Characterization of the oriI and oriII Origins of Replication in Phage-Plasmid P4

Abstract

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 α andcnr genes and the ori1 and crrsites; the oriII replicon is composed of the α gene, with the internal ori2 site, and the crr region. The P4 α 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 α 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 forori1 and ori2, respectively. By deletion mapping, we defined the minimal ori1 and ori2regions 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 α protein did not bind ori2. Thus, the α protein appears to act differently at the two origins of replication.

Extrachromosomal circular elements targeted by CRISPR-Cas in Dehalococcoides mccartyi are linked to mobilization of reductive dehalogenase genes

“…Bifunctional primase/polymerase is related to the phage P4 family of primases/polymerases. These are encoded in a single multifunctional gene with primase, [59][60][61]. Doublestranded DNA repair proteins, although used in the maintenance of genomes, can also be used by prokaryotes during homologous recombination [62,63].…”

Strong minor groove base conservation in sequence logos implies DNA distortion or base flipping during replication and transcription initiation

“…Plasmid P4 α protein binding sites. The phage-plasmid P4 replicates in E.coli by the binding of the α protein to 11 iterons in ori1 and crr sites (56,57). The left half (positions -2 to +1) of the sequence logo (Fig.…”

Cnr interferes with dimerization of the replication protein alpha in phage-plasmid P4

“…The formation of the multimeric protein-DNA complexes (handcuffing; 12) inhibits replication initiation and allows plasmid copy number control. However in P4, unlike the above model, crr is positively required in cis for replication and does not appear to be involved in P4 DNA replication control (4,7,13).…”

“…Two sites in the P4 genome, ori1 and crr, are essential in cis for replication. Both contain several direct and inverted repeats of a decameric sequence, the type I iterons (6,7), which are bound by the α protein (8). Although essential for replication (6,7), crr is not an origin of replication (4,9).…”

“…Both contain several direct and inverted repeats of a decameric sequence, the type I iterons (6,7), which are bound by the α protein (8). Although essential for replication (6,7), crr is not an origin of replication (4,9). In several iteron-containing plasmids (such as P1, R6K, RK2) (10,11) the replication protein binds to specific sites and DNA looping and/or intermolecular pairing of DNA molecules, mediated by protein-protein interactions, occurs.…”