Naktinis V, Onrust R, Fang L, O'Donnell M
Microbiology Department, Cornell University Medical College, New York, New York 10021, USA.
J Biol Chem. 1995 Jun 2;270(22):13358-65.
The Escherichia coli replicase, DNA polymerase III holoenzyme, derives its processivity from the beta subunit sliding clamp that encircles DNA and tethers the replicase to the template. The beta dimer is assembled around DNA by the gamma complex clamp loader in an ATP-dependent reaction. In this report, the essential contact between the clamp loader and beta is identified as mediated through the delta subunit of the gamma complex. The delta subunit appears to contact the face of the beta dimer ring that contains the two C termini. Surprisingly, ATP is required for the gamma complex to bind beta, but not for delta to bind beta. This indicates that delta is buried in the gamma complex and suggests a role for ATP in exposing delta for interaction with beta. A protease protection assay has been developed to specifically probe the delta subunit within the gamma complex. The results of the assay are consistent with an ATP-induced conformational change in the gamma complex that alters the state of the delta subunit within it. The implication of these key features to the clamp loading mechanism of the gamma complex is discussed.
大肠杆菌复制酶，即DNA聚合酶III全酶，其持续合成能力源于β亚基滑动夹，该滑动夹环绕DNA并将复制酶系于模板上。β二聚体由γ复合体夹装器在ATP依赖反应中围绕DNA组装而成。在本报告中，夹装器与β之间的关键接触被确定为由γ复合体的δ亚基介导。δ亚基似乎与β二聚体环中包含两个C末端的面接触。令人惊讶的是，γ复合体结合β需要ATP，但δ结合β则不需要。这表明δ埋于γ复合体中，并提示ATP在使δ暴露以与β相互作用方面的作用。已开发出一种蛋白酶保护试验，以特异性探测γ复合体中的δ亚基。试验结果与γ复合体中由ATP诱导的构象变化一致，该变化改变了其中δ亚基的状态。讨论了这些关键特征对γ复合体夹装机制的影响。
