Hingorani M M, O'Donnell M
Rockefeller University, New York, New York 10021, USA.
J Biol Chem. 1998 Sep 18;273(38):24550-63. doi: 10.1074/jbc.273.38.24550.
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 (gamma2 delta delta'chi psi) binds two ATP molecules (one per gamma subunit in the complex) with high affinity (Kd = 1-2. 5 x 10(-6) M) or two adenosine 5'-O-(3-thiotriphosphate)(ATPgammaS) molecules with slightly lower affinity (Kd = 5-6.5 x 10(-6) M). Experiments performed prior to the first ATP turnover (kcat = 4 x 10(-3) s-1 at 4 degreesC), or in the presence of ATPgammaS (kcat = 1 x 10(-4) s-1 at 37 degreesC), 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.
大肠杆菌γ复合物作为一种夹子加载器，在DNA复制过程中催化β蛋白夹子以ATP依赖的方式组装到引发的DNA模板上。这些环形夹子将DNA聚合酶III全酶拴系到模板上，促进快速且持续的DNA合成。本报告聚焦于γ复合物在夹子加载过程中催化的ATP结合与水解的作用。我们发现，ATP与γ复合物结合所产生的能量为夹子加载途径中的几个初始事件提供动力。γ复合物（γ2δδ'χψ）以高亲和力（Kd = 1 - 2.5×10⁻⁶ M）结合两个ATP分子（复合物中每个γ亚基结合一个），或以稍低亲和力（Kd = 5 - 6.5×10⁻⁶ M）结合两个腺苷5'-O-(3-硫代三磷酸)(ATPγS)分子。在首次ATP周转之前（4℃时kcat = 4×10⁻³ s⁻¹）或存在ATPγS的情况下（37℃时kcat = 1×10⁻⁴ s⁻¹）进行的实验表明，γ复合物与ATP相互作用后会发生构象变化。这种结合了ATP的γ复合物与β结合并在二聚体界面处打开环。仍在ATP水解之前，γ复合物与开放的β环的复合物以高亲和力结合到引物 - 模板DNA上。因此，ATP结合为夹子加载途径中直至γ复合物 - 开放β环 - DNA中间体组装的所有步骤提供动力，为环闭合和夹子加载器的周转奠定基础，这些步骤可能与随后的ATP水解相关。
