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

Protocol
Part of the Methods in Molecular Biology book series (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.

Key words

Single-molecule TIRF Optical tweezers Fluorescence localization Helicase UvrD NS3 Translocation Unwinding 

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Copyright information

© Springer Science+Business Media New York 2017

Authors and Affiliations

  1. 1.Department of Biophysics and Biophysical ChemistryJohns Hopkins UniversityBaltimoreUSA
  2. 2.Department of Biophysics and Biophysical Chemistry, Thomas C. Jenkins Department of Biophysics and Department of Biomedical EngineeringJohns Hopkins UniversityBaltimoreUSA
  3. 3.Howard Hughes Medical InstituteBaltimoreUSA

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