Fu T J, Sanders G M, O'Donnell M, Geiduschek E P
Department of Biology, University of California, San Diego, La Jolla 92093-0634, USA.
EMBO J. 1996 Aug 15;15(16):4414-22.
Bacteriophage T4 gene 45 protein (gp45) and Escherichia coli beta are DNA-tracking sliding-clamp proteins that increase processivity by tethering their conjugate DNA polymerases to DNA. gp45 also activates T4 late transcription. DNA loading of gp45 and beta requires ATP or dATP hydrolysis; efficient loading at primer-template junctions is assisted by single-stranded DNA-binding proteins. The kinetics of gp45 loading and tracking have been examined by DNase I footprinting of linear DNA with one blunt end, one primer-template junction, and binding sites for proteins that block gp45 tracking. DNA loading of gp45 can also be interrupted by adding the non-hydrolyzable ATP analog ATP-gamma-S. At saturation, DNA is very closely packed with gp45 or beta. When gp45 loading is interrupted, or when a segment of the track is blocked off, the gp45 footprint dissipates within seconds, but the DNA-tracking state of beta is much more stable. The stability of the tracking state of gp45 is, however, increased by the macromolecular crowding agent polyethylene glycol. We suggest that labile gp45 catenation directly generates the coupling of late transcription to DNA replication during bacteriophage T4 multiplication.
噬菌体T4基因45蛋白（gp45）和大肠杆菌β蛋白是DNA追踪滑动夹蛋白，它们通过将其共轭DNA聚合酶与DNA相连来提高持续合成能力。gp45还激活T4晚期转录。gp45和β蛋白的DNA加载需要ATP或dATP水解；单链DNA结合蛋白有助于在引物-模板连接处高效加载。通过对具有一个平端、一个引物-模板连接处以及阻断gp45追踪的蛋白结合位点的线性DNA进行DNase I足迹分析，研究了gp45加载和追踪的动力学。添加不可水解的ATP类似物ATP-γ-S也可中断gp45的DNA加载。在饱和状态下，DNA与gp45或β蛋白紧密堆积。当gp45加载中断或一段追踪路径被阻断时，gp45足迹在数秒内消散，但β蛋白的DNA追踪状态则稳定得多。然而，大分子拥挤剂聚乙二醇可增加gp45追踪状态的稳定性。我们认为，不稳定的gp45连环直接在噬菌体T4增殖过程中产生晚期转录与DNA复制的偶联。
