Ortega Marcos E, Catalano Carlos E
Department of Biochemistry and Molecular Genetics, University of Colorado Health Sciences Center, Denver, Colorado 80262, USA.
Biochemistry. 2006 Apr 25;45(16):5180-9. doi: 10.1021/bi052284b.
Terminase enzymes are common to both prokaryotic and eukaryotic double-stranded DNA viruses and are responsible for packaging viral DNA into the confines of an empty procapsid shell. In all known cases, the holoenzymes are heteroligomers composed of a large subunit that possesses the catalytic activities required for genome packaging and a small subunit that is responsible for specific recognition of viral DNA. In bacteriophage lambda, the DNA recognition protein is gpNu1. The gpNu1 subunit interacts with multiple recognition elements within cos, the packaging initiation site in viral DNA, to site-specifically assemble the packaging machinery. Motor assembly is modulated by the Escherichia coli integration host factor protein (IHF), which binds to a consensus sequence also located within cos. On the basis of a variety of biochemical data and the recently solved NMR structure of the DNA binding domain of gpNu1, we proposed a novel DNA binding mode that predicts significant bending of duplex DNA by gpNu1 (de Beer et al. (2002) Mol. Cell 9, 981-991). We further proposed that gpNu1 and IHF cooperatively bind and bend viral DNA to regulate the assembly of the packaging motor. Here, we characterize cooperative gpNu1 and IHF binding to the cos site in lambda DNA using a quantitative electrophoretic mobility shift (EMS) assay. These studies provide direct experimental support for the long presumed cooperative assembly of gpNu1 and IHF at the cos sequence of lambda DNA. Further, circular permutation experiments demonstrate that the viral and host proteins each introduce a strong bend in cos-containing DNA, but not nonspecific DNA substrates. Thus, specific recognition of viral DNA by the packaging apparatus is mediated by both DNA sequence information and by structural alteration of the duplex. The relevance of these results with respect to the assembly of a viral DNA-packaging motor is discussed.
末端酶在原核生物和真核生物的双链DNA病毒中都很常见，负责将病毒DNA包装到空的原衣壳壳体内。在所有已知情况下，全酶都是异源寡聚体，由一个具有基因组包装所需催化活性的大亚基和一个负责病毒DNA特异性识别的小亚基组成。在噬菌体λ中，DNA识别蛋白是gpNu1。gpNu1亚基与病毒DNA包装起始位点cos内的多个识别元件相互作用，以位点特异性方式组装包装机制。运动组装由大肠杆菌整合宿主因子蛋白（IHF）调节，该蛋白与也位于cos内的共有序列结合。基于各种生化数据以及最近解析的gpNu1 DNA结合结构域的核磁共振结构，我们提出了一种新的DNA结合模式，预测gpNu1会使双链DNA发生显著弯曲（德·比尔等人，《分子细胞》，2002年，第9卷，第981 - 991页）。我们进一步提出，gpNu1和IHF协同结合并弯曲病毒DNA以调节包装马达的组装。在这里，我们使用定量电泳迁移率变动分析（EMS）来表征gpNu1和IHF与λ噬菌体DNA中cos位点的协同结合。这些研究为长期以来推测的gpNu1和IHF在λ噬菌体DNA的cos序列处的协同组装提供了直接的实验支持。此外，环形排列实验表明，病毒蛋白和宿主蛋白都会使含cos的DNA产生强烈弯曲，但不会使非特异性DNA底物弯曲。因此，包装装置对病毒DNA的特异性识别是由DNA序列信息和双链结构改变共同介导的。我们还讨论了这些结果与病毒DNA包装马达组装的相关性。
