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Detection of oriC-Independent Replication in Escherichia coli Cells

  • Makisha Martel
  • Aurélien Balleydier
  • Julien Brochu
  • Marc Drolet
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1703)

Abstract

In bacteria, replication of the chromosome is normally initiated following the binding of DnaA proteins to the oriC region. However, under certain circumstances, replication can also be initiated independent of the oriC/DnaA system. This is the case, for example, in Escherichia coli cells lacking RNase HI (rnha mutants) or type 1A topoisomerase activity (topA topB mutants). Here, we present a protocol in which replication from the oriC/DnaA system is first inhibited by the addition of the protein synthesis inhibitor, spectinomycin, to exponentially growing bacterial cell cultures. The thymidine analog, 5-ethynyl-2′-deoxyurdine (EdU) is then added to the cells, and after 1 h the cells are fixed and the Alexa Fluor® 488 dye is conjugated to EdU by the click-iT® reaction. The oriC-independent replication is detected in fixed cells by flow cytometry and can be visualized by fluorescence microscopy.

Key words

DNA replication EdU Bacteria R-loop oriC Topoisomerases 

Notes

Acknowledgments

This work was supported by a Discovery Grant from the Natural Sciences and Engineering Research Council of Canada (NSERC) to MD. AB and MM were supported by an Undergraduate Student Research Award from the NSERC.

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Copyright information

© Springer Science+Business Media, LLC 2018

Authors and Affiliations

  • Makisha Martel
    • 1
  • Aurélien Balleydier
    • 1
  • Julien Brochu
    • 1
  • Marc Drolet
    • 1
  1. 1.Département de microbiologie, infectiologie et immunologieUniversité de MontréalMontréalCanada

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