Repair on the go: E. coli maintains a high proliferation rate while repairing a chronic DNA double-strand break

Abstract

DNA damage checkpoints exist to promote cell survival and the faithful inheritance of genetic information. It is thought that one function of such checkpoints is to ensure that cell division does not occur before DNA damage is repaired. However, in unicellular organisms, rapid cell multiplication confers a powerful selective advantage, leading to a dilemma. Is the activation of a DNA damage checkpoint compatible with rapid cell multiplication? By uncoupling the initiation of DNA replication from cell division, the Escherichia coli cell cycle offers a solution to this dilemma. Here, we show that a DNA double-strand break, which occurs once per replication cycle, induces the SOS response. This SOS induction is needed for cell survival due to a requirement for an elevated level of expression of the RecA protein. Cell division is delayed, leading to an increase in average cell length but with no detectable consequence on mutagenesis and little effect on growth rate and viability. The increase in cell length caused by chronic DNA double-strand break repair comprises three components: two types of increase in the unit cell size, one independent of SfiA and SlmA, the other dependent of the presence of SfiA and the absence of SlmA, and a filamentation component that is dependent on the presence of either SfiA or SlmA. These results imply that chronic checkpoint induction in E. coli is compatible with rapid cell multiplication. Therefore, under conditions of chronic low-level DNA damage, the SOS checkpoint operates seamlessly in a cell cycle where the initiation of DNA replication is uncoupled from cell division.

Figure 1. SOS-induced level of RecA is required for viability following DNA cleavage at a palindrome.

Effect of SbcCD expression on the viability of lexA3 and lexA3 recAo^{C 281} mutant strains encoding P_BAD-sbcDC and containing or not the chromosomal 246 bp interrupted palindrome (PAL). Spot tests of ten-fold dilution series were carried out on LB plates complemented with either 0.2% of arabinose (SbcCD⁺) or 0.5% of glucose (SbcCD^-).

Figure 2. Cleavage of the 246 bp palindrome by SbcCD induces the SOS response.

Fluorescence distribution profiles of average Gray value in pixels of populations of sbcDC ⁺ or ΔsbcDC cells containing or not the chromosomal 246 bp interrupted palindrome (PAL) and carrying a plasmid encoding a P_sfiA-gfp fusion (pGB150). Error bars show the standard error of the mean of 4 independent experiments investigating 350 cells each. (A) In a wild-type background. (B) In a ΔslmA background.

Figure 3. Cleavage of a palindrome by SbcCD has no detectable impact on cell growth.

Graphs showing the growth level of sbcDC ⁺ and ΔsbcDC E. coli strains containing or not the chromosomal 246 bp interrupted palindrome (PAL). Error bars show the standard error of the mean of 3 independent experiments. (A) Dilution-adjusted optical density at 600 nm of cultures kept in exponential phase. (B) Dilution-adjusted average number of cells per microliter of cultures kept in exponential phase as counted by flow cytometry.

Figure 4. Effect of the cleavage of a palindrome by SbcCD quantified by competition experiments.

Competition experiments were carried out between PAL ⁺ yfp ⁺ and PAL ⁻ yfp ⁻ strains on one hand and PAL ⁺ yfp ⁻ and PAL ⁻ yfp ⁺ strains on the other hand. Cells were either allowed to reach stationary phase at night time (ON^stat; grown for more than 80 generations) or constantly kept in exponential phase (ON^4°C; grown for more than 60 generations). (A) Example of a graph showing the percentage of cells containing the chromosomal 246 bp interrupted palindrome (PAL) in function of the number of generations of co-culture with a strain that does not contain the palindrome. These results are from the second replicate of the competition experiments. (B) Table presenting the average percentage of loss per generation of strains containing the chromosomal 246 bp interrupted palindrome (PAL) during these competition experiments. Errors indicated between brackets are the standard error of the mean of 3 independent experiments.

Figure 5. SOS induction following the cleavage of a palindrome by SbcCD does not induce mutagenesis.

Fluctuation analysis measuring the rate of mutagenesis (mutation to rifampicin resistance cells) in sbcDC ⁺ and ΔsbcDC E. coli strains containing or not the chromosomal 246 bp interrupted palindrome (PAL). Error bars show the 95% confidence intervals.

Figure 6. Role of SfiA and SlmA in the delay of cell division following a chronic DSB.

Graphs displaying the cell length distribution in micrometers of sbcDC ⁺ and ΔsbcDC E. coli cells containing or not the chromosomal 246 bp interrupted palindrome (PAL). Error bars show the standard error of the mean of 4 independent experiments investigating 350 cells each. (A) In a wild-type background. (B) In a ΔsfiA background. (C) In a ΔslmA background. (D) In a ΔsfiA ΔslmA background.