Single-molecule visualization of fast polymerase turnover in the bacterial replisome
- University of Wollongong, Australia
- University of Wisconsin-Madison, United States
- University of Groningen, Netherlands
Abstract
The Escherichia coli DNA replication machinery has been used as a road map to uncover design rules that enable DNA duplication with high efficiency and fidelity. Although the enzymatic activities of the replicative DNA Pol III are well understood, its dynamics within the replisome are not. Here we test the accepted view that the Pol III holoenzyme remains stably associated within the replisome. We use in vitro single-molecule assays with fluorescently labeled polymerases to demonstrate that the Pol III* complex (holoenzyme lacking the β2 sliding clamp), is rapidly exchanged during processive DNA replication. Nevertheless, the replisome is highly resistant to dilution in the absence of Pol III* in solution. We further show similar exchange in live cells containing labeled clamp loader and polymerase. These observations suggest a concentration-dependent exchange mechanism providing a balance between stability and plasticity, facilitating replacement of replisomal components dependent on their availability in the environment.
Article and author information
Author details
Antoine M van Oijen
Funding
Australian Research Council (DP150100956)
- Nicholas E Dixon
- Antoine M van Oijen
Australian Research Council (FL140100027)
- Antoine M van Oijen
National Institute of Health (GM32335)
- Michael M Cox
Fundamenteel onderzoek der materie (12CMCE03)
- Lisanne M Spenkelink
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: December 6, 2016
- Accepted: April 20, 2017
- Accepted Manuscript published: April 22, 2017 (version 1)
- Version of Record published: May 5, 2017 (version 2)
Copyright
© 2017, Lewis 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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Single-molecule visualization of fast polymerase turnover in the bacterial replisome
eLife 6:e23932.
https://doi.org/10.7554/eLife.23932
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