Identification of residues within two regions involved in self-association of viral histone-like protein p6 from phage theta29.
Protein p6 of Bacillus subtilis phage theta29 is involved in the initiation of viral DNA replication and transcription by forming a multimeric nucleoprotein complex with the phage DNA. Based on this, together with its abundance and its capacity to bind to the whole viral genome, it has been proposed to be a viral histone-like protein. Protein p6 is in a monomer-dimer-oligomer equilibrium association. We have identified protein p6 mutants deficient in self-association by testing random mutants obtained by degenerated polymerase chain reaction in an in vivo assay for dimer formation. The mutations were mainly clustered in two regions located at the N terminus, and the central part of the protein. Site-directed single mutants, corresponding to those found in vivo, have been constructed and purified. Mutant p6A44V, located at the central part of the protein, showed an impaired dimer formation ability, and a reduced capacity to bind DNA and to activate the initiation of O29 DNA replication. Mutant p6I8T has at least 10-fold reduced self-association capacity, does not bind DNA nor activate O29 DNA initiation of replication. C-terminal deletion mutants showed an enhanced dimer formation capacity. The highly acidic tail, removed in these mutants, is proposed to modulate the protein p6 self-association.
Abril
A M
AM
Centro de Biologia Molecular "Severo Ochoa" (Consejo Superior de Investigaciones Cientificas-Universidad Autónoma de Madrid), Universidad Autónoma, Canto Blanco, 28049 Madrid, Spain.
Salas
M
M
Hermoso
J M
JM
D000595:Amino Acid Sequence; D017103:Bacillus Phages / Q000235:genetics / Q000502:physiology*; D002942:Circular Dichroism; D004261:DNA Replication; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*; D014779:Virus Replication
D014764:Viral Proteins; C413452:phage phi29 protein p6