DNA replication in phage P4: characterization of replicon II

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Francesca Magnoni1, Claudia Sala, Francesca Forti

  • 1Dipartimento di Scienze Biomolecolari e Biotecnologie, Università degli Studi di Milano, Via Celoria 26, 20133 Milano, Italy.

Plasmid
|August 16, 2006
PubMed

Insights

The P4 phage

Area of Science:

  • Molecular Biology
  • Virology
  • Bacteriology

Background:

  • The P4 phage is a genetic element that replicates in Escherichia coli as both a satellite phage and a plasmid.
  • It possesses two overlapping replicons, replicon I and replicon II, each with specific replication requirements.
  • The P4 alpha gene product, a protein with primase and helicase activities, is crucial for the replication of both replicons.

Purpose of the Study:

  • To investigate the replication mechanism of replicon II in P4 phage.
  • To identify the essential DNA regions and their functional roles in replicon II replication.
  • To explore the interaction of host proteins with the cis-acting elements of replicon II.

Main Methods:

  • Site-directed mutagenesis to identify essential replication regions.
  • Electrophoretic mobility shift assays (EMSAs) to study protein-DNA interactions.
  • Replication assays to assess the functional impact of mutations and DNA regions.

Main Results:

  • The relative orientation of ori2 and crr sites is critical for replicon II replication.
  • The ori2 region was delimited to a 22 bp sequence within the alpha gene, essential for replication.
  • Mutagenesis of ori2 abolished replicon II replication, indicating its functional importance.
  • EMSAs revealed specific binding of host proteins to ori1 and crr, but not to ori2, suggesting a unique replication initiation mechanism for replicon II.

Conclusions:

  • Replicon II replication in P4 phage is dependent on the ori2 and crr sites, with their orientation being crucial.
  • The ori2 region, located within the alpha gene, is a key cis-acting element for replicon II replication.
  • Unlike ori1 and crr, ori2 does not appear to bind P4 alpha or other bacterial proteins, implying a distinct initiation mechanism.

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