A novel decatenation assay for DNA topoisomerases using a singly-linked catenated substrate

Waraich, N. F., Jain, S., Colloms, S. D. , Stark, W. M. , Burton, N. P. and Maxwell, A. (2020) A novel decatenation assay for DNA topoisomerases using a singly-linked catenated substrate. BioTechniques, 69(5), pp. 356-362. (doi: 10.2144/btn-2020-0059) (PMID:33000631)

Abstract

Decatenation is a crucial in vivo reaction of DNA topoisomerases in DNA replication and is frequently used in in vitro drug screening. Usually this reaction is monitored using kinetoplast DNA as a substrate, although this assay has several limitations. Here we have engineered a substrate for Tn3 resolvase that generates a singly-linked catenane that can readily be purified from the DNA substrate after restriction enzyme digestion and centrifugation. We show that this catenated substrate can be used with high sensitivity in topoisomerase assays and drug-inhibition assays.

• Item Type: Articles
• Additional Information: Work in A Maxwell's laboratory was supported by the Biotechnology and Biosciences Research Council (UK) Institute Strategic Programme Grant BB/P012523/1, and the Wellcome Trust (Investigator Award 110072/Z/15/Z). NF Waraich was funded by a BBSRC-CASE studentship (BB/M011216/1) supported by Inspiralis Ltd. SD Colloms was funded by a Leverhulme Trust grant (RP2013-K-017).
• Status: Published
• Refereed: Yes
• Glasgow Author(s) Enlighten ID: Stark, Professor Marshall and Colloms, Dr Sean
• Authors: Waraich, N. F., Jain, S., Colloms, S. D., Stark, W. M., Burton, N. P., and Maxwell, A.
• College/School: College of Medical Veterinary and Life Sciences > School of Molecular Biosciences
• Journal Name: BioTechniques
• Publisher: Future Science Group
• ISSN: 0736-6205
• ISSN (Online): 1940-9818
• Published Online: 01 October 2020
• Copyright Holders: Copyright © 2020 Anthony Maxwell
• First Published: First published in BioTechniques 69(5):356-362
• Publisher Policy: Reproduced under a Creative Commons license