IDENTIFICATION OF 2 REPLICONS IN PHAGE-PLASMID P4

DNA replication of phage-plasmid P4 proceeds bidirectionally from the ori1 site (previously named ori), but requires a second cis-acting reg ion, crr. Replication depends on the product of the P4 alpha gene, a p rotein with primase and helicase activity, that binds both ori1 and cr r. A negative regulator of P4 DNA replication, the Cnr protein, is req uired for copy number control of plasmid P4. Using a plasmid complemen tation test for replication, we found that two replicons, both depende nt on the ct gene product, coexist in P4. The first replicon is made b y the cnr and alpha genes and the ori1 and crr sites. The second is li mited to the a and err region. Thus, in the absence of the ori1 region , replication can initiate at a different site. By deletion mapping, a cis-acting region, ori2, essential for replication of the alpha-crr r eplicon was mapped within a 270-bp fragment in the first half of the a gene. The ori2 site was found to be dispensable in a replicon that co ntains ori1. A construct that besides err and a carries also the cnr g ene was unable to replicate, suggesting that Cnr not only controls rep lication from ori1, but also silences ori2. (C) 1998 Academic Press.