Abstract
DNA double-strand break (DSB) is one of the most genotoxic lesions, and unrepaired DSBs can lead to chromosomal instability and eventually cause cell death. Quantitative markers, such as phosphorylated histone H2AX (γ-H2AX) and p53-binding protein 1 (53BP1) foci in mammalian cells, are not available for the detection of DSBs in prokaryotes. Therefore, as an alternative method, pulsed-field gel electrophoresis (PFGE) is widely used to analyze broken DNA molecules by separating them from intact DNA. Here, we examined the accumulation of bleomycin (BLM)-induced DSBs by PFGE, using a rotating gel electrophoresis (RGE) system. We defined two sets of parameters with distinct advantages; the first one focuses on the analysis of the size of the broken DNA fragments, whereas the second allows for the direct comparison of the accumulation of DSBs among strains and treatments. This method represents a powerful tool for the study of genomic integrity and the characterization of genotoxic substances.
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References
Hanada K, Yamashita T, Shobuike Y, Ikeda H (2001) Role of DnaB helicase in UV-induced illegitimate recombination in Escherichia coli. J Bacteriol 183:4964–4969
Shiraishi K, Ogata Y, Hanada K, Kano Y, Ikeda H (2007) Roles of the DNA binding proteins H-NS and StpA in homologous recombination and repair of bleomycin-induced damage in Escherichia coli. Genes Genet Syst 82:433–439
Michel B, Sinha AK, Leach DRF (2018) Replication fork breakage and restart in Escherichia coli. Microbiol Mol Biol Rev 82
Seigneur M, Bidnenko V, Ehrlich SD, Michel B (1998) RuvAB acts at arrested replication forks. Cell 95:419–430
Handa N, Kobayashi I (2003) Accumulation of large non-circular forms of the chromosome in recombination-defective mutants of Escherichia coli. BMC Mol Biol 4:5
Kowalczykowski SC (2015) An Overview of the molecular mechanisms of recombinational DNA repair. Cold Spring Harb Perspect Biol 7:a016410
Hanada K, Ukita T, Kohno Y, Saito K, Kato J, Ikeda H (1997) RecQ DNA helicase is a suppressor of illegitimate recombination in Escherichia coli. Proc Natl Acad Sci U S A 94:3860–3865
Hanada K, Iwasaki M, Ihashi S, Ikeda H (2000) UvrA and UvrB suppress illegitimate recombination: synergistic action with RecQ helicase. Proc Natl Acad Sci U S A 97:5989–5994
Courcelle J, Wendel BM, Livingstone DD, Courcelle CT (2015) RecBCD is required to complete chromosomal replication: implications for double-strand break frequencies and repair mechanisms. DNA Repair (Amst) 32:86–95
Smith GR (2012) How RecBCD enzyme and Chi promote DNA break repair and recombination: a molecular biologist’s view. Microbiol Mol Biol Rev 76(2):217–228
Bell JC, Kowalczykowski SC (2016) RecA: regulation and mechanism of a molecular search engine. Trends Biochem Sci 41:491–507
Muller B, Jones C, West SC (1990) T7 endonuclease I resolves Holliday junctions formed in vitro by RecA protein. Nucleic Acids Res 18:5633–5636
West SC (1997) Processing of recombination intermediates by the RuvABC proteins. Annu Rev Genet 31:213–244
Rogakou EP, Boon C, Redon C, Bonner WM (1999) Megabase chromatin domains involved in DNA double-strand breaks in vivo. J Cell Biol 146:905–916
Huyen Y, Zgheib O, Ditullio RA Jr, Gorgoulis VG, Zacharatos P, Petty TJ, Sheston EA, Mellert HS, Stavridi ES, Halazonetis TD (2004) Methylated lysine 79 of histone H3 targets 53BP1 to DNA double-strand breaks. Nature 432:406–411
Michel B, Ehrlich SD, Uzest M (1997) DNA double-strand breaks caused by replication arrest. EMBO J 16:430–438
Xu T, Brown W, Marinus MG (2012) Bleomycin sensitivity in Escherichia coli is medium-dependent. PLoS One 7:e33256
Gutteridge JM, West M, Eneff K, Floyd RA (1990) Bleomycin-iron damage to DNA with formation of 8-hydroxydeoxyguanosine and base propenals. Indications that xanthine oxidase generates superoxide from DNA degradation products. Free Radic Res Commun 10:159–165
Chen J, Ghorai MK, Kenney G, Stubbe J (2008) Mechanistic studies on bleomycin-mediated DNA damage: multiple binding modes can result in double-stranded DNA cleavage. Nucleic Acids Res 36:3781–3790
Acknowledgments
All bacterial strains used in this study were provided by the National BioResource Project (NBRP) E. coli Strain collection (Microbial Genetics Laboratory, National Institute of Genetics, Mishima, Japan).
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Inoue, N., Narahara, H., Nishida, Y., Hanada, K. (2020). Detection of Bleomycin-Induced DNA Double-Strand Breaks in Escherichia coli by Pulsed-Field Gel Electrophoresis Using a Rotating Gel Electrophoresis System. In: Hanada, K. (eds) DNA Electrophoresis. Methods in Molecular Biology, vol 2119. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0323-9_14
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DOI: https://doi.org/10.1007/978-1-0716-0323-9_14
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