Bravo A, Salas M
Centro de Biología Molecular Severo Ochoa (CSIC-UAM), Universidad Autónoma Cantoblanco, Madrid, Spain.
J Mol Biol. 1997 May 30;269(1):102-12. doi: 10.1006/jmbi.1997.1032.
Initiation of in vitro phage phi29 DNA replication requires the formation of a heterodimer between a free molecule of terminal protein (TP), which acts as primer, and the viral DNA polymerase. We have analyzed membrane vesicles from phi29-infected Bacillus subtilis cells by quantitative immunoblot techniques. During phage DNA synthesis, large amounts of the viral proteins p1 and free TP were recovered in membrane fractions, as well as a low percentage of the total viral DNA polymerase. Interestingly, the amount of DNA polymerase in membrane fractions increased when viral DNA replication was blocked. Both protein p1 and free TP showed affinity for membranes in the absence of viral DNA. The association of protein p1 with membranes was abolished when the C-terminal 43 amino acid residues were deleted. The above results, together with the critical role of protein p1 for in vivo phi29 DNA replication, led us to conclude that a preliminary stage in the initiation of in vivo phi29 DNA replication could be the assembly of a membrane-associated multiprotein complex containing at least protein p1, free TP and DNA polymerase. Membrane-attachment of this complex could be directly mediated by both protein p1 and free TP. The ability of free TP to bind to membranes and to prime phi29 DNA replication would enable a nascent viral DNA molecule to become membrane-associated when its synthesis begins. We postulate that a general function of the TPs covalently linked to linear DNA genomes in prokaryotes might be, in addition to act as primer, to anchor the linear DNA molecule to the bacterial membrane.
体外噬菌体phi29 DNA复制的起始需要末端蛋白（TP）的游离分子（其作为引物）与病毒DNA聚合酶之间形成异二聚体。我们通过定量免疫印迹技术分析了来自phi29感染的枯草芽孢杆菌细胞的膜囊泡。在噬菌体DNA合成过程中，大量的病毒蛋白p1和游离TP以及总病毒DNA聚合酶的一小部分在膜组分中被回收。有趣的是，当病毒DNA复制受阻时，膜组分中DNA聚合酶的量增加。在没有病毒DNA的情况下，蛋白p1和游离TP都显示出对膜的亲和力。当C末端的43个氨基酸残基被删除时，蛋白p1与膜的结合被消除。上述结果，连同蛋白p1对体内phi29 DNA复制的关键作用，使我们得出结论，体内phi29 DNA复制起始的一个初步阶段可能是组装一个至少包含蛋白p1、游离TP和DNA聚合酶的膜相关多蛋白复合物。该复合物的膜附着可能直接由蛋白p1和游离TP介导。游离TP结合膜并引发phi29 DNA复制的能力将使新生的病毒DNA分子在其合成开始时与膜相关。我们推测，原核生物中与线性DNA基因组共价连接的TP的一个普遍功能可能是，除了作为引物外，还将线性DNA分子锚定到细菌膜上。
