Fig. 1: High-throughput imaging of DSB repair.
From: RecA finds homologous DNA by reduced dimensionality search
a, Left, cartoon showing a mother machine device used to grow cells. Right, montage of a single growth channel showing SOS activation after induction of DSBs. b, Cartoon showing formation, processing and repair of a DSB. Cas9 binds to the cut site and creates a DSB. Next, RecBCD binds to DNA ends at the DSB site and begins end processing, ejecting ParB proteins and generating a 3′ ssDNA tail to which RecA binds and induces the SOS response. The RecA–ssDNA filament searches for homology, and after homology is located the DSB is repaired. c, A cell undergoing DSB repair. Loss and recovery of ParB focus are annotated. Cell outlines are displayed with white lines. d, The time of SOS induction in individual cells, defined as the time from the start of the Cas9 induction to the time at which the CFP signal from the SOS reporter reached half of the maximum signal. Only cells that increased CFP signal by more than fourfold were plotted. Data were aggregated from four (top) or three (middle, bottom) experiments. e, Localization of ParB foci along the cell length during DSB repair. Cells were oriented so that the remaining ParB focus was positioned on top. Time was aligned by ParB focus loss, as annotated in c. Insert shows a ParB channel of a single cell overlaid with outline and backbone (top), and mapping of the foci position along the cell’s backbone (bottom), n = 717 cells from 4 experiments. f, Generation time (top left), size at the division (top right) and growth rates (bottom) of cells undergoing repair of a single DSB and their descendants. The reference (no DSB) sample consists of the cells without a DSB that were born during the time window of maximal DSB induction. The line represents the median and error bars show the first and the third quartile, n = 60,405 cells from 4 experiments. g, DSB repair times, with gamma distribution fitted to the data, n = 765 cells from 4 experiments.
