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Protein-nucleic acid interactions in bacteriophage phi 29 DNA replication

M Salas1, R Freire, M S Soengas

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

FEMS Microbiology Reviews
|August 1, 1995
PubMed
Summary

This study details the phi 29 DNA replication mechanism, highlighting protein priming and the roles of viral proteins p6 and p5. The research demonstrates efficient in vitro amplification of phi 29 DNA using these proteins.

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Area of Science:

  • Molecular Biology
  • Virology
  • Biochemistry

Background:

  • Phi 29 DNA replication initiates via a protein priming mechanism at DNA ends.
  • The process involves a terminal protein-dAMP initiation complex and a sliding-back mechanism for sequence maintenance.

Purpose of the Study:

  • To elucidate the structure-function relationships of phi 29 DNA polymerase domains.
  • To investigate the role of viral proteins p6 and p5 in phi 29 DNA replication.
  • To demonstrate the feasibility of in vitro amplification of the phi 29 DNA molecule.

Main Methods:

  • Site-directed mutagenesis of conserved amino acids in phi 29 DNA polymerase.
  • Structural determination of the protein p6-DNA complex.
  • Biochemical characterization of the phi 29 single-stranded DNA binding (SSB) protein (p5).

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Main Results:

  • The N-terminal domain of phi 29 DNA polymerase possesses 3'-5' exonuclease and strand-displacement activities, while the C-terminal domain handles initiation and polymerization.
  • Viral protein p6, through an alpha-helical structure in its N-terminal region, binds DNA via the minor groove, stimulating replication initiation.
  • Viral protein p5 functions as an SSB protein, binding displaced ssDNA and unwinding duplex DNA.
  • In vitro amplification of phi 29 DNA by 4000-fold was achieved using terminal protein, DNA polymerase, p6, and p5, yielding infective DNA.

Conclusions:

  • The distinct domains of phi 29 DNA polymerase are crucial for its diverse functions.
  • Viral proteins p6 and p5 play essential, coordinated roles in phi 29 DNA replication.
  • A complete system of viral proteins enables highly efficient in vitro amplification of phi 29 DNA with retained infectivity.