Serrano-Heras Gemma, Salas Margarita, Bravo Alicia
Instituto de Biología Molecular Eladio Viñuela (CSIC), Centro de Biología Molecular Severo Ochoa (CSIC-UAM), Universidad Autónoma, Cantoblanco, 28049 Madrid, Spain.
J Biol Chem. 2003 Oct 17;278(42):40771-7. doi: 10.1074/jbc.M306935200. Epub 2003 Aug 6.
The mechanisms underlying compartmentalization of prokaryotic DNA replication are largely unknown. In the case of the Bacillus subtilis phage 29, the viral protein p1 enhances the rate of in vivo viral DNA replication. Previous work showed that p1 generates highly ordered structures in vitro. We now show that protein p1, like integral membrane proteins, has an amphiphilic nature. Furthermore, immunoelectron microscopy studies reveal that p1 has a peripheral subcellular location. By combining in vivo chemical cross-linking and cell fractionation techniques, we also demonstrate that p1 assembles in infected cells into multimeric structures that are associated with the bacterial membrane. These structures exist both during viral DNA replication and when 29 DNA synthesis is blocked due to the lack of viral replisome components. In addition, protein p1 encoded by plasmid generates membrane-associated multimers and supports DNA replication of a p1-lacking mutant phage, suggesting that the pre-assembled structures are functional. We propose that a phage structure assembled on the cell membrane provides a specific site for 29 DNA replication.
原核生物DNA复制区室化的潜在机制在很大程度上尚不清楚。就枯草芽孢杆菌噬菌体29而言，病毒蛋白p1可提高体内病毒DNA的复制速率。先前的研究表明，p1在体外能形成高度有序的结构。我们现在发现，蛋白p1与整合膜蛋白一样，具有两亲性。此外，免疫电子显微镜研究显示，p1位于亚细胞外周位置。通过结合体内化学交联和细胞分级分离技术，我们还证明，p1在受感染细胞中组装成与细菌膜相关的多聚体结构。这些结构在病毒DNA复制期间以及由于缺乏病毒复制体成分导致29 DNA合成受阻时均存在。此外，由质粒编码的蛋白p1可生成与膜相关的多聚体，并支持缺乏p1的突变噬菌体的DNA复制，这表明预先组装的结构具有功能。我们提出，在细胞膜上组装的噬菌体结构为29 DNA复制提供了一个特定位点。
