CRYOELECTRON MICROSCOPIC VISUALIZATION OF FUNCTIONAL SUBASSEMBLIES OF THE BACTERIOPHAGE-T4 DNA-REPLICATION COMPLEX
CRYOELECTRON MICROSCOPIC VISUALIZATION OF FUNCTIONAL SUBASSEMBLIES OF THE BACTERIOPHAGE-T4 DNA-REPLICATION COMPLEX
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DOI：
10.1016/0022-2836(92)91003-8
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发表时间：
1992-03-20
影响因子：
5.6
通讯作者：
VONHIPPEL, PH
中科院分区：
文献类型：
--
作者：
GOGOL, EP;YOUNG, MC;VONHIPPEL, PH
一个特定的复合物的蛋白质参与噬菌体T4复制已被可视化的冷冻电子显微镜作为独特的结构与DNA。这些结构的形成需要T4聚合酶辅助蛋白（T4基因44、45和62的产物）、ATP和适当的DNA辅因子的存在，我们将其与DNA结合的特征性外观称为“散列标记”。由DNA刺激的ATP酶活性的辅助蛋白的ATP水解是必要的哈希标记结构的可视化。如果ATP水解被Mg 2+的螯合作用、不可水解的ATP类似物的稀释或ATP供应的耗尽所停止，则DNA相关结构在数秒至数分钟内消失，表明它们具有有限且相对较短的寿命。结构的不稳定性使得它们的研究更传统的方法，电子显微镜，以及大多数其他结构的方法，困难，如果不是不可能的。T4基因32蛋白的添加增加了散列标记结构的数量，以及增加了ATP水解的速率。使用质粒DNA在一个天然的（超螺旋）或酶修饰的状态，我们已经表明，有切口或缺口的DNA需要作为一个辅助因子的散列标记的形成。辅助蛋白的ATP酶活性的刺激具有类似的辅因子要求。这些用于形成和可视化结构的条件与这些复合物在促进T4 DNA聚合酶的酶活性以及晚期T4基因的转录中的作用所需的条件平行。散列标记中的亚结构已经通过图像分析进行了检查，其揭示了包括结构的亚基的投影密度的变化。的三维尺寸的散列标记，建模为一个坚实的椭圆体，是一致的基因44 - 62蛋白质亚复合物。密度的变化表明，从DNA轴的不同角度观察，亚基的排列是四边形或三角形。散列标记结构通常以簇的形式出现，即使是在只有一个切口的DNA中。我们将这种分布解释为散列标记沿着DNA一维移位的结果，这些标记在它们依赖于ATP的初始结合并在缺口或缺口处注入DNA中。
A specific complex of proteins involved in bacteriophage T4 replication has been visualized by cryoelectron microscopy as distinctive structures in association with DNA. Formation of these structures, which we term “hash-marks” for their characteristic appearance in association with DNA, requires the presence of the T4 polymerase accessory proteins (the products of T4 genes 44, 45 and 62), ATP and appropriate DNA cofactors. ATP hydrolysis by the DNA-stimulated ATPase activity of the accessory proteins is required for visualization of the hash-mark structures. If ATP hydrolysis is stopped by chelation of Mg 2+, by dilution with a non-hydrolyzable ATP analogue, or by exhaustion of the ATP supply, the DNA-associated structures disappear within seconds to minutes, indicating that they have a finite and relatively short lifetime. The labile nature of the structures makes their study by more conventional methods of electron microscopy, as well as by most other structural approaches, difficult if not impossible. Addition of T4 gene 32 protein increases the number of hash-mark structures, as well as increasing the rate of ATP hydrolysis. Using plasmid DNA in either a native (supercoiled) or enzymatically modified state, we have shown that nicked or gapped DNA is required as a cofactor for hash-mark formation. Stimulation of the ATPase activity of the accessory proteins has a similar cofactor requirement. These conditions for the formation and visualization of the structures parallel those required for the action of these complexes in promoting the enzymatic activity of the T4 DNA polymerase, as well as the transcription of late T4 genes. Substructure in the hash-marks has been examined by image analysis, which reveals a variation in the projected density of the subunits comprising the structures. The three-dimensional size of the hash-marks, modeled as a solid ellipsoid, is consistent with that of the gene 44 62 protein subcomplex. Density variations suggest an arrangement of subunits, either tetragonal or trigonal, viewed from a variety of angles about the DNA axis. The hash-mark structures often appear in clusters, even in DNA that has a single nick. We interpret this distribution as the result of one-dimensional translocation of the hash-marks along the DNA after their ATP-dependent initial association with, and injection into, the DNA at nicks or gaps.