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

  • Natalia FiliEmail author
  • Christopher P. Toseland
  • Mark S. Dillingham
  • Martin R. Webb
  • Justin E. Molloy
Protocol
Part of the Methods in Molecular Biology book series (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.

Key words

DNA unwinding Helicase SSB Single molecule TIRF microscopy 

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Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Natalia Fili
    • 1
    • 2
    Email author
  • Christopher P. Toseland
    • 1
    • 3
  • Mark S. Dillingham
    • 4
  • Martin R. Webb
    • 1
  • Justin E. Molloy
    • 1
  1. 1.MRC National Institute for Medical ResearchLondonUK
  2. 2.École Polytechnique Fédérale de LausanneLausanneSwitzerland
  3. 3.Institut für Zelluläre Physiologie, Physiologisches InstitutLudwig Maximilians UniversitätMunichGermany
  4. 4.DNA–Protein Interactions Unit, Department of Biochemistry, School of Medical SciencesUniversity of BristolBristolUK

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