Mechanistic aspects of DnaA-RepA interaction as revealed by yeast forward and reverse two-hybrid analysis

R. Sharma, A. Kachroo, D. Bastia

Research output: Contribution to journalArticlepeer-review

25 Scopus citations

Abstract

Using yeast forward and reverse two-hybrid analysis and biochemical techniques, we present novel and definitive in vivo and in vitro evidence that both the N-terminal domain I and C-terminal domain IV of the host-encoded DnaA initiator protein of Escherichia coli interact physically with plasmid-encoded RepA initiator of pSC101. The N-terminal, but not the C-terminal, region of RepA interacted with DnaA in vitro. These protein-protein interactions are critical for two very early steps of replication initiation, namely origin unwinding and helicase loading. Neither domain I nor IV of DnaA could individually collaborate with RepA to promote pSC101 replication. However, when the two domains are co-expressed within a common cell milieu and allowed to associate non-covalently with each other via a pair of leucine zippers, replication of the plasmid was supported in vivo. Thus, the result shows that physical tethering, either non-covalent or covalent, of domain I and IV of DnaA and interaction of both domains with RepA, are critical for replication initiation. The results also provide the molecular basis for a novel, potential, replication-based bacterial two-hybrid system.

Original languageEnglish
Pages (from-to)4577-4587
Number of pages11
JournalEMBO Journal
Volume20
Issue number16
DOIs
StatePublished - Aug 15 2001

Keywords

  • Helicase loading
  • Ori unwinding
  • Protein-protein interaction
  • Replication initiation
  • Yeast reverse two-hybrid

ASJC Scopus subject areas

  • Neuroscience (all)
  • Molecular Biology
  • Biochemistry, Genetics and Molecular Biology (all)
  • Immunology and Microbiology (all)

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