Compartmentalization of phage phi29 DNA replication: interaction between the primer terminal protein and the membrane-associated protein p1.
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.
Bravo
A
A
Centro de Biología Molecular 'Severo Ochoa' (CSIC-UAM), Universidad Autónoma, Cantoblanco, 28049 Madrid, Spain.
Illana
B
B
Salas
M
M
D000249:Adenosine Monophosphate / Q000031:analogs & derivatives / Q000378:metabolism; D017103:Bacillus Phages / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D001412:Bacillus subtilis / Q000821:virology*; D003432:Cross-Linking Reagents / Q000378:metabolism; D004261:DNA Replication / Q000235:genetics; D004279:DNA, Viral / Q000096:biosynthesis / Q000235:genetics; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D007700:Kinetics; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D017384:Sequence Deletion / Q000235:genetics; D013388:Succinimides / Q000378:metabolism; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014779:Virus Replication / Q000235:genetics*
D003432:Cross-Linking Reagents; D004279:DNA, Viral; D008565:Membrane Proteins; D013388:Succinimides; D014764:Viral Proteins; C040784:terminal protein, Bacillus phage phi29; D000249:Adenosine Monophosphate; C035759:3,3'-dithiobis(sulfosuccinimidyl propionate); D004259:DNA-Directed DNA Polymerase