Direct Visualization of Helicase Dynamics Using Fluorescence Localization and Optical Trapping

C. T. Lin, Taekjip Ha

Research output: Chapter in Book/Report/Conference proceedingChapter

Abstract

Helicases control the accessibility of single-stranded (ss) nucleic acid (NA) generated as a transient intermediate during almost every step in cells related to nucleic acid metabolisms. For subsequent processing, however, helicases need to adjust the pace of unwinding adequately to avoid ssNA exposure to nucleases. Therefore, understanding how the unwinding process of helicases is regulated is crucial to address genome integrity and repair mechanisms. Using single-molecule fluorescence-force spectroscopy with fluorescence localization, we recently observed the stoichiometry of UvrD helicase, which determines the functions of UvrD: translocation and unwinding. For the first time, we provide direct evidence that a UvrD dimer is required to initiate the unwinding pathway. Moreover, with subpixel precision of fluorescence localization, the dynamic parameters of helicases can be obtained directly. Here, we present detailed single-molecule assays for observing the biochemical activities of helicases in real time and revealing how mechanical forces are involved in protein–nucleic acid interactions. These single-molecule approaches are generally applicable to many other protein–nucleic acid systems.

Original languageEnglish (US)
Title of host publicationMethods in Enzymology
PublisherAcademic Press Inc.
Pages121-136
Number of pages16
Volume582
DOIs
StatePublished - 2017

Publication series

NameMethods in Enzymology
Volume582
ISSN (Print)00766879
ISSN (Electronic)15577988

Keywords

  • Fluorescence localization
  • Helicase
  • Optical tweezers
  • TIRF
  • UvrD

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

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