High-throughput assays for DNA gyrase and other topoisomerases

High-throughput assays for DNA gyrase and other topoisomerases

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...

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Main Authors: Maxwell, Anthony, Burton, Nicolas P., O'Hagan, Natasha
Format: Online
Language:English
Published: Oxford University Press 2006
Online Access:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1616945/
id pubmed-1616945
recordtype oai_dc
spelling pubmed-16169452006-10-27 High-throughput assays for DNA gyrase and other topoisomerases Maxwell, Anthony Burton, Nicolas P. O'Hagan, Natasha Methods Online 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. Oxford University Press 2006-09 2006-08-26 /pmc/articles/PMC1616945/ /pubmed/16936317 http://dx.doi.org/10.1093/nar/gkl504 Text en © 2006 The Author(s)
repository_type Open Access Journal
institution_category Foreign Institution
institution US National Center for Biotechnology Information
building NCBI PubMed
collection Online Access
language English
format Online
author Maxwell, Anthony
Burton, Nicolas P.
O'Hagan, Natasha
spellingShingle Maxwell, Anthony
Burton, Nicolas P.
O'Hagan, Natasha
High-throughput assays for DNA gyrase and other topoisomerases
author_facet Maxwell, Anthony
Burton, Nicolas P.
O'Hagan, Natasha
author_sort Maxwell, Anthony
title High-throughput assays for DNA gyrase and other topoisomerases
title_short High-throughput assays for DNA gyrase and other topoisomerases
title_full High-throughput assays for DNA gyrase and other topoisomerases
title_fullStr High-throughput assays for DNA gyrase and other topoisomerases
title_full_unstemmed High-throughput assays for DNA gyrase and other topoisomerases
title_sort high-throughput assays for dna gyrase and other topoisomerases
description 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.
publisher Oxford University Press
publishDate 2006
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1616945/
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