Compartmentalization of phage phi29 DNA replication: interaction between the primer terminal protein and the membrane-associated protein p1

EMBO J. 2000 Oct 16;19(20):5575-84. doi: 10.1093/emboj/19.20.5575.

Abstract

The bacteriophage phi29 replication protein p1 (85 amino acids) is membrane associated in Bacillus subtilis-infected cells. The C-terminal 52 amino acid residues of p1 are sufficient for assembly into protofilament sheet structures. Using chemical cross-linking experiments, we demonstrate here that p1DeltaC43, a C-terminally truncated p1 protein that neither associates with membranes in vivo nor self-interacts in vitro, can interact with the primer terminal protein (TP) in vitro. Like protein p1, plasmid-encoded protein p1DeltaC43 reduces the rate of phi29 DNA replication in vivo in a dosage-dependent manner. We also show that truncated p1 proteins that retain the N-terminal 42 amino acids, when present in excess, interfere with the in vitro formation of the TP.dAMP initiation complex in a reaction that depends on the efficient formation of a primer TP-phi29 DNA polymerase heterodimer. This interference is suppressed by increasing the concentration of either primer TP or phi29 DNA polymerase. We propose a model for initiation of in vivo phi29 DNA replication in which the viral replisome attaches to a membrane-associated p1-based structure.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Adenosine Monophosphate / analogs & derivatives
  • Adenosine Monophosphate / metabolism
  • Bacillus Phages / genetics*
  • Bacillus Phages / growth & development
  • Bacillus Phages / metabolism
  • Bacillus subtilis / virology*
  • Cross-Linking Reagents / metabolism
  • DNA Replication / genetics
  • DNA, Viral / biosynthesis
  • DNA, Viral / genetics
  • DNA-Directed DNA Polymerase / metabolism
  • Kinetics
  • Membrane Proteins / chemistry
  • Membrane Proteins / genetics
  • Membrane Proteins / metabolism*
  • Plasmids / genetics
  • Protein Binding
  • Protein Structure, Quaternary
  • Sequence Deletion / genetics
  • Succinimides / metabolism
  • Viral Proteins / chemistry
  • Viral Proteins / genetics
  • Viral Proteins / metabolism*
  • Virus Replication / genetics*

Substances

  • Cross-Linking Reagents
  • DNA, Viral
  • Membrane Proteins
  • Succinimides
  • Viral Proteins
  • terminal protein, Bacillus phage phi29
  • Adenosine Monophosphate
  • 3,3'-dithiobis(sulfosuccinimidyl propionate)
  • DNA-Directed DNA Polymerase