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Spontaneous DNA breakage in single living Escherichia coli cells

Nature Genetics volume 39, pages 797–802 (2007)Cite this article

A Corrigendum to this article was published on 01 September 2007

This article has been updated

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 evolution1,2,3. 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 ∼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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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.
Figure 2: Efficient SOS induction by chromosomal DSBs.
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.
Figure 4: Reduction of spontaneous SOS induction from DSEs in cells having DNA Pol III with altered function.

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  • 29 August 2007

    In the version of this article initially published, our estimate of the rate of formation of spontaneous DNA double-strand breaks (DSBs) in E. coli proportional to DNA content in humans should read that it differs from that of Vilenchik and Knudson (Proc. Natl. Acad. Sci. USA 100, 12871–12876; 2003) by fourfold, not 'approximately tenfold' (page 800, line 3, and page 800, line 59). We estimated that there are 0.01 DSBs per E. coli genome replication. Because E. coli has approximately 4.7 x 106 bp per genome (Blattner, F.R. et al., Science 277, 1453–1474; 1997), we estimate that approximately 2 x 10–9 DSBs per bp are replicated, or about fourfold fewer than the estimate of about 0.8 x 10–8 DSBs per bp replicated in human somatic cells (or 50 DSBs per diploid human genome replication) from Vilenchik and Knudson (Proc. Natl. Acad. Sci. USA 100, 12871–12876; 2003). This would bring the number of DSBs per human genome replication down to approximately 13, if it were proportional to that in E. coli. Our error arose from calculating the human equivalent based on haploid, not diploid, human genome size. This error has been corrected in the HTML and PDF versions of the article.

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Acknowledgements

We thank the Baylor College of Medicine Flow Cytometry Core and S.A. Sincock and J.P. Robinson (Purdue Cytometry Laboratories) for help with flow cytometry; P.J. Hastings, D.B. Magner, S. Plon and G. Weinstock for discussions and D. Bates, A. Bielinsky, M.M. Cox, C. Herman, M.N. Hersh, G. Ira, M.-J. Lombardo, G.J. McKenzie, X. Pan, R. Rothstein and J.D. Wang for comments on the manuscript. This work was supported by a US Department of Defense Breast Cancer Research Program predoctoral fellowship (J.M.P.) and by US National Institutes of Health grants R01-GM53158 and R01-CA85777, equally.

Author information

Authors and Affiliations

  1. Interdepartmental Program in Cell and Molecular Biology and Department of Molecular and Human Genetics, Houston, 77030-3411, Texas, USA

    Jeanine M Pennington & Susan M Rosenberg

  2. Department of Biochemistry and Molecular Biology, Department of Molecular Virology and Microbiology and Dan L. Duncan Cancer Center, Baylor College of Medicine, One Baylor Plaza, Houston, 77030-3411, Texas, USA

    Susan M Rosenberg

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Contributions

S.M.R. conceived the study; J.M.P. and S.M.R. designed the study, analyzed the data and wrote the paper and J.M.P. performed the experiments.

Corresponding author

Correspondence to Susan M Rosenberg.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

One chromosome in most dnaA46(TS) cells at the restrictive temperature. (PDF 52 kb)

Supplementary Fig. 2

Chronic SOS induction in dnaA46(TS) cells. (PDF 54 kb)

Supplementary Fig. 3

Similar frequencies of spontaneous SOS induction during early, mid- and late-logarithmic phases of growth. (PDF 53 kb)

Supplementary Fig. 4

Low frequencies of inviable, propidium iodide-stained cells among spontaneously SOS-induced green cells. (PDF 78 kb)

Supplementary Table 1

E. coli K12 strains. (PDF 151 kb)

Supplementary Methods (PDF 183 kb)

Supplementary Note (PDF 230 kb)

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Pennington, J., Rosenberg, S. Spontaneous DNA breakage in single living Escherichia coli cells. Nat Genet 39, 797–802 (2007). https://doi.org/10.1038/ng2051

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