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Related Experiment Videos

Structural changes in RepA, a plasmid replication initiator, upon binding to origin DNA.

Teresa Díaz-López1, Marta Lages-Gonzalo, Ana Serrano-López

  • 1Department of Molecular Microbiology, Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Cientificas (CSIC), C/Velázquez, 144, 28006 Madrid, Spain.

The Journal of Biological Chemistry
|March 15, 2003
PubMed
Summary
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Pseudomonas plasmid pPS10 initiator protein RepA dissociates into monomers upon binding iteron DNA, revealing an allosteric mechanism. Mutations in its leucine zipper motif mimic this monomerization, offering insights into DNA replication initiation.

Area of Science:

  • Molecular Biology
  • Biochemistry
  • Genetics

Background:

  • RepA protein initiates DNA replication for Pseudomonas plasmid pPS10.
  • RepA functions as a dimer repressing its own synthesis and as a monomer initiating replication.
  • RepA possesses two winged-helix (WH) domains, similar to eukaryotic and archaeal initiators.

Purpose of the Study:

  • To investigate the mechanism of RepA monomerization and conformational changes upon DNA binding.
  • To explore the role of the leucine zipper-like motif in RepA's N-terminal WH domain.
  • To understand the molecular basis of plasmid DNA replication initiation.

Main Methods:

  • In vitro DNA binding assays with RepA protein.
  • Analysis of RepA conformation and oligomerization state.

Related Experiment Videos

  • Site-directed mutagenesis of the putative leucine zipper in RepA.

    • Main Results:

    • Iteron DNA binding in vitro induces RepA dimer dissociation into monomers and alters protein conformation, indicating an allosteric effect.
    • Mutations replacing leucine residues with alanine in the leucine zipper motif (RepA-2L2A) induce similar conformational changes.
    • The RepA-2L2A mutant may represent a folding intermediate towards active monomers.

    Conclusions:

    • RepA monomerization and conformational changes are triggered by iteron DNA binding via an allosteric mechanism.
    • The leucine zipper motif is crucial for maintaining RepA's dimeric structure and influences its transition to active monomers.
    • These findings contribute to understanding the initiation of plasmid DNA replication and the role of initiator proteins.