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Optical Tweezers

Volume 1486 of the series Methods in Molecular Biology pp 295-316

Date:

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

  • Chang-Ting LinAffiliated withDepartment of Biophysics and Biophysical Chemistry, Johns Hopkins University
  • , Taekjip HaAffiliated withDepartment of Biophysics and Biophysical Chemistry, Thomas C. Jenkins Department of Biophysics and Department of Biomedical Engineering, Johns Hopkins UniversityHoward Hughes Medical Institute Email author 

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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