Single-Molecule Magnetic Tweezer Analysis of Topoisomerases
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.
Key wordsSingle-molecule Magnetic tweezers Functionalized DNA Flow cell Noncovalent antibody attachment Bright-field microscopy Topoisomerases
We thank members of the Mondragón and Marko laboratories for discussions and assistance. Research was supported by the NIH (R01 GM051350 to A.M., and R01 GM105847 and U54 CA193419 (CR-PS-OC) to J.F.M.) and the NSF (MCB-1022117 and DMR-1206868 to J.F.M.). K.H.G. was supported by a Dr. John N. Nicholson Fellowship and an NRSA predoctoral training grant (T32 GM008382).
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