Spatial and temporal organization of RecA in the Escherichia coli DNA-damage response
- University of Wollongong, Australia
- University of Wisconsin-Madison, United States
- National Institutes of Health, United States
Abstract
The RecA protein orchestrates the cellular response to DNA damage via its multiple roles in the bacterial SOS response. Lack of tools that provide unambiguous access to the various RecA states within the cell have prevented understanding of the spatial and temporal changes in RecA structure/function that underlie control of the damage response. Here, we develop a monomeric C-terminal fragment of the l repressor as a novel fluorescent probe that specifically interacts with RecA filaments on single-stranded DNA (RecA*). Single-molecule imaging techniques in live cells demonstrate that RecA is largely sequestered in storage structures during normal metabolism. Upon DNA damage, the storage structures dissolve and the cytosolic pool of RecA rapidly nucleates to form early SOS-signaling complexes, maturing into DNA-bound RecA bundles at later time points. Both before and after SOS induction, RecA* largely appears at locations distal from replisomes. Upon completion of repair, RecA storage structures reform.
Data availability
All data generated or analysed during this study are included in the manuscript and supporting files. Codes used for analysis are publicly available (in GitHub as described in previous publications). Scripts using these codes are also now provided in this submission as Source Code files for the relevant figures.
Article and author information
Author details
Antoine M van Oijen
Funding
Australian Research Council (DP150100956)
- Antoine M van Oijen
National Institutes of Health (GM32335)
- Michael M Cox
Australian Research Council (FL140100027)
- Antoine M van Oijen
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, United States
Version history
- Received: October 18, 2018
- Accepted: January 22, 2019
- Accepted Manuscript published: February 5, 2019 (version 1)
- Version of Record published: February 5, 2019 (version 2)
Copyright
This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.
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Spatial and temporal organization of RecA in the Escherichia coli DNA-damage response
eLife 8:e42761.
https://doi.org/10.7554/eLife.42761
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