ATP binding to the Escherichia coli clamp loader powers opening of the ring-shaped clamp of DNA polymerase III holoenzyme
ATP binding to the Escherichia coli clamp loader powers opening of the ring-shaped clamp of DNA polymerase III holoenzyme
复制标题
DOI：
10.1074/jbc.273.38.24550
复制
发表时间：
1998-09-18
影响因子：
4.8
通讯作者：
O'Donnell, M
中科院分区：
文献类型：
--
作者：
Hingorani, MM;O'Donnell, M
大肠杆菌伽马复合体作为钳位加载器，在DNA复制过程中催化依赖于ATP的β蛋白钳位组装到启动的DNA模板上。这些环状的夹子将DNA聚合酶III的全酶连接到模板上，促进了DNA的快速和连续合成。本报告重点研究了在钳夹负荷过程中，伽玛复合体催化的ATP结合和水解酶的作用。我们证明了ATP与伽马复合体结合的能量为钳制加载途径中的几个初始事件提供动力。伽马复合体((Gamma2)Delta‘(Chi)psi)与两个高亲和力(K-d=1-2.5x10(-6)M)或两个亲和力稍低(K-d=5-6.5x10(-6)nr)的腺苷5’-O-(3-硫代三磷酸)(ATPGamma S)分子结合。在第一次三磷酸腺苷翻转(k(CAT)=4×10(-3)S(-1)在4摄氏度)之前进行的实验，或在三磷酸腺苷伽玛S(k(CAT)=1×10(-4)S(-1)在37摄氏度)的存在下进行的实验表明，与三磷酸腺苷相互作用时，伽马复合体发生构象变化。这种与ATP结合的伽马复合体与β结合，并在二聚体界面打开环。在ATP水解之前，伽马复合体和开放的β环与引物-模板DNA具有高亲和力。因此，ATP结合为钳位加载途径中的所有步骤提供动力，导致伽马复合体开放的β环的组装。DNA中间体，为钳位加载器的环关闭和翻转设置阶段，这些步骤可能与随后的ATP水解有关。
The Escherichia coli gamma complex serves as a clamp loader, catalyzing ATP-dependent assembly of beta protein clamps onto primed DNA templates during DNA replication. These ring-shaped clamps tether DNA polymerase III holoenzyme to the template, facilitating rapid and processive DNA synthesis. This report focuses on the role of ATP binding and hydrolysis catalyzed by the gamma complex during clamp loading. We show that the energy from ATP binding to gamma complex powers several initial events in the clamp loading pathway. The gamma complex ((gamma 2)delta delta'(chi)psi) binds two ATP molecules (one per gamma subunit in the complex) with high affinity (K-d = 1-2.5 x 10(-6) M) or two adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S) molecules with slightly lower affinity (K-d = 5-6.5 x 10(-6) nr). Experiments performed prior to the first ATP turnover (k(cat) = 4 x 10(-3) s(-1) at 4 degrees C), or in the presence of ATP gamma S (k(cat) = 1 x 10(-4) s(-1) at 37 degrees C), demonstrate that upon interaction with ATP the gamma complex undergoes a change in conformation. This ATP-bound gamma complex binds beta and opens the ring at the dimer interface. Still prior to ATP hydrolysis, the composite of gamma complex and the open beta ring binds with high affinity to primer-template DNA. Thus ATP binding powers all the steps in the clamp loading pathway leading up to the assembly of a gamma complex open beta ring.DNA intermediate, setting the stage for ring closing and turnover of the clamp loader, steps that may be linked to subsequent hydrolysis of ATP.