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Nucleic Acids Research 2005 33(18):5945-5953; doi:10.1093/nar/gki906
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Published online 19 October 2005

© The Author 2005. Published by Oxford University Press. All rights reserved
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Article

Atomic force microscopy shows that vaccinia topoisomerase IB generates filaments on DNA in a cooperative fashion

Fernando Moreno-Herrero, Laurent Holtzer, Daniel A. Koster, Stewart Shuman1, Cees Dekker and Nynke H. Dekker*

Kavli Institute of Nanoscience, Faculty of Applied Sciences, Delft University of Technology Lorentzweg 1, 2628 CJ Delft, The Netherlands 1Molecular Biology Program, Sloan-Kettering Institute New York, NY 10021, USA

*To whom correspondence should be addressed. Tel: +31 (0) 15 278 3219; Fax: +31 (0) 15 278 1202; Email: Nynke.Dekker{at}mb.tn.tudelft.nl

Received August 5, 2005. Revised September 29, 2005. Accepted September 29, 2005.

Type IB DNA topoisomerases cleave and rejoin one strand of the DNA duplex, allowing for the removal of supercoils generated during replication and transcription. In addition, electron microscopy of cellular and viral TopIB–DNA complexes has suggested that the enzyme promotes long-range DNA–DNA crossovers and synapses. Here, we have used the atomic force microscope to visualize and quantify the interaction between vaccinia topoisomerase IB (vTopIB) and DNA. vTopIB was found to form filaments on nicked-circular DNA by intramolecular synapsis of two segments of a single DNA molecule. Measuring the filament length as a function of protein concentration showed that synapsis is a highly cooperative process. At high protein:DNA ratios, synapses between distinct DNA molecules were observed, which led to the formation of large vTopIB-induced DNA clusters. These clusters were observed in the presence of Mg2+, Ca2+ or Mn2+, suggesting that the formation of intermolecular vTopIB-mediated DNA synapsis is favored by screening of the DNA charge.

Present address: Laurent Holtzer, Department of Biophysics, Leiden Institute of Physics, Leiden University, Niels Bohrweg 2, 2333 CA Leiden, The Netherlands


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