Single-Molecule Magnetic Tweezer Analysis of Topoisomerases

Overview

Journal Methods Mol Biol

Specialty Molecular Biology

Date 2017 Nov 28

PMID 29177739

Citations 3

Authors

Kathryn H Gunn

John F Marko

Alfonso Mondragon

Affiliations

Soon will be listed here.

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.

Citing Articles

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Unravelling the mechanisms of Type 1A topoisomerases using single-molecule approaches.

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