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High-Throughput Microtitre Plate-Based Assay for DNA Topoisomerases

  • James A. Taylor
  • Nicolas P. Burton
  • Anthony MaxwellEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 815)

Abstract

We have developed a rapid, high-throughput assay for measuring the catalytic activity (DNA supercoiling or relaxation) of DNA topoisomerases. The assay utilizes intermolecular triplex formation between an immobilized triplex-forming oligo (TFO) and a triplex-forming region inserted into the plasmid substrate (pNO1), and capitalizes on the observation that supercoiled DNA forms triplexes more readily than relaxed DNA. Thus, supercoiled DNA is preferentially retained by the TFO under triplex-forming conditions while relaxed DNA can be washed away. Due to its high speed of sample analysis and reduced sample handling over conventional gel-based techniques, this assay can be used to screen chemical libraries for novel inhibitors of topoisomerases.

Key words

Topoisomerase DNA gyrase Triplex formation Supercoiling Relaxation High-throughput screening 

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Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • James A. Taylor
    • 1
  • Nicolas P. Burton
    • 2
  • Anthony Maxwell
    • 1
    Email author
  1. 1.Department of Biological ChemistryJohn Innes CentreColneyUK
  2. 2.Inspiralis Ltd, Norwich Bioincubator, Norwich Research ParkColneyUK

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