Thompson Jennifer A, Paschall Christopher O, O'Donnell Mike, Bloom Linda B
Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, Florida 32610-0245, USA.
J Biol Chem. 2009 Nov 13;284(46):32147-57. doi: 10.1074/jbc.M109.045997. Epub 2009 Sep 15.
In Escherichia coli, the gamma complex clamp loader loads the beta-sliding clamp onto DNA. The beta 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, gamma complex binds beta and DNA to form a ternary complex. Binding to primed template DNA triggers gamma complex to hydrolyze ATP and release the clamp onto DNA. Here, we investigated the kinetics of forming a ternary complex by measuring rates of gamma complex binding beta and DNA. A fluorescence intensity-based beta binding assay was developed in which the fluorescence of pyrene covalently attached to beta increases when bound by gamma complex. Using this assay, an association rate constant of 2.3 x 10(7) m(-1) s(-1) for gamma complex binding beta was determined. The rate of beta binding was the same in experiments in which gamma complex was preincubated with ATP before adding beta or added directly to beta and ATP. In contrast, when gamma complex is preincubated with ATP, DNA binding is faster than when gamma 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 beta binding and DNA binding differ. The slow ATP-induced conformational change that precedes DNA binding may provide a kinetic preference for gamma complex to bind beta before DNA during the clamp loading reaction cycle.
在大肠杆菌中，γ复合体钳加载器将β滑动钳加载到DNA上。β钳将DNA聚合酶III与DNA相连，并通过提高DNA合成的持续合成能力来增强复制效率。在ATP存在的情况下，γ复合体与β和DNA结合形成三元复合物。与引发的模板DNA结合会触发γ复合体水解ATP并将钳释放到DNA上。在此，我们通过测量γ复合体与β和DNA的结合速率来研究三元复合物形成的动力学。我们开发了一种基于荧光强度的β结合测定法，其中当与γ复合体结合时，与β共价连接的芘的荧光会增强。使用该测定法，确定了γ复合体与β结合的缔合速率常数为2.3×10⁷ m⁻¹ s⁻¹。在将γ复合体与ATP预孵育后再添加β或直接将其添加到β和ATP的实验中，β的结合速率是相同的。相比之下，当γ复合体与ATP预孵育时，DNA结合比γ复合体同时添加到DNA和ATP时更快。在没有ATP预孵育的情况下DNA结合缓慢是限速ATP诱导的构象变化的结果。我们的结果强烈表明，促进β结合和DNA结合的ATP诱导的构象变化是不同的。在DNA结合之前缓慢的ATP诱导的构象变化可能在钳加载反应循环中为γ复合体在DNA之前结合β提供了动力学偏好。
