrium between a stacked and unstacked state in both the noncovalent complex and the covalent phosphotyrosine complex. These NMR observations are supported by the selective sensitivity of the +1 T and +1 5-F-dUrd to KMnO4 oxidation. A role for localized DNA distortion in the topoisomerase I mechanism is suggested.",
author = "Keehwan Kwon and Jiang, {Yu Lin} and Fenhong Song and James Stivers",
year = "2002",
month = "1",
day = "4",
doi = "10.1074/jbc.M109450200",
language = "English (US)",
volume = "277",
pages = "353--358",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "1",

}

TY - JOUR

T1 - 19F NMR studies of vaccinia type IB topoisomerase. Conformational dynamics of the bound DNA substrate

AU - Kwon, Keehwan

AU - Jiang, Yu Lin

AU - Song, Fenhong

AU - Stivers, James

PY - 2002/1/4

Y1 - 2002/1/4

N2 - The site-specific DNA cleavage and religation activities of the vaccinia virus type IB topoisomerase at (C/T)CCTT+1X-1 sites in duplex DNA have allowed detailed investigations of the chemical and conformational steps on the reaction pathway of this enzyme (see accompanying article (Kwon, K., and Stivers, J. T. (2002) J. Biol. Chem. 277, 345-352)). To extend these studies to the DNA substrate, we have performed 19F NMR experiments using substrates in which the +1 T has been replaced with the NMR-sensitive thymidine base analogue 5-fluoro-2′-deoxyuridine (5-F-dUrd). Substitution of 5-F-dUrd has little effect on the binding affinity of topoisomerase I for DNA, results in small changes in the cleavage and religation rate constants, and produces a net 3-fold decrease in the cleavage equilibrium constant as compared with the CCCTT consensus DNA. One-dimensional 19F NMR experiments show that the +1 5-F-dUrd is in a dynamic equilibrium between a stacked and unstacked state in both the noncovalent complex and the covalent phosphotyrosine complex. These NMR observations are supported by the selective sensitivity of the +1 T and +1 5-F-dUrd to KMnO4 oxidation. A role for localized DNA distortion in the topoisomerase I mechanism is suggested.

AB - The site-specific DNA cleavage and religation activities of the vaccinia virus type IB topoisomerase at (C/T)CCTT+1X-1 sites in duplex DNA have allowed detailed investigations of the chemical and conformational steps on the reaction pathway of this enzyme (see accompanying article (Kwon, K., and Stivers, J. T. (2002) J. Biol. Chem. 277, 345-352)). To extend these studies to the DNA substrate, we have performed 19F NMR experiments using substrates in which the +1 T has been replaced with the NMR-sensitive thymidine base analogue 5-fluoro-2′-deoxyuridine (5-F-dUrd). Substitution of 5-F-dUrd has little effect on the binding affinity of topoisomerase I for DNA, results in small changes in the cleavage and religation rate constants, and produces a net 3-fold decrease in the cleavage equilibrium constant as compared with the CCCTT consensus DNA. One-dimensional 19F NMR experiments show that the +1 5-F-dUrd is in a dynamic equilibrium between a stacked and unstacked state in both the noncovalent complex and the covalent phosphotyrosine complex. These NMR observations are supported by the selective sensitivity of the +1 T and +1 5-F-dUrd to KMnO4 oxidation. A role for localized DNA distortion in the topoisomerase I mechanism is suggested.

UR - http://www.scopus.com/inward/record.url?scp=0037016262&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0037016262&partnerID=8YFLogxK

U2 - 10.1074/jbc.M109450200

DO - 10.1074/jbc.M109450200

M3 - Article

C2 - 11689573

AN - SCOPUS:0037016262

VL - 277

SP - 353

EP - 358

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 1

ER -