Freire R, Serrano M, Salas M, Hermoso J M
Centro de Biología Molecular "Severo Ochoa" (CSIC-UAM), Universidad Autónoma, Cantoblanco, 28049 Madrid, Spain.
J Biol Chem. 1996 Nov 29;271(48):31000-7. doi: 10.1074/jbc.271.48.31000.
Protein p6 of Bacillus subtilis phage phi29 activates the initiation of viral DNA replication by forming a multimeric nucleoprotein complex at the origins of replication, located at both ends of the linear genome. This activation requires a precise positioning of the protein p6 array with respect to the initiation site. To investigate this activation mechanism, we have purified the phi29 protein p6 counterparts from the related phages Nf and GA-1 and analyzed the formation of complexes with DNA. In the homologous protein p6-DNA complexes the phi29 and Nf protein arrays showed an identical positioning, different than that of the GA-1 protein array. In contrast, in the heterologous complexes the protein showed a different arrangement except in the case of the Nf protein-phi29 DNA complex. We have also purified the proteins involved in the initiation of replication (terminal protein and DNA polymerase) from phages Nf and GA-1 and measured the ability of the different p6 proteins to activate homologous and heterologous replication origins. The results obtained indicate that the activation requires not only the formation of a specific nucleoprotein complex but also its specific recognition by the proteins involved in the initiation of DNA replication.
枯草芽孢杆菌噬菌体phi29的蛋白质p6通过在位于线性基因组两端的复制起点处形成多聚体核蛋白复合物来激活病毒DNA复制的起始。这种激活需要蛋白质p6阵列相对于起始位点的精确定位。为了研究这种激活机制，我们从相关噬菌体Nf和GA-1中纯化了phi29蛋白质p6的对应物，并分析了与DNA形成的复合物。在同源蛋白质p6-DNA复合物中，phi29和Nf蛋白质阵列显示出相同的定位，与GA-1蛋白质阵列不同。相反，在异源复合物中，除了Nf蛋白质-phi29 DNA复合物外，蛋白质呈现出不同的排列。我们还从噬菌体Nf和GA-1中纯化了参与复制起始的蛋白质（末端蛋白和DNA聚合酶），并测量了不同p6蛋白质激活同源和异源复制起点的能力。所得结果表明，激活不仅需要形成特定的核蛋白复合物，还需要参与DNA复制起始的蛋白质对其进行特异性识别。
