Figure 5. mCI stains RecA bundles after UV-damage.

(A) Montage of a single recA-gfp cell exhibiting large, dynamic RecA-GFP structures at late time points from Figure 5—video 1. Note that the immobilized cell divides at t = 1 hr. The daughter cell is carried away by flow. Cell outlines are provided as a guide to the eye. Time is indicated as hh:mm. (B) At late time points in the DNA damage response, PAmCherry-mCI forms large bundles in recA⁺ cells. Shown here is an example of an overlay of the mCI signal (magenta) and replisomal ϵ foci (cyan) at t = 45 min after 20 Jm⁻² UV (N = 4 independent experiments). Yellow arrows point to RecA bundles. For purposes of illustration, peaks in the ϵ images were enhanced using a discoidal average filter. (C) YPet-mCI also forms bundles (indicated by red arrows) in response to UV-damage in recA⁺ cells (N = 3 independent experiments). (D) Cells carrying the recA1 allele do not exhibit foci or bundle formation upon UV-irradiation under identical conditions as in panel (n > 250 cells from 12 fields of view at each of the 37 time points). Scale bar s correspond to 5 μm. See also Figure 5—figure supplement 1. Cell outlines provided as a guide to the eye.

Figure 5—figure supplement 1. Detection of RecA bundles using mCI in ΔuvrD cells.

Related to Figure 5. Fluorescence image of MG1655 ΔuvrD cells carrying pBAD-YPet-mcI (N = 1 repeat). Cells exhibit constitutive SOS and the presence of large RecA bundles (A) before and (B) 15 min after UV (20 Jm⁻²). Scale bar represents 5 μm. n > 100 cells for each condition.

Figure 5—video 1. Visualization of RecA-GFP bundles in recA-gfp cells after UV exposure.

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DOI: 10.7554/eLife.42761.014

Related to Figure 5. Cells were imaged in the 488 channel every 5 min for three hours following UV irradiation at t = 0 min. Here a video of a single cell growing under constant flow of growth medium is presented. Note the large, dynamic structure (termed a ‘RecA bundle’) that appears at later time points. Video is relayed at 15 fps.