Bertram J G, Bloom L B, Turner J, O'Donnell M, Beechem J M, Goodman M F
Department of Biological Sciences, Hedco Molecular Biology Laboratories, University of Southern California, Los Angeles, California 90089-1340, USA.
J Biol Chem. 1998 Sep 18;273(38):24564-74. doi: 10.1074/jbc.273.38.24564.
The beta protein, a dimeric ring-shaped clamp essential for processive DNA replication by Escherichia coli DNA polymerase III holoenzyme, is assembled onto DNA by the gamma complex. This study examines the clamp loading pathway in real time, using pre-steady state fluorescent depolarization measurements to investigate the loading reaction and ATP requirements for the assembly of beta onto DNA. Two beta dimer interface mutants, L273A and L108A, and a nonhydrolyzable ATP analog, adenosine 5'-O-(3-thiotriphosphate) (ATPgammaS), have been used to show that ATP binding is required for gamma complex and beta to associate with DNA, but that a gamma complex-catalyzed ATP hydrolysis is required for gamma complex to release the beta.DNA complex and complete the reaction. In the presence of ATP and gamma complex, the beta mutants associate with DNA as efficiently as wild type beta. However, completion of the reaction is much slower with the beta mutants because of decreased ATP hydrolysis by the gamma complex, resulting in a much slower release of the mutants onto DNA. The effects of mutations in the dimer interface were similar to the effects of replacing ATP with ATPgammaS in reactions using wild type beta. Thus, the assembly of beta around DNA is coupled tightly to the ATPase activity of the gamma complex, and completion of the assembly process requires ATP hydrolysis for turnover of the catalytic clamp loader.
β蛋白是一种二聚体环状夹子，对大肠杆菌DNA聚合酶III全酶进行持续性DNA复制至关重要，它由γ复合体组装到DNA上。本研究利用稳态前荧光去极化测量来实时检测夹子加载途径，以研究β蛋白组装到DNA上的加载反应和ATP需求。两个β二聚体界面突变体L273A和L108A，以及一种不可水解的ATP类似物腺苷5'-O-(3-硫代三磷酸)(ATPγS)，已被用于表明ATP结合是γ复合体和β蛋白与DNA结合所必需的，但γ复合体催化的ATP水解是γ复合体释放β-DNA复合体并完成反应所必需的。在ATP和γ复合体存在的情况下，β突变体与DNA结合的效率与野生型β蛋白相同。然而，由于γ复合体的ATP水解减少，β突变体的反应完成要慢得多，导致突变体释放到DNA上的速度慢得多。在使用野生型β蛋白的反应中，二聚体界面突变的影响与用ATPγS替代ATP的影响相似。因此，β蛋白围绕DNA的组装与γ复合体的ATP酶活性紧密偶联，组装过程的完成需要ATP水解以使催化夹子加载器周转。
