1. Structural Biology and Molecular Biophysics
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Hexameric helicase G40P unwinds DNA in single base pair steps

  1. Michael Schlierf  Is a corresponding author
  2. Ganggang Wang
  3. Xiaojiang S Chen
  4. Taekjip Ha  Is a corresponding author
  1. TU Dresden, Germany
  2. University of Southern California, United States
  3. Johns Hopkins University, United States
Research Article
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Cite this article as: eLife 2019;8:e42001 doi: 10.7554/eLife.42001

Abstract

Most replicative helicases are hexameric, ring-shaped motor proteins that translocate on and unwind DNA. Despite extensive biochemical and structural investigations, how their translocation activity is utilized chemo-mechanically in DNA unwinding is poorly understood. We examined DNA unwinding by G40P, a DnaB-family helicase, using a single-molecule fluorescence assay with a single base pair resolution. The high-resolution assay revealed that G40P by itself is a very weak helicase that stalls at barriers as small as a single GC base pair and unwinds DNA with the step size of a single base pair. Binding of a single ATPgS could stall unwinding, demonstrating highly coordinated ATP hydrolysis between six identical subunits. We observed frequent slippage of the helicase, which is fully suppressed by the primase DnaG. We anticipate that these findings allow a better understanding on the fine balance of thermal fluctuation activation and energy derived from hydrolysis.

Data availability

All data generated or analysed during this study are included in the manuscript and supporting files. Due to their large size (~200Gb total), raw video data files are available upon request.

Article and author information

Author details

  1. Michael Schlierf

    B CUBE - Center for Molecular Bioengineering, TU Dresden, Dresden, Germany
    For correspondence
    michael.schlierf@tu-dresden.de
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-6209-2364

  2. Ganggang Wang

    Molecular and Computational Biology, Department of Biological Sciences, University of Southern California, Los Angeles, United States
    Competing interests
    No competing interests declared.

  3. Xiaojiang S Chen

    Molecular and Computational Biology, Department of Biological Sciences, University of Southern California, Los Angeles, United States
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-9574-0551

  4. Taekjip Ha

    Department of Biophysics and Biophysical Chemistry, Johns Hopkins University, Baltimore, United States
    For correspondence
    tjha@jhu.edu
    Competing interests
    Taekjip Ha, Reviewing editor, eLife.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-2195-6258

Funding

National Institutes of Health (GM065367)

  • Taekjip Ha

National Science Foundation (PHY-082261)

  • Taekjip Ha

Deutsche Forschungsgemeinschaft (SCHL1896/1-1)

  • Michael Schlierf

Bundesministerium für Bildung und Forschung (03Z2EN11)

  • Michael Schlierf

Howard Hughes Medical Institute

  • Taekjip Ha

National Institutes of Health (137405)

  • Xiaojiang S Chen

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Reviewing Editor

  1. Sebastian Deindl, Uppsala University, Sweden

Publication history

  1. Received: September 14, 2018
  2. Accepted: January 21, 2019
  3. Accepted Manuscript published: January 28, 2019 (version 1)
  4. Version of Record published: February 11, 2019 (version 2)

Copyright

© 2019, Schlierf et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

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