Abstract
Homologous recombination is essential for the accurate repair of double-stranded DNA breaks (DSBs)1. Initially, the RecBCD complex2 resects the ends of the DSB into 3′ single-stranded DNA on which a RecA filament assembles3. Next, the filament locates the homologous repair template on the sister chromosome4. Here we directly visualize the repair of DSBs in single cells, using high-throughput microfluidics and fluorescence microscopy. We find that, in Escherichia coli, repair of DSBs between segregated sister loci is completed in 15 ± 5 min (mean ± s.d.) with minimal fitness loss. We further show that the search takes less than 9 ± 3 min (mean ± s.d) and is mediated by a thin, highly dynamic RecA filament that stretches throughout the cell. We propose that the architecture of the RecA filament effectively reduces search dimensionality. This model predicts a search time that is consistent with our measurement and is corroborated by the observation that the search time does not depend on the length of the cell or the amount of DNA. Given the abundance of RecA homologues5, we believe this model to be widely conserved across living organisms.
Highlights
Formation of a DSB is followed by end processing by RecBCD, which removes ParB markers close to the break[8], and by activation of the SOS-response reporter (Fig. 1b)
To visualize the dynamics of the break site, we used a set of MalI markers integrated at distances that are not processed by RecBCD8,9 (−25 kb or +170 kb from the cut site) (Fig. 2b)
As the sister locus is no different from any other chromosomal locus until it has been located through search, we concluded that the colocalization of sister loci implies completed homology search (Extended Data Fig. 4e)
Summary
We propose that the stretched RecA–ssDNA filament—in a simple and elegant way—positions at least one ssDNA segment in the proximity of its homologous sister, such that the homologous dsDNA segment can find the ssDNA segment using a fast, short-range search. RecA is a prototypic member of the strand-exchange protein family, which is found in all forms of life and shares a common mechanism[3,5]. C. RecBCD enzyme and the repair of double-stranded DNA breaks. Direct observation of end resection by RecBCD during double-stranded DNA break repair in vivo. Quantitative genomic analysis of RecA protein binding during DNA double-strand break repair reveals RecBCD action in vivo. J. RecA bundles mediate homology pairing between distant sisters during DNA break repair. L. Dynamic formation of RecA filaments at DNA double strand break repair centers in live cells. Dynamics of RecA-mediated repair of replication-dependent DNA breaks. C. Single-molecule imaging of DNA pairing by RecA reveals a three-dimensional homology search. B. On the mechanism of homology search by RecA protein filaments. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/
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