Figure 2.

Efficient SOS induction by chromosomal DSBs. (a–c) Representative flow cytometry histograms and (d–f) quantification of multiple experiments, showing GFP⁺ cells after induction of I-SceI endonuclease in the presence of a single chromosomal I-SceI cut site. Horizontal bars in a–c represent GFP⁺ gate (Supplementary Methods). (a) SOS induction by I-SceI in log-phase cells with a single chromosomal cut site. (b) Efficient detection of a chromosomal DSB repaired (and removed) by homologous recombination with an F′ episome. (c) Detection of cells with only two DSEs (a single chromosome with an I-SceI-induced DSB) in dnaA(TS) cells with chromosome counts reduced to one (Supplementary Fig. 1 and Supplementary Note). Isogenic dnaA⁺ cells assayed in parallel produced profiles similar to those in a. Here and in a, there is a small enzyme- and cut site–dependent contribution to green cells independent of intentional induction, which probably reflects difficulty of repressing I-SceI expression⁸. In b, there is a low-level, repeatable F′-specific and I-SceI induction–specific SOS induction in all cells, perhaps suggesting F′-specific binding of I-SceI that induces a low-level response. (d–f) Multiple experiments assessing SOS and GFP induction and their dependence on RecA and RecB in response to I-SceI-mediated DSBs. Strains as in a–c, carrying a single chromosomal I-SceI cut site in the absence (d) or in the presence (e) of the F′ episome carrying the 20-kb homologous region for recombinational repair or in dnaA46(TS) cells (f) at restrictive temperature (one chromosome per cell; Supplementary Fig. 1). White and gray bars indicate percentages of cells within the green gate (shown in a–c) after repression and induction, respectively, of I-SceI. ‘DSB’ indicates that both I-SceI and a cut site were present. Shown are mean ± s.e.m. of three (d), three to seven (e) and three (f) experiments. Strain designated ‘F′ cut site’ (e) has a cut site disrupting the homology on the F′. This control shows that the reduction of green signal when homology is supplied for repair is caused by repair, not extraneous aspects of the F′.