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Single-molecule analysis of DNA uncoiling by a type II topoisomerase


Type II DNA topoisomerases are ubiquitous ATP-dependent enzymes capable of transporting a DNA through a transient double-strand break in a second DNA segment1. This enables them to untangle DNA2,3,4,5,6 and relax the interwound supercoils (plectonemes) that arise in twisted DNA7. In vivo, they are responsible for untangling replicated chromosomes and their absence at mitosis or meiosis ultimately causes cell death8,9. Here we describe a micromanipulation experiment in which we follow in real time a single Drosophila melanogaster topoisomerase II acting on a linear DNA molecule which is mechanically stretched and supercoiled10,11,12,13. By monitoring the DNA's extension in the presence of ATP, we directly observe the relaxation of two supercoils during a single catalytic turnover. By controlling the force pulling on the molecule, we determine the variation of the reaction rate with the applied stress. Finally, in the absence of ATP, we observe the clamping of a DNA crossover by a single topoisomerase on at least two different timescales (configurations). These results show that single molecule experiments are a powerful new tool for the study of topoisomerases.

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Figure 1: Extension versus supercoiling behaviour for stretched DNA.
Figure 2: Individual time courses for the topo II-mediated relaxation of positively supercoiled DNA stretched at F = 0.7 pN.
Figure 3: Reaction dependence on ATP concentration and force.
Figure 4: Evidence for topo II clamping a crossover between two DNA segments (no ATP).


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We thank B. Maier and J.-F. Allemand for helpful comments and O. Hyrien, J.-L. Sikorav, M. Duguet, V. Rybenkov, N. Crisona and N. Cozzarelli for stimulating conversations. We also thank N. Cozzarelli for a gift of cloned topo II. T.R.S. was supported by a CNRS BDI fellowship.

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Strick, T., Croquette, V. & Bensimon, D. Single-molecule analysis of DNA uncoiling by a type II topoisomerase . Nature 404, 901–904 (2000).

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