Molecular and General Genetics MGG

, Volume 218, Issue 1, pp 50–56

Initiation of DNA replication in Escherichia coli after overproduction of the DnaA protein

  • Kirsten Skarstad
  • Anders Løbner-Olesen
  • Tove Atlung
  • Kaspar von Meyenburg
  • Erik Boye
Article

DOI: 10.1007/BF00330564

Cite this article as:
Skarstad, K., Løbner-Olesen, A., Atlung, T. et al. Mol Gen Genet (1989) 218: 50. doi:10.1007/BF00330564

Summary

Flow cytometry was used to study initiation of DNA replication in Escherichia coli K12 after induced expression of a plasmid-borne dnaA+ gene. When the dnaA gene was induced from either the plac or the λpL promoter initiation was stimulated, as evidenced by an increase in the number of origins and in DNA content per mass unit. During prolonged growth under inducing conditions the origin and DNA content per mass unit were stabilized at levels significantly higher than those found before induction or in similarly treated control cells. The largest increase was observed when using the stronger promoter λpL compared to plac. Synchrony of initiation was reasonably well maintained with elevated DnaA protein concentrations, indicating that simultaneous initiation of all origins was still preferred under these conditions. A reduced rate of replication fork movement was found in the presence of rifampin when the DnaA protein was overproduced. We conclude that increased synthesis levels or increased concentrations of the DnaA protein stimulate initiation of DNA replication. The data suggest that the DnaA protein may be the limiting factor for initiation under normal physiological conditions.

Key words

Escherichia coli Initiation of DNA replication DnaA protein Flow cytometry 

Copyright information

© Springer-Verlag 1989

Authors and Affiliations

  • Kirsten Skarstad
    • 1
  • Anders Løbner-Olesen
    • 1
    • 2
  • Tove Atlung
    • 2
  • Kaspar von Meyenburg
    • 2
  • Erik Boye
    • 1
  1. 1.Department of Biophysics, Institute of Cancer ResearchThe Norwegian Radium HospitalOslo 3Norway
  2. 2.Department of MicrobiologyThe Technical University of DenmarkLyngbyDenmark
  3. 3.Department of BiochemistryStanford University School of MedicineStanfordUSA
  4. 4.Ciba-Geigy AGBaselSwitzerland

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