Tracking sliding clamp opening and closing during bacteriophage T4 DNA polymerase holoenzyme assembly
Tracking sliding clamp opening and closing during bacteriophage T4 DNA polymerase holoenzyme assembly
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DOI：
10.1021/bi992377r
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发表时间：
2000-03-21
期刊：
影响因子：
2.9
通讯作者：
Benkovic, SJ
中科院分区：
文献类型：
--
作者：
Alley, SC;Abel-Santos, E;Benkovic, SJ
噬菌体T4 DNA聚合酶全酶由DNA聚合酶（gp 43）、滑动夹（gp 45）和夹加载器（gp 44/62）组成，在ATP依赖性多步反应中加载到DNA上。三聚体的环状gp 45被加载到DNA上，使得DNA穿过环的中心。gp 43与该复合物结合，从而与DNA形成拓扑连接并增加其持续合成能力。利用停流荧光共振能量转移，我们研究了全酶组装过程中gp 45环的打开和关闭。位于gp 45亚基界面相对侧的两个氨基酸，用香豆素标记的W 91和V162 C，分别用作荧光供体和受体。在溶液中游离，gp 45具有两个封闭的亚基界面，W 91至V162-香豆素距离为19埃，和一个开放的亚基界面，W 91至V162 C-香豆素距离为40埃。假设两个封闭子单元之间的距离。虽然在全酶组装过程中开放亚基界面的距离没有变化，但我们发现在10步组装机制中，开放亚基界面的距离首先增加到大于45埃，然后减少到30埃。gp 45环在全酶复合物中不完全闭合，这与先前的证据一致，表明gp 43的C-末端插入gp 45亚基界面中。出乎意料的是，ATP水解事件耦合到只有一小部分的总距离变化，与构象变化连接到结合DNA和gp 43耦合到大部分的总距离变化。使用不可水解的ATP类似物ATP-γ-S导致非生产性gp 45 gp 44/62复合物的形成;然而，向该非生产性复合物中加入过量的ATP导致快速的ATP/ATP-γ-S交换，从而在几秒钟内产生生产性gp 45 gp 44/62复合物。
The bacteriophage T4 DNA polymerase holoenzyme, consisting of the DNA polymerase (gp43), the sliding clamp (gp45), and the clamp loader (gp44/62), is loaded onto DNA in an ATP-dependent, multistep reaction. The trimeric, ring-shaped gp45 is loaded onto DNA such that the DNA passes through the center of the ring. gp43 binds to this complex, thereby forming a topological link with the DNA and increasing its processivity. Using stopped-flow fluorescence-resonance energy transfer, we have investigated opening and closing of the gp45 ring during the holoenzyme assembly process. Two amino acids that lie on opposite sides of the gp45 subunit interface, W91 and V162C labeled with coumarin, were used as the fluorescence donor and acceptor, respectively Free in solution, gp45 has two closed subunit interfaces with W91 to V162-coumarin distances of 19 Angstrom and one open subunit interface with a W91 to V162C-coumarin distance of 40 Angstrom. Making the assumption that the distance across the two closed subunit. interfaces is unchanged during the holoenzyme assembly process, we have found that the distance across the open subunit interface is first increased to greater than 45 Angstrom and is then decreased to 30 Angstrom during a 10-step assembly mechanism. The gp45 ring is not completely closed in the holoenzyme complex, consistent with previous evidence suggesting that the C-terminus of gp43 is inserted into the gp45 subunit interface. Unexpectedly, ATP-hydrolysis events are coupled to only a fraction of the total distance change, with conformational changes linked to binding DNA and gp43 coupled to the majority of the total distance change. Using the nonhydrolyzable ATP analogue ATP-gamma-S results in formation of a nonproductive gp45 gp44/62 complex; however, adding an excess of ATP to this nonproductive complex results in rapid ATP/ATP-gamma-S exchange to yield a productive gp45 gp44/62 complex within seconds.