Polymerization of bacteriophage phi 29 replication protein p1 into protofilament sheets.
Protein p1 (85 amino acids) of the Bacillus subtilis phage phi29 is a membrane-associated protein required for in vivo viral DNA replication. In the present study, we have constructed two fusion proteins, maltose-binding protein (MalE)-p1 and MalE-p1DeltaN33. By using both sedimentation assays and negative-stain electron microscopy analysis, we demonstrated that MalE-p1 molecules self-associated into long filamentous structures, which did not assemble further into larger arrays. These structures were constituted by a core of protein p1 surrounded by MalE subunits. After removal of the MalE component by cleavage with protease factor Xa, the resulting protein p1 filaments tended to associate, forming bundles. The MalE-p1DeltaN33 fusion protein, however, did not self-interact in solution. Nevertheless, after being separated from the MalE domain by factor Xa digestion, protein p1DeltaN33 assembled into long protofilaments that associated in a highly ordered, parallel array forming large two-dimensional sheets. These structures resemble eukaryotic tubulin and bacterial FtsZ polymers. In addition, we show that protein p1 influences the rate of in vivo phi29 DNA synthesis in a temperature-dependent manner. We propose that protein p1 is a component of a viral-encoded structure that associates with the bacterial membrane. This structure would provide an anchoring site for the viral DNA replication machinery.
Bravo
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Centro de Biología Molecular 'Severo Ochoa' (CSIC-UAM), Universidad Autónoma, Cantoblanco, 28049 Madrid, Spain.
Salas
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D000595:Amino Acid Sequence; D017103:Bacillus Phages / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D001412:Bacillus subtilis / Q000821:virology*; D002352:Carrier Proteins / Q000235:genetics; D004261:DNA Replication; D004279:DNA, Viral / Q000096:biosynthesis; D058619:Maltose-Binding Proteins; D008969:Molecular Sequence Data; D011108:Polymers / Q000378:metabolism*; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000235:genetics; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D019065:Virus Assembly
D002352:Carrier Proteins; D004279:DNA, Viral; D058619:Maltose-Binding Proteins; D011108:Polymers; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; C060516:phage phi29 protein p1