Physical and Functional Characterization of a Viral Genome Maturation Complex.
Physical and Functional Characterization of a Viral Genome Maturation Complex.
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病毒基因组成熟复合物的物理和功能表征。
DOI：
10.1016/j.bpj.2017.02.041
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发表时间：
2017
影响因子：
3.4
通讯作者：
Catalano,CarlosE
中科院分区：
文献类型：
--
作者：
Yang,Teng-Chieh;Ortiz,David;Yang,Qin;DeAngelis,RolandoW;Sanyal,SaurarshiJ;Catalano,CarlosE
基因组包装在复杂的双链DNA病毒中是高度保守的，包括疱疹病毒和许多噬菌体。在这些情况下，病毒DNA通过末端酶包装到原衣壳壳中。包装底物通常是由以头对尾方式连接的多个基因组组成的多联体，并且末端酶执行两个基本功能：1）从多联体切除单位长度的基因组（基因组成熟）和2）将双链体易位到原衣壳中（基因组包装）。虽然包装马达已被详细描述，但成熟复合物的特征仍不清楚。在这里，我们描述的组装，物理特性，和催化活性的λ-基因组成熟复合物。λ-末端酶原聚体由一个大的催化亚基和两个DNA识别亚基组成。分离的原聚体结合DNA弱，不区分非特异性DNA和双链体，包含包装起始序列，cos。大肠杆菌整合宿主因子蛋白（IHF）是λ-体在体内高效发育所必需的，在incos中发现了一个特异性的IHF识别序列。我们发现，IHF和末端酶的原体合作组装在thecosite和小末端酶亚基起主导作用，在复杂的组装。超电泳分析表明，成熟复合物由四个原体和一个IHF异源二聚体组成。四聚体组装激活酶的半切割核酸酶活性，其使基因组末端成熟以准备包装。的化学计量和催化活性的复杂的是让人想起的IIE和IIF型限制性内切酶和两个系统可能共享的机械功能。据我们所知，这项研究提供了我们第一次详细了解病毒基因组成熟复合物的结构和功能特征，这是复杂dsDNA病毒发展的重要中间体。
Genome packaging is strongly conserved in the complex double-stranded DNA viruses, including the herpesviruses and many bacteriophages. In these cases, viral DNA is packaged into a procapsid shell by a terminase enzyme. The packaging substrate is typically a concatemer composed of multiple genomes linked in a head-to-tail fashion, and terminase enzymes perform two essential functions: 1) excision of a unit length genome from the concatemer (genome maturation) and 2) translocation of the duplex into a procapsid (genome packaging). While the packaging motors have been described in some detail, the maturation complexes remain ill characterized. Here we describe the assembly, physical characteristics, and catalytic activity of theλ-genome maturation complex. Theλ-terminase protomer is composed of one large catalytic subunit tightly associated with two DNA recognition subunits. The isolated protomer binds DNA weakly and does not discriminate between nonspecific DNA and duplexes that contain the packaging initiation sequence,cos. TheEscherichia coliintegration host factor protein (IHF) is required for efficientλ-development in vivo and a specific IHF recognition sequence is found withincos. We show that IHF and the terminase protomer cooperatively assemble at thecossite and that the small terminase subunit plays the dominant role in complex assembly. Analytical ultracentrifugation analysis reveals that the maturation complex is composed of four protomers and one IHF heterodimer bound at thecossite. Tetramer assembly activates thecos-cleavage nuclease activity of the enzyme, which matures the genome end in preparation for packaging. The stoichiometry and catalytic activity of the complex is reminiscent of the type IIE and IIF restriction endonucleases and the two systems may share mechanistic features. This study, to our knowledge, provides our first detailed glimpse into the structural and functional features of a viral genome maturation complex, an essential intermediate in the development of complex dsDNA viruses.