Synergy between RecBCD subunits is essential for efficient DNA unwinding
- Technion - Israel Institute of Technology, Israel
Abstract
The subunits of the bacterial RecBCD act in coordination, rapidly and processively unwinding DNA at the site of a double strand break. RecBCD is able to displace DNA binding proteins, suggesting that it generates high forces, but the specific role of each subunit in the force generation is unclear. Here, we present a novel optical tweezers assay that allows monitoring the activity of RecBCD's individual subunits, when they are part of an intact full complex. We show that RecBCD and its subunits are able to generate forces up to 25-40 pN without a significant effect on their velocity. Moreover, the isolated RecD translocates fast, but is a weak helicase with limited processivity. Experiments at a broad range of [ATP] and forces suggest that RecD unwinds DNA as a Brownian ratchet, rectified by ATP binding, and that the presence of the other subunits shifts the ratchet equilibrium towards the post-translocation state.
Data availability
All data generated during this study have been deposited in Dryad under accession code doi:10.5061/dryad.jb10510
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Data from: Synergy between RecBCD subunits is essential for efficient DNA unwindingDryad, 10.5061/dryad.jb10510.
Article and author information
Author details
Funding
Israel Science Foundation (1782/17)
- Ariel Kaplan
Israel Centers of Research Excellence (1902/12)
- Ariel Kaplan
Elyhau Pen Research Fund
- Ariel Kaplan
Israel Science Foundation (1403705/11)
- Arnon Henn
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Reviewing Editor
- Taekjip Ha, Johns Hopkins University School of Medicine, United States
Version history
- Received: August 6, 2018
- Accepted: January 1, 2019
- Accepted Manuscript published: January 2, 2019 (version 1)
- Version of Record published: January 18, 2019 (version 2)
Copyright
© 2019, Zananiri 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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Synergy between RecBCD subunits is essential for efficient DNA unwinding
eLife 8:e40836.
https://doi.org/10.7554/eLife.40836
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