Fig. 6. Mu insertions generate replication-dependent DSBs in their vicinity.

Mu::TetO is detected by TetR-mCherry (labeled Mu-mCherry) and DSBs by either Gam-GFP or RecA-GFP in WT (BW25113) or its DnaEts derivative (SJ005). (A) Left, snapshot of Mu-mCherry foci relative to Gam-GFP foci upon Mu infection of WT. Right, quantitation of the position of the green and red foci. GFP foci were scored as ‘at Mu’ when they completely overlapped with mCherry foci, ‘near Mu’ when at least their edges touched, and ‘random’ when they did not touch. Background fluorescence with no detectable foci was scored ‘diffuse’. (B) As in A, but with a RecA-GFP expressing strain. Foci with large or aberrant morphology are classified ‘other’. Using microbeTracker software (Sliusarenko et al., 2011) the average area of a cell and of a fluorescent focus were calculated to be 1.78 μm² and 0.0675 μm² respectively, yielding a total of 26 independent sites that any focus can occupy in the cell. Two foci that touch occupy 4 times the area of one focus. The probability that any two foci will be stochastically near each other is then modeled by 4/26² or slightly less than 0.6%, well below the experimentally observed 50%, which include foci that completely overlap. (C) Left, replication-arrested cells were infected with Mu and monitored for Mu-mCherry and Gam-GFP foci (top panel). The same frame of cells was photographed after releasing the replication block by shifting cells to 25°C (bottom panel). White arrow points to GFP focus that appeared after the temperature shift. Right, quantitation of foci after fork release, as in A. See also Fig. S6. See Experimental Procedures for details.