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A high-throughput fluorescence anisotropy-based assay for human topoisomerase II β-catalyzed ATP-dependent supercoiled DNA relaxation

Lookup NU author(s): Professor Caroline AustinORCiD

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This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License (CC BY-NC-ND).


Abstract

Because of their essentiality for DNA replication, transcription, and repair, type II topoisomerases are targets for antibacterial and anticancer drugs. There are two type II topoisomerases in humans, topoisomerase II alpha (TOP2A) and topoisomerase II beta (TOP2B), and two in bacteria, gyrase and topoisomerase IV. Inhibition of one or both of the human type II topoisomerases by antibacterial compounds targeting their bacterial counterparts could result in toxicity. In addition, side effects of anticancer drugs targeting TOP2A could result from inhibition of TOP2B. A simple and rapid biochemical assay for the activity of TOP2A and TOP2B would be advantageous for screening for novel inhibitors, testing them for selectivity for one enzyme over the other, and testing for potential toxicity of antibacterial type II topoisomerases mediated by human topoisomerase II inhibition. In this paper, we show that a previously reported high-throughput, fluorescence anisotropy-based assay for ATP-dependent relaxation of supercoiled DNA by human TOP2A can also be used under identical conditions for human TOP2B. We used this assay to compare the potencies versus both enzymes of 19 compounds reported in the literature to inhibit human and/or bacterial type II topoisomerases. We also used the assay to investigate the effect of ATP concentration on inhibitor potencies. (C) 2013 The Authors. Published by Elsevier Inc. All rights reserved.


Publication metadata

Author(s): Shapiro AB, Austin CA

Publication type: Article

Publication status: Published

Journal: Analytical Biochemistry

Year: 2014

Volume: 448

Pages: 23-29

Print publication date: 01/03/2014

Online publication date: 03/12/2013

Acceptance date: 24/11/2013

Date deposited: 12/05/2014

ISSN (print): 0003-2697

ISSN (electronic): 1096-0309

Publisher: Elsevier Inc.

URL: http://dx.doi.org/10.1016/j.ab.2013.11.029

DOI: 10.1016/j.ab.2013.11.029


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Funding

Funder referenceFunder name
LLR12031Leukaemia Lymphoma Research grant

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