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Single Molecule Enzymology pp 193–214Cite as

A Single-Molecule Approach to Visualize the Unwinding Activity of DNA Helicases

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 778))

Abstract

Almost all aspects of DNA metabolism involve separation of double-stranded DNA catalyzed by helicases. Observation and measurement of the dynamics of these events at the single-molecule level provide important mechanistic details of helicase activity and give the opportunity to probe aspects that are not revealed in bulk solution measurements. The assay, presented here, provides information about helicase unwinding rates and processivity. Visualization is achieved by using a fluorescent single-stranded DNA-binding protein (SSB), which allows the time course of individual DNA unwinding events to be observed using total internal reflection fluorescence microscopy. Observation of a prototypical helicase, Bacillus subtilis AddAB, shows that the unwinding process consists of bursts of unwinding activity, interspersed with periods of pausing.

An erratum to this chapter is available at http://dx.doi.org/10.1007/978-1-61779-261-8_19

An erratum to this chapter can be found at http://dx.doi.org/10.1007/978-1-61779-261-8_19

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Author information

Authors and Affiliations

  1. MRC National Institute for Medical Research, London, UK

    Natalia Fili, Christopher P. Toseland, Martin R. Webb & Justin E. Molloy

  2. École Polytechnique Fédérale de Lausanne, Lausanne, Switzerland

    Natalia Fili

  3. Institut für Zelluläre Physiologie, Physiologisches Institut, Ludwig Maximilians Universität, Munich, Germany

    Christopher P. Toseland

  4. DNA–Protein Interactions Unit, Department of Biochemistry, School of Medical Sciences, University of Bristol, Bristol, UK

    Mark S. Dillingham

Authors
  1. Natalia Fili
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  2. Christopher P. Toseland
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  3. Mark S. Dillingham
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  4. Martin R. Webb
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  5. Justin E. Molloy
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Corresponding author

Correspondence to Natalia Fili .

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Editors and Affiliations

  1. , Division of Physical Biochemistry, National Institute for Medical Research, The Ridgeway, Mill Hill, London, NW7 1AA, United Kingdom

    Gregory I. Mashanov

  2. Physiologisches Institute, Zelluläre Physiologie, Ludwig-Maximilian Universität München, Schillerstrasse 44, Munich, 80336, Germany

    Christopher Batters

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Fili, N., Toseland, C.P., Dillingham, M.S., Webb, M.R., Molloy, J.E. (2011). A Single-Molecule Approach to Visualize the Unwinding Activity of DNA Helicases . In: Mashanov, G., Batters, C. (eds) Single Molecule Enzymology. Methods in Molecular Biology, vol 778. Humana Press. https://doi.org/10.1007/978-1-61779-261-8_13

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  • DOI: https://doi.org/10.1007/978-1-61779-261-8_13

  • Published:

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-61779-260-1

  • Online ISBN: 978-1-61779-261-8

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