Hexameric helicase G40P unwinds DNA in single base pair steps

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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.

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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

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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.

"This ORCID iD identifies the author of this article:" 0000-0002-6209-2364
Ganggang Wang

Molecular and Computational Biology, Department of Biological Sciences, University of Southern California, Los Angeles, United States

Competing interests
No competing interests declared.
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.

"This ORCID iD identifies the author of this article:" 0000-0001-9574-0551
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.

"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.

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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.