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Nucleic Acids Research Advance Access published online on August 26, 2006
Nucleic Acids Research, doi:10.1093/nar/gkl504
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© 2006 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Methods Online

High-throughput assays for DNA gyrase and other topoisomerases

Anthony Maxwell*, Nicolas P. Burton1 and Natasha O'Hagan

Department of Biological Chemistry, John Innes Centre Norwich Research Park, Colney,Norwich NR4 7UH, UK 1 John Innes Enterprises Ltd, John Innes Centre Norwich Research Park, Colney,Norwich NR4 7UH, UK

*To whom correspondence should be addressed. Tel: +44 1603 450771; Fax: +44 1603 450018; Email: tony.maxwell{at}bbsrc.ac.uk

Received March 24, 2006. Revised May 5, 2006. Accepted June 30, 2006.

We have developed high-throughput microtitre plate-based assays for DNA gyrase and other DNA topoisomerases. These assays exploit the fact that negatively supercoiled plasmids form intermolecular triplexes more efficiently than when they are relaxed. Two assays are presented, one using capture of a plasmid containing a single triplex-forming sequence by an oligonucleotide tethered to the surface of a microtitre plate and subsequent detection by staining with a DNA-specific fluorescent dye. The other uses capture of a plasmid containing two triplex-forming sequences by an oligonucleotide tethered to the surface of a microtitre plate and subsequent detection by a second oligonucleotide that is radiolabelled. The assays are shown to be appropriate for assaying DNA supercoiling by Escherichia coli DNA gyrase and DNA relaxation by eukaryotic topoisomerases I and II, and E.coli topoisomerase IV. The assays are readily adaptable to other enzymes that change DNA supercoiling (e.g. restriction enzymes) and are suitable for use in a high-throughput format.

Present addresses: Natasha O'Hagan, School of Biological Sciences, University of Edinburgh, Mayfield Road, Edinburgh, EH9 3JR, UK

Nicolas Burton, Inspiralis Ltd, Norwich Bioincubator, Norwich Research Park, Colney, Norwich, NR4 7UH, UK


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