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Elife. 2019 Feb 5;8. pii: e42761. doi: 10.7554/eLife.42761.

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

Author information

1
Molecular Horizons and School of Chemistry and Molecular Bioscience, University of Wollongong, Wollongong, Australia.
2
Illawarra Health and Medical Research Institute, Wollongong, Australia.
3
Department of Biochemistry, University of Wisconsin-Madison, Madison, United States.
4
Laboratory of Genomic Integrity, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, 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 λ 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.

KEYWORDS:

DNA damage; DNA repair intermediates; E. coli; RecA; SOS response; live cell imaging; molecular biophysics; physics of living systems; single molecule imaging; structural biology

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