Phage phi29 proteins p1 and p17 are required for efficient binding of architectural protein p6 to viral DNA in vivo.
Bacteriophage phi29 protein p6 is a viral architectural protein, which binds along the whole linear phi29 DNA in vivo and is involved in initiation of DNA replication and transcription control. Protein p1 is a membrane-associated viral protein, proposed to attach the viral genome to the cell membrane. Protein p17 is involved in pulling phi29 DNA into the cell during the injection process. We have used chromatin immunoprecipitation and real-time PCR to analyze in vivo p6 binding to DNA in cells infected with phi29 sus1 or sus17 mutants; in both cases p6 binding is significantly decreased all along phi29 DNA. phi29 DNA is topologically constrained in vivo, and p6 binding is highly increased in the presence of novobiocin, a gyrase inhibitor that produces a loss of DNA negative superhelicity. Here we show that, in cells infected with phi29 sus1 or sus17 mutants, the increase of p6 binding by novobiocin is even higher than in cells containing p1 and p17, alleviating the p6 binding deficiency. Therefore, proteins p1 and p17 could be required to restrain the proper topology of phi29 DNA, which would explain the impaired DNA replication observed in cells infected with sus1 or sus17 mutants.
González-Huici
Víctor
V
Instituto de Biología Molecular Eladio Viñuela (CSIC), Centro de Biología Molecular Severo Ochoa (CSIC-UAM), Universidad Autónoma, Canto Blanco, 28049 Madrid, Spain.
Alcorlo
Martín
M
Salas
Margarita
M
Hermoso
José M
JM
D017103:Bacillus Phages / Q000235:genetics / Q000378:metabolism*; D001412:Bacillus subtilis / Q000821:virology*; D047369:Chromatin Immunoprecipitation; D004261:DNA Replication; D004279:DNA, Viral / Q000378:metabolism*; D015967:Gene Expression Regulation, Viral; D009154:Mutation; D016133:Polymerase Chain Reaction; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
D004279:DNA, Viral; D014764:Viral Proteins; C413452:phage phi29 protein p6; C117368:phage phi29, protein p17; C060516:phage phi29 protein p1