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Probing Single Helicase Dynamics on Long Nucleic Acids Through Fluorescence-Force Measurement

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Optical Tweezers

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1486))

Abstract

Helicases are nucleic acid-dependent ATPases which can bind and remodel nucleic acids, protein–nucleic acid complexes, or both. They are involved in almost every step in cells related to nucleic acid metabolisms, including DNA replication and repair, transcription, RNA maturation and splicing, and nuclear export processes. Using single-molecule fluorescence-force spectroscopy, we have previously directly observed helicase translocation on long single-stranded DNA and revealed that two monomers of UvrD helicase are required for the initiation of unwinding function. Here, we present the details of fluorescence-force spectroscopy instrumentation, calibration, and activity assays in detail for observing the biochemical activities of helicases in real time and revealing how mechanical forces are involved in protein–nucleic acid interaction. These single-molecule approaches are generally applicable to many other protein–nucleic acid systems.

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Acknowledgements

T. H. is an Investigator of the Howard Hughes Medical Institute. This work is supported by NIH grant GM065367 and NSF grants PHY-1430124 to T. H. We would like to thank Olivia Yang for proofreading the manuscript and Dr. Kyung Suk Lee for constructing the original instrument and training.

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Authors and Affiliations

  1. Department of Biophysics and Biophysical Chemistry, Johns Hopkins University, Baltimore, MD, USA

    Chang-Ting Lin

  2. Department of Biophysics and Biophysical Chemistry, Thomas C. Jenkins Department of Biophysics and Department of Biomedical Engineering, Johns Hopkins University, Baltimore, MD, USA

    Taekjip Ha

  3. Howard Hughes Medical Institute, Baltimore, MD, USA

    Taekjip Ha

Authors
  1. Chang-Ting Lin
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  2. Taekjip Ha
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Correspondence to Taekjip Ha .

Editor information

Editors and Affiliations

  1. Department of Anatomy and Structural Biology & Gruss-Lipper Biophotonics Center, Albert Einstein College of Medicine, Bronx, New York, USA

    Arne Gennerich

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Lin, CT., Ha, T. (2017). Probing Single Helicase Dynamics on Long Nucleic Acids Through Fluorescence-Force Measurement. In: Gennerich, A. (eds) Optical Tweezers. Methods in Molecular Biology, vol 1486. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6421-5_11

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  • DOI: https://doi.org/10.1007/978-1-4939-6421-5_11

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-6419-2

  • Online ISBN: 978-1-4939-6421-5

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