Lefebvre S D, Wong M L, Morrical S W
Department of Biochemistry, University of Vermont College of Medicine, Burlington, Vermont 05405, USA.
J Biol Chem. 1999 Aug 6;274(32):22830-8. doi: 10.1074/jbc.274.32.22830.
The T4 gp59 protein is the major accessory protein of the phage's replicative DNA helicase, gp41. gp59 helps load gp41 at DNA replication forks by promoting its assembly onto single-stranded (ss) DNA covered with cooperatively bound molecules of gp32, the T4 single-strand DNA binding protein (ssb). A gp59-gp32-ssDNA ternary complex is an obligatory intermediate in this helicase loading mechanism. Here, we characterize the properties of gp59-gp32-ssDNA complexes and reveal some of the biochemical interactions that occur within them. Our results indicate the following: (i) gp59 is able to co-occupy ssDNA pre-saturated with either gp32 or gp32-A (a truncated gp32 species lacking interactions with gp59); (ii) gp59 destabilizes both gp32-ssDNA and (gp32-A)-ssDNA interactions; (iii) interactions of gp59 with the A-domain of gp32 alter the ssDNA-binding properties of gp59; and (iv) gp59 organizes gp32-ssDNA versus (gp32-A)-ssDNA into morphologically distinct complexes. Our results support a model in which gp59-gp32 interactions are non-essential for the co-occupancy of both proteins on ssDNA but are essential for the formation of structures competent for helicase assembly. The data argue that specific "cross-talk" between gp59 and gp32, involving conformational changes in both, is a key feature of the gp41 helicase assembly pathway.
T4噬菌体的gp59蛋白是噬菌体复制性DNA解旋酶gp41的主要辅助蛋白。gp59通过促进gp41组装到覆盖有协同结合的gp32分子（T4单链DNA结合蛋白，即ssb）的单链（ss）DNA上，帮助其在DNA复制叉处加载。gp59 - gp32 - ssDNA三元复合物是这种解旋酶加载机制中的一个必需中间体。在此，我们对gp59 - gp32 - ssDNA复合物的特性进行了表征，并揭示了其中发生的一些生化相互作用。我们的结果表明：（i）gp59能够与预先被gp32或gp32 - A（一种缺乏与gp59相互作用的截短型gp32）饱和的ssDNA共同占据；（ii）gp59使gp32 - ssDNA和（gp32 - A）- ssDNA相互作用不稳定；（iii）gp59与gp32的A结构域的相互作用改变了gp59的ssDNA结合特性；（iv）gp59将gp32 - ssDNA与（gp32 - A）- ssDNA组织成形态上不同的复合物。我们的结果支持了一个模型，其中gp59 - gp32相互作用对于两种蛋白质在ssDNA上的共同占据并非必需，但对于形成能够进行解旋酶组装的结构是必需的。数据表明，gp59和gp32之间涉及两者构象变化的特定“串扰”是gp41解旋酶组装途径的一个关键特征。
