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 α and cnr genes and the ori1 and crr sites; 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 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 α protein did not bind ori2. Thus, the α protein appears to act differently at the two origins of replication.

在大肠杆菌（Escherichia coli）噬菌体质粒P4中，存在两种部分重叠的复制子（replicon），二者均带有二分复制起始位点（origin of replication，简称ori）。oriI型复制子的必需组分包括α、cnr基因以及ori1与crr位点；oriII型复制子则由携带内部ori2位点的α基因与crr区域构成。P4的α蛋白兼具引物酶与解旋酶活性，可特异性结合存在于ori1与crr中的I型迭代子（iterons）。通过质粒复制互补实验，我们证实两种复制子均依赖α蛋白的引物酶与解旋酶活性；此外，任一复制子均无需宿主的DnaA、DnaG与Rep蛋白功能。两种复制子的二分复制起始位点共享crr位点，二者分别以ori1与ori2作为各自的差异元件。通过缺失定位法，我们确定了足以支持复制的最小ori1与ori2区域。ori1位点被限定在一段123 bp的区域内，该区域包含6个间距与螺旋周期性高度吻合的I型迭代子，以及一段35 bp的AT富集区。缺失一个或多个I型迭代子会使oriI型复制子失活；此外，在ori1区域内插入6或10 bp碱基同样会消除其复制能力，这表明迭代子的相对排布对复制起始至关重要。ori2位点被限定在一段36 bp的P4区域内，该区域不含I型迭代子。体外实验中，α蛋白无法结合ori2。因此，α蛋白在两种复制起始位点的作用机制存在差异。