Characterization of the oriI and oriII origins of replication in phage-plasmid P4.
In the Escherichia coli phage-plasmid P4, two partially overlapping replicons with bipartite ori sites coexist. The essential components of the oriI replicon are the alpha and cnr genes and the ori1 and crr sites; the oriII replicon is composed of the alpha gene, with the internal ori2 site, and the crr region. The P4 alpha protein has primase and helicase activities and specifically binds type I iterons, present in ori1 and crr. Using a complementation test for plasmid replication, we demonstrated that the two replicons depend on both the primase and helicase activities of the alpha protein. Moreover, neither replicon requires the host DnaA, DnaG, and Rep functions. The bipartite origins of the two replicons share the crr site and differ for ori1 and ori2, respectively. By deletion mapping, we defined the minimal ori1 and ori2 regions sufficient for replication. The ori1 site was limited to a 123-bp region, which contains six type I iterons spaced regularly close to the helical periodicity, and a 35-bp AT-rich region. Deletion of one or more type I iterons inactivated oriI. Moreover, insertion of 6 or 10 bp within the ori1 region also abolished replication ability, suggesting that the relative arrangement of the iterons is relevant. The ori2 site was limited to a 36-bp P4 region that does not contain type I iterons. In vitro, the alpha protein did not bind ori2. Thus, the alpha protein appears to act differently at the two origins of replication.
Tocchetti
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A
Dipartimento di Genetica e di Biologia dei Microrganismi, Università di Milano, 20133 Milan, Italy.
Galimberti
G
G
Dehò
G
G
Ghisotti
D
D
D001426:Bacterial Proteins / Q000235:genetics; D001483:Base Sequence; D003090:Coliphages / Q000235:genetics*; D004265:DNA Helicases / Q000235:genetics / Q000378:metabolism; D019915:DNA Primase / Q000235:genetics / Q000378:metabolism; D004279:DNA, Viral; D004268:DNA-Binding Proteins / Q000235:genetics; D004926:Escherichia coli / Q000821:virology; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010957:Plasmids; D018741:Replication Origin; D012093:Replicon; D015534:Trans-Activators / Q000235:genetics; D014764:Viral Proteins / Q000378:metabolism; D014779:Virus Replication
D001426:Bacterial Proteins; D004279:DNA, Viral; D004268:DNA-Binding Proteins; C066312:DnaA protein, Bacteria; D015534:Trans-Activators; D014764:Viral Proteins; C045264:replication initiator protein; D019915:DNA Primase; D004265:DNA Helicases