Spatial and temporal organization of RecA in the Escherichia coli DNA-damage response

Abstract
Data availability
Article and author information
Metrics

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

Harshad Ghodke

School of Chemistry and Molecular Bioscience, University of Wollongong, Wollongong, Australia

Competing interests
The authors declare that no competing interests exist.

"This ORCID iD identifies the author of this article:" 0000-0002-6628-876X
Bishnu P Paudel

School of Chemistry and Molecular Bioscience, University of Wollongong, Wollongong, Australia

Competing interests
The authors declare that no competing interests exist.
Jacob S Lewis

School of Chemistry and Molecular Bioscience, University of Wollongong, Wollongong, Australia

Competing interests
The authors declare that no competing interests exist.
Slobodan Jergic

School of Chemistry and Molecular Bioscience, University of Wollongong, Wollongong, Australia

Competing interests
The authors declare that no competing interests exist.
Kamya Gopal

Department of Biochemistry, University of Wisconsin-Madison, Madison, United States

Competing interests
The authors declare that no competing interests exist.
Zachary J Romero

Department of Biochemistry, University of Wisconsin-Madison, Madison, United States

Competing interests
The authors declare that no competing interests exist.
Elizabeth A Wood

Department of Biochemistry, University of Wisconsin-Madison, Madison, United States

Competing interests
The authors declare that no competing interests exist.
Roger Woodgate

Laboratory of Genomic Integrity, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, United States

Competing interests
The authors declare that no competing interests exist.
Michael M Cox

Department of Biochemistry, University of Wisconsin-Madison, Madison, United States

Competing interests
The authors declare that no competing interests exist.

"This ORCID iD identifies the author of this article:" 0000-0003-3606-5722
Antoine M van Oijen

School of Chemistry and Molecular Bioscience, University of Wollongong, Wollongong, Australia

For correspondence
vanoijen@uow.edu.au

Competing interests
The authors declare that no competing interests exist.

"This ORCID iD identifies the author of this article:" 0000-0002-1794-5161

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.

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.