Bloom L B, Turner J, Kelman Z, Beechem J M, O'Donnell 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. 1996 Nov 29;271(48):30699-708. doi: 10.1074/jbc.271.48.30699.
A "minimal" DNA primer-template system, consisting of an 80-mer template and 30-mer primer, supports processive DNA synthesis by DNA polymerase III core in the presence of the beta sliding clamp, gamma complex clamp loader, and single-stranded binding protein from Escherichia coli. This primer-template system was used to measure the loading of the beta sliding clamp by the gamma complex in an ATP-dependent reaction. Bound protein-DNA complexes were detected by monitoring fluorescence depolarization of DNA. Steady state and time-resolved anisotropies were measured, and stopped-flow pre-steady state fluorescence measurements allowed visualization of the loading reactions in real time. The rate of loading beta onto DNA was 12 s-1, demonstrating that clamp assembly is rapid on the time scale required for lagging strand Okazaki fragment synthesis. The association rate appears to be limited by an intramolecular step occurring prior to the clamp-loading reaction, possibly the opening of the toroidal beta dimer.
一个“最小化”的DNA引物-模板系统，由一个80聚体模板和30聚体引物组成，在存在β滑动夹、γ复合物夹装载器和来自大肠杆菌的单链结合蛋白的情况下，支持DNA聚合酶III核心进行持续性DNA合成。这个引物-模板系统被用于在依赖ATP的反应中测量γ复合物对β滑动夹的装载。通过监测DNA的荧光去极化来检测结合的蛋白质-DNA复合物。测量了稳态和时间分辨各向异性，并且停流预稳态荧光测量允许实时观察装载反应。将β装载到DNA上的速率为12 s⁻¹，表明在滞后链冈崎片段合成所需的时间尺度上，夹组装是快速的。缔合速率似乎受到夹装载反应之前发生的分子内步骤的限制，可能是环形β二聚体的打开。
