title: Bacteriophage SPP1 DNA replication strategies promote viral and disable host replication in vitro.
abstract: Complex viruses that encode their own initiation proteins and subvert the host's elongation apparatus have provided valuable insights into DNA replication. Using purified bacteriophage SPP1 and Bacillus subtilis proteins, we have reconstituted a rolling circle replication system that recapitulates genetically defined protein requirements. Eleven proteins are required: phage-encoded helicase (G40P), helicase loader (G39P), origin binding protein (G38P) and G36P single-stranded DNA-binding protein (SSB); and host-encoded PolC and DnaE polymerases, processivity factor (β(2)), clamp loader (τ-δ-δ') and primase (DnaG). This study revealed a new role for the SPP1 origin binding protein. In the presence of SSB, it is required for initiation on replication forks that lack origin sequences, mimicking the activity of the PriA replication restart protein in bacteria. The SPP1 replisome is supported by both host and viral SSBs, but phage SSB is unable to support B. subtilis replication, likely owing to its inability to stimulate the PolC holoenzyme in the B. subtilis context. Moreover, phage SSB inhibits host replication, defining a new mechanism by which bacterial replication could be regulated by a viral factor.
authors.affiliation: Departamento de Biotecnología Microbiana, Centro Nacional, Centro Nacional de Biotecnología, CSIC, 28049 Madrid, Spain.
authors.forename: Elena M
authors.initials: EM
authors.lastname: Seco
authors.forename: John C
authors.initials: JC
authors.lastname: Zinder
authors.forename: Carol M
authors.initials: CM
authors.lastname: Manhart
authors.forename: Ambra
authors.initials: A
authors.lastname: Lo Piano
authors.forename: Charles S
authors.initials: CS
authors.lastname: McHenry
authors.forename: Silvia
authors.initials: S
authors.lastname: Ayora
mesh_terms: D017103:Bacillus Phages / Q000235:genetics*; D001412:Bacillus subtilis / Q000235:genetics*; D001426:Bacterial Proteins / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D004265:DNA Helicases / Q000378:metabolism; D004258:DNA Polymerase III / Q000378:metabolism; D019915:DNA Primase / Q000378:metabolism; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins; D017361:Viral Nonstructural Proteins / Q000378:metabolism; D014764:Viral Proteins / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D002352:Carrier Proteins; D004268:DNA-Binding Proteins; C119220:G38P protein, Bacteriophage SPP1; C119221:G39P protein, Bacteriophage SPP1; D047908:Intracellular Signaling Peptides and Proteins; C022262:Okazaki fragments; D017361:Viral Nonstructural Proteins; D014764:Viral Proteins; D004247:DNA; D019915:DNA Primase; C074904:DNA polymerase III, alpha subunit; C446929:PolC protein, bacteria; D004258:DNA Polymerase III; D004259:DNA-Directed DNA Polymerase; D004265:DNA Helicases