Assembly and subunit stoichiometry of the functional helicase-primase (primosome) complex of bacteriophage T4
Assembly and subunit stoichiometry of the functional helicase-primase (primosome) complex of bacteriophage T4
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DOI：
10.1073/pnas.1210040109
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发表时间：
2012-08-21
影响因子：
11.1
通讯作者：
von Hippel, Peter H.
中科院分区：
文献类型：
--
作者：
Jose, Davis;Weitzel, Steven E.;von Hippel, Peter H.
利用物理生化技术建立噬菌体T4DNA复制系统的原始体复合体的结构、亚单位化学计量学和组装途径。分析超速离心法和荧光各向异性方法表明，功能性的T4原粒由6个gp41解旋酶亚基组成，它们组装成一个六边形，由位于亚基间界面并稳定的6个NTP(或6个不可水解的GTP-S类似物)与一个紧密结合的gp61启动子亚基的结合驱动。将原始体的组件组装到模型DNA复制叉上是一个多步骤的过程，但不能沿着所有混合路径达到平衡。生产功能复合体需要在添加底物酶之前在DNA复制叉状结构的存在下组装解旋酶六聚体，以避免形成亚稳定的DNA-蛋白质聚集体。在没有底物酶的情况下，gp41解旋酶六聚体与分叉DNA结合很弱，但形成一个更稳定的原粒复合体，当以6：1的底物与gp61底物酶亚基结合时，该复合体表达完整的和功能性的底物酶(和底物酶)活性。额外的底物酶亚单位的存在不会改变原始体的分子质量或解旋酶活性，但显著抑制其底物的活性。我们为T4原始体的结构和功能开发了一个组装途径和一个最小机械模型，这可能也与高等生物的复制原始体亚组件的组装和功能有关。
Physical biochemical techniques are used to establish the structure, subunit stoichiometry, and assembly pathway of the primosome complex of the bacteriophage T4 DNA replication system. Analytical ultracentrifugation and fluorescence anisotropy methods show that the functional T4 primosome consists of six gp41 helicase subunits that assemble into a hexagon, driven by the binding of six NTPs (or six nonhydrolyzable GTP gamma S analogues) that are located at and stabilize the intersubunit interfaces, together with a single tightly bound gp61 primase subunit. Assembling the components of the primosome onto a model DNA replication fork is a multistep process, but equilibrium cannot be reached along all mixing pathways. Producing a functional complex requires that the helicase hexamer be assembled in the presence of the DNA replication fork construct prior to the addition of the primase to avoid the formation of metastable DNA-protein aggregates. The gp41 helicase hexamer binds weakly to fork DNA in the absence of primase, but forms a much more stable primosome complex that expresses full and functional helicase (and primase) activities when bound to a gp61 primase subunit at a helicase: primase subunit ratio of 6: 1. The presence of additional primase subunits does not change the molecular mass or helicase activity of the primosome, but significantly inhibits its primase activity. We develop both an assembly pathway and a minimal mechanistic model for the structure and function of the T4 primosome that are likely to be relevant to the assembly and function of the replication primosome subassemblies of higher organisms as well.