Single-molecule magnetic tweezer analysis of topoisomerases

Kathryn H. Gunn, John F. Marko, Alfonso Mondragón*

*Corresponding author for this work

Research output: Chapter in Book/Report/Conference proceedingChapter

Abstract

Magnetic tweezers (MT) provide a powerful single-molecule approach to study the mechanism of topoisomerases, giving the experimenter the ability to change and read out DNA topology in real time. By using diverse DNA substrates, one can study different aspects of topoisomerase function and arrive at a better mechanistic understanding of these fascinating enzymes. Here we describe methods for the creation of three different DNA substrates used in MT experiments with topoisomerases: double-stranded DNA (dsDNA) tethers, “braided” (intertwined or catenated) DNA tether pairs, and dsDNA tethers with single-stranded DNA (ssDNA) regions. Additionally, we discuss how to build flow cells for bright-field MT microscopy, as well as how to noncovalently attach anti-digoxigenin to the coverslip surface for tethering digoxigenin-labeled DNAs. Finally, we describe procedures for the identification of a suitable DNA substrate for MT study and data collection.

Original languageEnglish (US)
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Pages139-152
Number of pages14
DOIs
StatePublished - Jan 1 2018

Publication series

NameMethods in Molecular Biology
Volume1703
ISSN (Print)1064-3745

Keywords

  • Bright-field microscopy
  • Flow cell
  • Functionalized DNA
  • Magnetic tweezers
  • Noncovalent antibody attachment
  • Single-molecule
  • Topoisomerases

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

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  • Cite this

    Gunn, K. H., Marko, J. F., & Mondragón, A. (2018). Single-molecule magnetic tweezer analysis of topoisomerases. In Methods in Molecular Biology (pp. 139-152). (Methods in Molecular Biology; Vol. 1703). Humana Press Inc.. https://doi.org/10.1007/978-1-4939-7459-7_10