Black Lindsay W, Peng Guihong
Department of Biochemistry and Molecular Biology, University of Maryland Medical School, Baltimore, Maryland 21201-1503, USA.
J Biol Chem. 2006 Sep 1;281(35):25635-43. doi: 10.1074/jbc.M602093200. Epub 2006 Jun 28.
Regulation of the terminal stage of viral DNA development, DNA packaging, is poorly understood. A new phage T4 in vitro DNA packaging assay employed purified proheads, terminase (gp17 + gp16), and ATP to encapsidate DNA resistant to nuclease. Mature phage T4 DNA and linearized plasmid DNAs containing or lacking a cloned T4 gene were packaged with high (approximately 10%) efficiency. Supercoiled, relaxed covalently closed, and nicked circular plasmid DNAs were packaged inefficiently, if at all, by these components. However, efficient packaging is achieved for nicked circular plasmid DNA, but not covalently closed plasmid DNA, upon addition to packaging mixtures of the purified T4 late transcription-replication machinery proteins: gp45 (sliding clamp), gp44/gp62 (clamp loader complex), gp55 (late sigma-factor), and gp33 (transcriptional co-activator). The small terminase subunit (gp16) is inhibitory for packaging linear DNAs, but enhances the transcription-replication protein packaging of nicked plasmid DNA. Taken together with genetic and biochemical evidence of a requirement for gp55 for concatemer packaging to assemble active wild-type phage particles (1), the plasmid packaging results show that initiation of phage T4 packaging on "endless" concatemeric DNA in vivo by terminase depends upon interaction with the DNA loaded gp45 coupled late transcription-replication machinery. The results suggest a close mechanistic connection in vivo between DNA packaging and developmentally concurrent replication-dependent late transcription.
病毒DNA发育的终末阶段，即DNA包装，目前了解甚少。一种新的噬菌体T4体外DNA包装试验使用纯化的原头部、末端酶（gp17 + gp16）和ATP来包裹抗核酸酶的DNA。成熟的噬菌体T4 DNA以及含有或缺乏克隆的T4基因的线性化质粒DNA以高（约10%）效率被包装。超螺旋、松弛共价闭合和带切口的环状质粒DNA若被这些组分包装，则效率很低，甚至根本不被包装。然而，在向包装混合物中添加纯化的T4晚期转录 - 复制机制蛋白：gp45（滑动夹钳）、gp44/gp62（夹钳加载复合物）、gp55（晚期σ因子）和gp33（转录共激活因子）后，带切口的环状质粒DNA能被高效包装，但共价闭合的质粒DNA则不能。小末端酶亚基（gp16）对线性DNA的包装有抑制作用，但能增强带切口质粒DNA的转录 - 复制蛋白包装。结合关于gp55对串联体包装以组装活性野生型噬菌体颗粒的必要性的遗传学和生物化学证据（1），质粒包装结果表明，在体内，末端酶在“无尽”的串联体DNA上启动噬菌体T4包装取决于与加载了DNA的gp45偶联的晚期转录 - 复制机制的相互作用。这些结果表明，在体内DNA包装与发育过程中同时发生的依赖复制的晚期转录之间存在密切的机制联系。
