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Single-Molecule Magnetic Tweezer Analysis of Topoisomerases

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Book cover DNA Topoisomerases

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1703))

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.

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References

  1. Charvin G, Strick TR, Bensimon D, Croquette V (2005) Tracking topoisomerase activity at the single-molecule level. Annu Rev Biophys Biomol Struct 34:201–219. https://doi.org/10.1146/annurev.biophys.34.040204.144433

    Article  CAS  PubMed  Google Scholar 

  2. Dekker NH, Rybenkov VV, Duguet M, Crisona NJ, Cozzarelli NR, Bensimon D, Croquette V (2002) The mechanism of type IA topoisomerases. Proc Natl Acad Sci U S A 99(19):12126–12131. https://doi.org/10.1073/pnas.132378799

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Baker NM, Rajan R, Mondragon A (2009) Structural studies of type I topoisomerases. Nucleic Acids Res 37(3):693–701. https://doi.org/10.1093/nar/gkn1009

    Article  CAS  PubMed  Google Scholar 

  4. Taneja B, Schnurr B, Slesarev A, Marko JF, Mondragon A (2007) Topoisomerase V relaxes supercoiled DNA by a constrained swiveling mechanism. Proc Natl Acad Sci U S A 104(37):14670–14675. https://doi.org/10.1073/pnas.0701989104

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Basu A, Parente AC, Bryant Z (2016) Structural dynamics and mechanochemical coupling in DNA gyrase. J Mol Biol 428(9 Pt B):1833–1845. https://doi.org/10.1016/j.jmb.2016.03.016

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Koster DA, Croquette V, Dekker C, Shuman S, Dekker NH (2005) Friction and torque govern the relaxation of DNA supercoils by eukaryotic topoisomerase IB. Nature 434(7033):671–674. https://doi.org/10.1038/nature03395

    Article  CAS  PubMed  Google Scholar 

  7. Strick TR, Croquette V, Bensimon D (2000) Single-molecule analysis of DNA uncoiling by a type II topoisomerase. Nature 404(6780):901–904. https://doi.org/10.1038/35009144

    Article  CAS  PubMed  Google Scholar 

  8. Ogawa T, Yogo K, Furuike S, Sutoh K, Kikuchi A, Kinosita K Jr (2015) Direct observation of DNA overwinding by reverse gyrase. Proc Natl Acad Sci U S A 112(24):7495–7500. https://doi.org/10.1073/pnas.1422203112

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Terekhova K, Gunn KH, Marko JF, Mondragon A (2012) Bacterial topoisomerase I and topoisomerase III relax supercoiled DNA via distinct pathways. Nucleic Acids Res 40(20):10432–10440. https://doi.org/10.1093/nar/gks780

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Terekhova K, Marko JF, Mondragon A (2014) Single-molecule analysis uncovers the difference between the kinetics of DNA decatenation by bacterial topoisomerases I and III. Nucleic Acids Res 42(18):11657–11667. https://doi.org/10.1093/nar/gku785

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Lionnet T, Allemand JF, Revyakin A, Strick TR, Saleh OA, Bensimon D, Croquette V (2012) Magnetic trap construction. Cold Spring Harb Protoc 2012(1):133–138. https://doi.org/10.1101/pdb.prot067496

    PubMed  Google Scholar 

  12. Gosse C, Croquette V (2002) Magnetic tweezers: micromanipulation and force measurement at the molecular level. Biophys J 82(6):3314–3329. https://doi.org/10.1016/S0006-3495(02)75672-5

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Bai H, Sun M, Ghosh P, Hatfull GF, Grindley ND, Marko JF (2011) Single-molecule analysis reveals the molecular bearing mechanism of DNA strand exchange by a serine recombinase. Proc Natl Acad Sci U S A 108(18):7419–7424. https://doi.org/10.1073/pnas.1018436108

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Strick TR, Allemand JF, Bensimon D, Bensimon A, Croquette V (1996) The elasticity of a single supercoiled DNA molecule. Science 271(5257):1835–1837. https://doi.org/10.1126/science.271.5257.1835

  15. Gunn KH, Marko JF, Mondragon A (2017) An orthogonal single-molecule experiment reveals multiple-attempt dynamics of type IA topoisomerases. Nat Struct Mol Biol 24(5):484–490. https://doi.org/10.1038/nsmb.3401

  16. Joo C, Ha T (2012) Preparing sample chambers for single-molecule FRET. Cold Spring Harb Protoc 2012(10):1104–1108. https://doi.org/10.1101/pdb.prot071530

    PubMed  Google Scholar 

  17. Burnham DR, De Vlaminck I, Henighan T, Dekker C (2014) Skewed brownian fluctuations in single-molecule magnetic tweezers. PLoS One 9(9):e108271. https://doi.org/10.1371/journal.pone.0108271

    Article  PubMed  PubMed Central  Google Scholar 

  18. Graham JS, Johnson RC, Marko JF (2011) Counting proteins bound to a single DNA molecule. Biochem Biophys Res Commun 415(1):131–134. https://doi.org/10.1016/j.bbrc.2011.10.029

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Seol Y, Neuman KC (2011) Single-molecule measurements of topoisomerase activity with magnetic tweezers. Methods Mol Biol 778:229–241. https://doi.org/10.1007/978-1-61779-261-8_15

    Article  CAS  PubMed  Google Scholar 

  20. Charvin G, Bensimon D, Croquette V (2003) Single-molecule study of DNA unlinking by eukaryotic and prokaryotic type-II topoisomerases. Proc Natl Acad Sci U S A 100(17):9820–9825. https://doi.org/10.1073/pnas.1631550100

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgments

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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Authors and Affiliations

  1. Department of Molecular Biosciences, Northwestern University, Evanston, IL, USA

    Kathryn H. Gunn & John F. Marko

  2. Department of Physics and Astronomy, Northwestern University, Evanston, IL, USA

    John F. Marko

  3. Department of Molecular Biosciences, Northwestern University, 2205 Tech Drive, Evanston, IL, 60208, USA

    Alfonso Mondragón

Authors
  1. Kathryn H. Gunn
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  2. John F. Marko
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  3. Alfonso Mondragón
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Corresponding author

Correspondence to Alfonso Mondragón .

Editor information

Editors and Affiliations

  1. Département de microbiologie, infectiologie et immunologie, Université de Montréal, Montréal, Québec, Canada

    Marc Drolet

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Gunn, K.H., Marko, J.F., Mondragón, A. (2018). Single-Molecule Magnetic Tweezer Analysis of Topoisomerases. In: Drolet, M. (eds) DNA Topoisomerases. Methods in Molecular Biology, vol 1703. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7459-7_10

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  • DOI: https://doi.org/10.1007/978-1-4939-7459-7_10

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7458-0

  • Online ISBN: 978-1-4939-7459-7

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