The in vivo function of phage phi29 nucleoid-associated protein p6 requires formation of dimers.
The Bacillus subtilis phage phi29 nucleoid-associated protein p6 (103 amino acids) is essential for in vivo viral DNA replication and control of transcription, and it has been proposed to play a role in genome organization and compaction. This protein self-associates in vitro from preformed dimers forming high-molecular-weight oligomers and binds to double-stranded DNA giving rise to multimeric nucleoprotein complexes. Site-directed mutants, p6I8T and p6A44V, were completely or partially inactive, respectively, in an in vitro dimerization assay. In this paper, and by in vivo crosslinking, we have detected dimers of protein p6 either in phage-infected cells or in protein p6 producing B. subtilis or Escherichia coli cells. Therefore, this self-association does not require viral DNA. We also show that mutants p6I8T and p6A44V are deficient in dimer formation, and they do not support phage DNA replication in a trans-complementation assay with phi29sus6 mutant-infected B. subtilis cells. We conclude that dimeric protein p6 is the active form of the protein in vivo, required for viral DNA replication.
Abril
Ana  M
A
Centro de Biología Molecular Severo Ochoa (CSIC-UAM), Universidad Autónoma, Canto Blanco, 28049 Madrid, Spain.
Salas
Margarita
M
Hermoso
José M
JM
D017103:Bacillus Phages / Q000235:genetics*; D001412:Bacillus subtilis / Q000821:virology*; D015153:Blotting, Western; D004279:DNA, Viral / Q000235:genetics; D019281:Dimerization; D005816:Genetic Complementation Test; D009154:Mutation; D013696:Temperature; D013997:Time Factors; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D014779:Virus Replication / Q000235:genetics
D004279:DNA, Viral; D014764:Viral Proteins; C413452:phage phi29 protein p6