Tracking enzymatic steps of DNA topoisomerases using single-molecule micromanipulation

Terence R. Strick; Gilles Charvin; Nynke H. Dekker; Jean-François Allemand; David Bensimon; Vincent Croquette

doi:10.1016/S1631-0705(02)01347-6

Biophysique à l'échelle de la molécule unique/single-molecule biophysics

Tracking enzymatic steps of DNA topoisomerases using single-molecule micromanipulation
[Observation des cycles enzymatiques des ADN topoisomérases par micromanipulation de molécules individuelles]

Terence R. Strick ¹ ; Gilles Charvin ² ; Nynke H. Dekker ² ; Jean-François Allemand ² ; David Bensimon ² ; Vincent Croquette ²

¹ Cold Spring Harbor Labs, Beckman Building, 1 Bungtown Road, Cold Spring Harbor, NY 11724, USA
² Laboratoire de physique statistique et département de biologie de l'École normale supérieure, UMR 8550 CNRS, associated with the Universities of Paris VI and Paris VII, 24, rue Lhomond, 75231 Paris cedex 05, France

Comptes Rendus. Physique, Volume 3 (2002) no. 5, pp. 595-618.

Résumé
Abstract

Dans cet article, nous décrivons des expériences sur des molécules individuelles utilisant des pinces magnétiques. Nous les utilisons pour caractériser les enzymes topoisomérases dont le rôle biologique est de démêler les molécules d'ADN. Dans un premier temps, l'élasticité d'une molécule d'ADN est mesurée en utilisant cette technique de micromanipulation. Nous montrons qu'il est facile de contrôler une contrainte de torsion sur une molécule d'ADN et que son effet sur son élasticité peut être mesuré avec précision. Nous décrivons ensuite l'observation de l'activité enzymatique à l'échelle de la molécule unique. Ceci nous permet d'accéder à la mesure des constantes réactionnelles de l'enzyme tel sa vitesse ou sa processivité. Nous passons en revue les résultats que nous avons obtenus en particulier sut la topoisomérase II, et nous montrons qu'il est possible d'enregistrer en temps réel les cycles de déroulement d'une molécule d'ADN sous torsion. Ceci nous permet une caractérisation précise de la biochimie de cette enzyme. La mesure directe des cycles enzymatiques ne peut se faire que lorsque le rapport signal sur bruit du dispositif expérimental est élevé. Nous discutons également les méthodes de traitement des données qui permettent d'accéder à la distribution des cycles enzymatiques en fonction de la qualité du rapport signal sur bruit.

In this article, we describe single-molecule assays using magnetic traps and we applied these assays to topoisomerase enzymes which unwind and disentangle DNA molecules. First, the elasticity of single DNA molecule is characterized using the magnetic trap. We show that a twisting constraint may be easily applied and that its effect upon DNA may be measured accurately. Then we describe how the topoisomerase activity may be observed at the single-molecule level giving direct access to the important biological parameters of the enzyme such as velocity and processivity. Furthermore, individual cycles of unwinding can be observed in real time. This permits an accurate characterization of the enzyme's biochemical cycle. The data treatment required to identify and analyze individual topoisomerization cycles will be presented in detail. This analysis is applicable to a wide variety of molecular motors.

Reçu le : 2002-05-10
Accepté le : 2002-05-20
Publié le : 2002-01-01

DOI : 10.1016/S1631-0705(02)01347-6

Keywords: micromanipulation, magnetic tweezers, topoisomerases
Mots-clés : micromanipulation, pinces magnetiques, topoisomerases

Affiliations des auteurs :

Terence R. Strick ¹ ; Gilles Charvin ² ; Nynke H. Dekker ² ; Jean-François Allemand ² ; David Bensimon ² ; Vincent Croquette ²

¹ Cold Spring Harbor Labs, Beckman Building, 1 Bungtown Road, Cold Spring Harbor, NY 11724, USA
² Laboratoire de physique statistique et département de biologie de l'École normale supérieure, UMR 8550 CNRS, associated with the Universities of Paris VI and Paris VII, 24, rue Lhomond, 75231 Paris cedex 05, France

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     author = {Terence R. Strick and Gilles Charvin and Nynke H. Dekker and Jean-Fran\c{c}ois Allemand and David Bensimon and Vincent Croquette},
     title = {Tracking enzymatic steps of {DNA} topoisomerases using single-molecule micromanipulation},
     journal = {Comptes Rendus. Physique},
     pages = {595--618},
     publisher = {Elsevier},
     volume = {3},
     number = {5},
     year = {2002},
     doi = {10.1016/S1631-0705(02)01347-6},
     language = {en},
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%A Jean-François Allemand
%A David Bensimon
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Terence R. Strick; Gilles Charvin; Nynke H. Dekker; Jean-François Allemand; David Bensimon; Vincent Croquette. Tracking enzymatic steps of DNA topoisomerases using single-molecule micromanipulation. Comptes Rendus. Physique, Volume 3 (2002) no. 5, pp. 595-618. doi : 10.1016/S1631-0705(02)01347-6. https://comptes-rendus.academie-sciences.fr/physique/articles/10.1016/S1631-0705(02)01347-6/

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