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Single-Molecule FRET Analysis of Replicative Helicases

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Molecular Motors

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1805))

Abstract

Over the recent years single-molecule fluorescence resonance energy transfer (smFRET) technique has proven to be one of the most powerful tools for revealing mechanistic insights into helicase activities. Here we describe details of single-molecule FRET assays for probing DNA unwinding activities as well as functional dynamics by replicative helicases in real time. The ability of smFRET to measure the behavior of biomolecules at a nanometer scale enabled us to address how the leading and lagging strand synthesis are coordinated during DNA replication, to resolve DNA unwinding steps of Bacteriophage T7 helicase, and to observe heterogeneous unwinding patterns modulated by the DNA binding domain of E1 helicase. These single-molecule FRET assays are generally applicable to other replicative and nonreplicative hexameric helicases.

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Acknowledgments

The authors would like to thank all the members of Ha laboratory for valuable discussions and experimental help. These studies were supported by grants from the National Institute of Health and the National Science Foundation. T.H. is an Investigator with the Howard Hughes Medical Institute.

Author information

Authors and Affiliations

  1. Department of Physics and Astronomy, Research Institute for Basic Sciences, Seoul National University, Seoul, South Korea

    Seung-Jae Lee

  2. Division of Abbott Diagnostics, Abbott Laboratories, Chicago, IL, USA

    Salman Syed

  3. Department of Biophysics and Biophysical Chemistry, Howard Hughes Medical Institute, Johns Hopkins University, Baltimore, MD, USA

    Taekjip Ha

  4. Department of Biophysics, Howard Hughes Medical Institute, Johns Hopkins University, Baltimore, MD, USA

    Taekjip Ha

  5. Department of Biomedical Engineering, Howard Hughes Medical Institute, Johns Hopkins University, Baltimore, MD, USA

    Taekjip Ha

Authors
  1. Seung-Jae Lee
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  2. Salman Syed
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  3. Taekjip Ha
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Corresponding author

Correspondence to Taekjip Ha .

Editor information

Editors and Affiliations

  1. National Museum of Natural History, CNRS UMR7196 / INSERM U1154, Paris, France

    Christophe Lavelle

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Lee, SJ., Syed, S., Ha, T. (2018). Single-Molecule FRET Analysis of Replicative Helicases. In: Lavelle, C. (eds) Molecular Motors. Methods in Molecular Biology, vol 1805. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8556-2_12

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  • DOI: https://doi.org/10.1007/978-1-4939-8556-2_12

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-8554-8

  • Online ISBN: 978-1-4939-8556-2

  • eBook Packages: Springer Protocols

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