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Mapping E. coli Topoisomerase IV Binding and Activity Sites

Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1703)

Abstract

This methods article described a protocol aiming at mapping E. coli Topoisomerase IV (Topo IV) binding and cleavage activity sites on the genome. The approach is readily applicable to any Type II topoisomerase on a broad variety of gram-positive and gram-negative bacterial species. Conventional ChIP-seq of flag tagged Topo IV subunits and a novel method aimed at trapping only DNA bound to active Topo IV (called NorfliP) are described. NorfliP relies on the ability of norfloxacin, a quinolone drug, to cross-link the 5′ ends of the DNA breaks with the catalytic tyrosine of bacterial Type II topoisomerases. These methods give complementary results and their combination brought important insights on both the function and regulation of Topo IV.

Key words

Topoisomerase IV ChIP-seq NorflIP Norfloxacin 

Notes

Acknowledgments

This research was supported by funding to O.E. from Agence Nationale pour la Recherche (HiResBaCS ANR-15-CE11-0023).

References

  1. 1.
    Zechiedrich EL, Khodursky AB, Cozzarelli NR (1997) Topoisomerase IV, not gyrase, decatenates products of site-specific recombination in Escherichia coli. Genes Dev 11:2580–2592CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Kato J, Nishimura Y, Imamura R, Niki H, Hiraga S, Suzuki H (1990) New topoisomerase essential for chromosome segregation in E. coli. Cell 63:393–404CrossRefPubMedGoogle Scholar
  3. 3.
    Wang X, Reyes-Lamothe R, Sherratt DJ (2008) Modulation of Escherichia coli sister chromosome cohesion by topoisomerase IV. Genes Dev 22:2426–2433CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Lesterlin C, Gigant E, Boccard F, Espéli O (2012) Sister chromatid interactions in bacteria revealed by a site-specific recombination assay. EMBO J 31:3468–3479CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Cebrián J, Castán A, Martínez V, Kadomatsu-Hermosa MJ, Parra C, Fernández-Nestosa MJ, Schaerer C, Hernández P, Krimer DB, Schvartzman JB (2015) Direct evidence for the formation of precatenanes during DNA replication. J Biol Chem 290:13725–13735CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Peter BJ, Ullsperger C, Hiasa H, Marians KJ, Cozzarelli NR (1998) The structure of supercoiled intermediates in DNA replication. Cell 94:819–827CrossRefPubMedGoogle Scholar
  7. 7.
    Joshi MC, Magnan D, Montminy TP, Lies M, Stepankiw N, Bates D (2013) Regulation of sister chromosome cohesion by the replication fork tracking protein SeqA. PLoS Genet 9:e1003673CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Espéli O, Borne R, Dupaigne P, Thiel A, Gigant E, Mercier R, Boccard F (2012) A MatP-divisome interaction coordinates chromosome segregation with cell division in E. coli. EMBO J 31:3198–3211CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Nolivos S, Upton AL, Badrinarayanan A, Müller J, Zawadzka K, Wiktor J, Gill A, Arciszewska L, Nicolas E, Sherratt D (2016) MatP regulates the coordinated action of topoisomerase IV and MukBEF in chromosome segregation. Nat Commun 7:10466CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Espeli O, Levine C, Hassing H, Marians KJ (2003) Temporal regulation of topoisomerase IV activity in E. coli. Mol Cell 11:189–201CrossRefPubMedGoogle Scholar
  11. 11.
    El Sayyed H, Le Chat L, Lebailly E, Vickridge E, Pages C, Cornet F, Cosentino Lagomarsino M, Espéli O (2016) Mapping topoisomerase IV binding and activity sites on the E. coli genome. PLoS Genet 12:e1006025CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Hojgaard A, Szerlong H, Tabor C, Kuempel P (1999) Norfloxacin-induced DNA cleavage occurs at the dif resolvase locus in Escherichia coli and is the result of interaction with topoisomerase IV. Mol Microbiol 33:1027–1036CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2018

Authors and Affiliations

  1. 1.Center for Interdisciplinary Research in Biology (CIRB)College de France, CNRS/UMR 7241 – INSERM U1050, PSL Research UniversityParis Cedex 05France

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