Bacteriophage lambda gpNu 1 and Escherichia coli IHF proteins cooperatively bind and bend viral DNA:: Implications for the assembly of a genome-packaging motor
Bacteriophage lambda gpNu 1 and Escherichia coli IHF proteins cooperatively bind and bend viral DNA:: Implications for the assembly of a genome-packaging motor
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DOI：
10.1021/bi052284b
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发表时间：
2006-04-25
期刊：
影响因子：
2.9
通讯作者：
Catalano, CE
中科院分区：
文献类型：
--
作者：
Ortega, ME;Catalano, CE
终止酶是原核和真核双链DNA病毒所共有的，负责将病毒DNA包装到空的原衣壳壳的范围内。在所有已知的情况下，全酶是由具有基因组包装所需的催化活性的大亚基和负责特异性识别病毒DNA的小亚基组成的异源寡聚体。在λ噬菌体中，DNA识别蛋白是gpNu 1。gpNu 1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，以位点特异性组装包装机器。马达组装是由大肠杆菌整合宿主因子蛋白（IHF），它结合到一个共识序列也位于COS。基于各种生物化学数据和最近解析的gpNu 1的DNA结合结构域的NMR结构，我们提出了一种新的DNA结合模式，其预测了gpNu 1对双链体DNA的显著弯曲（de Beer等人（2002）Mol. Cell 9，981-991）。我们进一步提出，gpNu 1和IHF协同结合和弯曲病毒DNA来调节包装马达的组装。在这里，我们的特点合作gpNu 1和IHF结合到COS网站在λ DNA使用定量电泳迁移率变动（EMS）测定。这些研究提供了直接的实验支持的长期假定的合作组装的gpNu 1和IHF在cos序列的λ DNA。此外，循环置换实验表明，病毒和宿主蛋白质各自在含cos的DNA中引入强弯曲，但不引入非特异性DNA底物。因此，包装装置对病毒DNA的特异性识别由DNA序列信息和双链体的结构改变介导。这些结果的相关性相对于组装的病毒DNA包装电机进行了讨论。
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.