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Initiation of bacteriophage phi29 DNA replication in vivo: assembly of a membrane-associated multiprotein complex

A Bravo1, M Salas

  • 1Centro de Biología Molecular Severo Ochoa (CSIC-UAM), Universidad Autónoma Cantoblanco, Madrid, Spain.

Journal of Molecular Biology
|May 30, 1997
PubMed

Insights

Phage phi29 DNA replication initiation involves a membrane-associated complex of proteins p1, terminal protein (TP), and DNA polymerase. This complex formation is crucial for anchoring the viral DNA to the bacterial membrane during replication.

Area of Science:

  • * Molecular Biology
  • * Virology
  • * Biochemistry

Background:

  • * Initiation of phage phi29 DNA replication requires a heterodimer of terminal protein (TP) and viral DNA polymerase.
  • * TP acts as the primer for viral DNA synthesis.
  • * Protein p1 is essential for in vivo phi29 DNA replication.

Purpose of the Study:

  • * To investigate the role of membrane association in phage phi29 DNA replication initiation.
  • * To identify viral proteins involved in membrane association during replication.
  • * To elucidate the mechanism of membrane anchoring of the viral DNA.

Main Methods:

  • * Quantitative immunoblotting techniques were used to analyze membrane vesicles from infected Bacillus subtilis cells.
  • * Analysis of viral protein association with membranes under different conditions (DNA synthesis, replication block).
  • * Investigated the role of specific protein domains (C-terminal residues of p1) in membrane binding.

Main Results:

  • * Viral proteins p1 and free TP, along with a portion of the DNA polymerase, were found in membrane fractions during phage DNA synthesis.
  • * Increased DNA polymerase presence in membrane fractions was observed when replication was blocked.
  • * Both protein p1 and free TP demonstrated membrane affinity independently of viral DNA, with p1's association dependent on its C-terminal domain.

Conclusions:

  • * A membrane-associated multiprotein complex, including p1, free TP, and DNA polymerase, is likely involved in the initiation of in vivo phi29 DNA replication.
  • * Protein p1 and free TP mediate the membrane attachment of this complex.
  • * Free TP's membrane-binding and priming abilities facilitate the association of nascent viral DNA with the bacterial membrane.
  • * Terminal proteins (TPs) in prokaryotes may serve a dual role: priming DNA replication and anchoring linear DNA genomes to the bacterial membrane.

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