Direct Fluorescent Imaging of Translocation and Unwinding by Individual DNA Helicases

T. L. Pavankumar, J. C. Exell, S. C. Kowalczykowski

Research output: Chapter in Book/Report/Conference proceedingChapter

2 Scopus citations

Abstract

The unique translocation and DNA unwinding properties of DNA helicases can be concealed by the stochastic behavior of enzyme molecules within the necessarily large populations used in ensemble experiments. With recent technological advances, the direct visualization of helicases acting on individual DNA molecules has contributed significantly to the current understanding of their mechanisms of action and biological functions. The combination of single-molecule techniques that enable both manipulation of individual protein or DNA molecules and visualization of their actions has made it possible to literally see novel and unique biochemical characteristics that were previously masked. Here, we describe the execution and use of single-molecule fluorescence imaging techniques, focusing on methods that include optical trapping in conjunction with epifluorescent imaging, and also surface immobilization in conjunction with total internal reflection fluorescence visualization. Combined with microchannel flow cells and microfluidic control, these methods allow individual fluorescently labeled protein and DNA molecules to be imaged and tracked, affording measurement of DNA unwinding and translocation at single-molecule resolution.

Original languageEnglish (US)
Title of host publicationMethods in Enzymology
PublisherAcademic Press Inc.
Pages1-32
Number of pages32
Volume581
DOIs
StatePublished - 2016

Publication series

NameMethods in Enzymology
Volume581
ISSN (Print)00766879
ISSN (Electronic)15577988

Keywords

  • DNA helicase
  • DNA motor protein
  • DNA translocation
  • DNA unwinding
  • Microfluidics
  • Optical trapping
  • Single-molecule imaging
  • Single-stranded DNA sensor
  • TIRF
  • Total internal reflection fluorescence

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

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    Pavankumar, T. L., Exell, J. C., & Kowalczykowski, S. C. (2016). Direct Fluorescent Imaging of Translocation and Unwinding by Individual DNA Helicases. In Methods in Enzymology (Vol. 581, pp. 1-32). (Methods in Enzymology; Vol. 581). Academic Press Inc.. https://doi.org/10.1016/bs.mie.2016.09.010