A Slow ATP-induced Conformational Change Limits the Rate of DNA Binding but Not the Rate of β Clamp Binding by the Escherichia coli γ Complex Clamp Loader*
A Slow ATP-induced Conformational Change Limits the Rate of DNA Binding but Not the Rate of β Clamp Binding by the Escherichia coli γ Complex Clamp Loader*
复制标题
缓慢的 ATP 诱导的构象变化限制了 DNA 结合速率，但不限制大肠杆菌 γ 复合夹加载器的 β 夹结合速率*
DOI：
--
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发表时间：
2009
影响因子：
4.8
通讯作者：
L. Bloom
中科院分区：
文献类型：
--
作者：
Jennifer A. Thompson;Christopher O. Paschall;M. O’Donnell;L. Bloom
在大肠杆菌中，γ复合物夹加载器将β-滑动夹加载到DNA上。β夹将DNA聚合酶III拴在DNA上，并通过增加DNA合成的持续合成能力来提高复制效率。在ATP存在下，γ复合物与β和DNA结合形成三元复合物。与引发的模板DNA结合触发γ复合物水解ATP并释放到DNA上的夹。在这里，我们通过测量γ复合物结合β和DNA的速率来研究形成三元复合物的动力学。建立了一种基于荧光强度的β结合测定法，其中与β共价连接的芘在与γ复合物结合时荧光增强。使用该测定法，确定γ复合物结合β的结合速率常数为2.3 × 107 m−1 s−1。在加入β之前与ATP预孵育或直接加入β和ATP的实验中，β结合率相同。与此相反，当γ复合物与ATP一起预孵育时，DNA结合比γ复合物同时加入DNA和ATP时更快。在没有ATP预孵育的情况下，缓慢的DNA结合是限速ATP诱导的构象变化的结果。我们的研究结果强烈表明，ATP诱导的构象变化，促进β结合和DNA结合不同。ATP诱导的构象变化先于DNA的结合，这可能为γ复合物在夹持加载反应周期中优先结合β而不是DNA提供了动力学优势。
In Escherichia coli, the γ complex clamp loader loads the β-sliding clamp onto DNA. The β clamp tethers DNA polymerase III to DNA and enhances the efficiency of replication by increasing the processivity of DNA synthesis. In the presence of ATP, γ complex binds β and DNA to form a ternary complex. Binding to primed template DNA triggers γ complex to hydrolyze ATP and release the clamp onto DNA. Here, we investigated the kinetics of forming a ternary complex by measuring rates of γ complex binding β and DNA. A fluorescence intensity-based β binding assay was developed in which the fluorescence of pyrene covalently attached to β increases when bound by γ complex. Using this assay, an association rate constant of 2.3 × 107 m−1 s−1 for γ complex binding β was determined. The rate of β binding was the same in experiments in which γ complex was preincubated with ATP before adding β or added directly to β and ATP. In contrast, when γ complex is preincubated with ATP, DNA binding is faster than when γ complex is added to DNA and ATP at the same time. Slow DNA binding in the absence of ATP preincubation is the result of a rate-limiting ATP-induced conformational change. Our results strongly suggest that the ATP-induced conformational changes that promote β binding and DNA binding differ. The slow ATP-induced conformational change that precedes DNA binding may provide a kinetic preference for γ complex to bind β before DNA during the clamp loading reaction cycle.
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DOI：
10.1016/s0021-9258(18)99166-0
发表时间：
1991-06
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
P. Stukenberg;P. S. Studwell-Vaughan;M. O’Donnell
通讯作者：
P. Stukenberg;P. S. Studwell-Vaughan;M. O’Donnell
从过量生产的菌株中对 DNA 聚合酶 III 全酶的 β 亚基进行化学表征和纯化。
DOI：
--
发表时间：
1986
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Johanson,KO;Haynes,TE;McHenry,CS
通讯作者：
McHenry,CS
DOI：
10.1073/pnas.0511263103
发表时间：
2006-02-21
影响因子：
11.1
作者：
Zhuang, ZH;Yoder, BL;Benkovic, SJ
通讯作者：
Benkovic, SJ
影响因子：
2.9
作者：
Millar, D;Trakselis, MA;Benkovic, SJ
通讯作者：
Benkovic, SJ
影响因子：
2.9
作者：
Kuzmic, P
通讯作者：
Kuzmic, P