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. 2007 Jun;39(6):797-802.
doi: 10.1038/ng2051. Epub 2007 May 27.

Spontaneous DNA breakage in single living Escherichia coli cells

Jeanine M Pennington  1 , Susan M Rosenberg
Affiliations

Spontaneous DNA breakage in single living Escherichia coli cells

Jeanine M Pennington et al. Nat Genet. 2007 Jun.

Erratum in

  • Nat Genet. 2007 Sep;39(9):1174

Abstract

Spontaneous DNA breakage is predicted to be a frequent, inevitable consequence of DNA replication and is thought to underlie much of the genomic change that fuels cancer and evolution. Despite its importance, there has been little direct measurement of the amounts, types, sources and fates of spontaneous DNA lesions in living cells. We present a direct, sensitive flow cytometric assay in single living Escherichia coli cells for DNA lesions capable of inducing the SOS DNA damage response, and we report its use in quantification of spontaneous DNA double-strand breaks (DSBs). We report efficient detection of single chromosomal DSBs and rates of spontaneous breakage approximately 20- to 100-fold lower than predicted. In addition, we implicate DNA replication in the origin of spontaneous DSBs with the finding of fewer spontaneous DSBs in a mutant with altered DNA polymerase III. The data imply that spontaneous DSBs induce genomic changes and instability 20-100 times more potently than previously appreciated. Finally, FACS demonstrated two main cell fates after spontaneous DNA damage: viability with or without resumption of proliferation.

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Figures

Figure 1
Figure 1
Steady-state levels of spontaneous RecB-dependent SOS induction demonstrate the presence of spontaneous DSBs and/or DSEs in a small cell subpopulation. (a–c) Representative flow cytometry histograms. (a) Positive control, GFP+ lexA51(Def) cells, and negative controls, lexA3(Ind−) and ΔrecA control cells. (b) Wild-type cells after exposure to ultraviolet C light. (c) Steady-state levels of SOS induction in wild-type, recA, recB and recB recA cells (full genotypes of all strains are given in Supplementary Methods and Supplementary Table 1). Horizontal bars represent the GFP+ gate. For b, ~ 0.9% of these cells are spontaneously green (see c,d), but the y-axis scale hides this shoulder to the main peak. For c, the recA and recB recA curves are both shown in gray, as the lines superimpose. (d) Quantification of steady-state levels of green wild-type cells (within the GFP+ gate) (mean ± s.e.m. of 18, 15, 7 and 4 experiments for wild-type, recB, recA and recB recA cells, respectively). In all figures (except where specified), sample groups with stars indicate statistical significance among all other starred sample groups using single-factor ANOVA and Tukey test, P < 0.01. Additionally, in Figure 1d, all strains are significantly different from all others (P < 0.01), except for recA and recB recA, which are not.
Figure 2
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 expression. 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′.
Figure 3
Figure 3
Induction of the LexA regulon of the SOS response is required for survival of cells (as determined by colony formation) with induced DSBs. (a) Irreparable DSBs. (b) Reparable DSBs. Note the much greater survival with homologous DNA present for repair (b). The fraction of cells surviving I-SceI induction in the presence of a single chromosomal I-SceI site is reduced in cells unable to induce expression of SOS genes, carrying the lexA3(Ind−) allele, and in cells lacking RecA and RecB proteins required for DSB repair (in addition to their roles in SOS induction). When irreparable DSBs are induced with I-SceI (a), constitutive high expression of LexA and SOS genes in lexA(Def) repressor-null mutant cells also improves survival (to 13% ± 1%) relative to wild-type (4% ± 0.4%). Data represent mean ± s.e.m. from four or five experiments. Stars indicate statistical significance calculated as in Figure 1 but versus wild-type cells only. In addition, the surviving fraction of wild-type cells with both I-SceI and cut site differs significantly from the surviving fraction of I-SceI-only cells and the surviving fraction of cut site–only cells, in both a and b. CFU, colony-forming units.
Figure 4
Figure 4
Reduction of spontaneous SOS induction from DSEs in cells having DNA Pol III with altered function. Labels are as in Figure 1. (a) Representative flow cytometry histograms of the isogenic strains indicated (Supplementary Table 1 for full genotypes). The dnaE915 antimutator allele of the gene encoding the DNA Pol III catalytic subunit is reviewed in the text. (b) Data represent mean ± s.e.m. from four experiments. As in Figure 1, sample groups marked with an asterisk differ significantly from all other sample groups with an asterisk (P < 0.01). Additionally, here, all sample groups differ significantly from all others (P < 0.01) except for recB versus dnaE915 recB, and recA versus dnaE915 recA, which do not differ significantly.
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Comment in

  • Scarce but scary.
    Bielinsky AK. Bielinsky AK. Nat Genet. 2007 Jun;39(6):707-8. doi: 10.1038/ng0607-707. Nat Genet. 2007. PMID: 17534363 No abstract available.

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