Zhuang Zhihao, Berdis Anthony J, Benkovic Stephen J
Department of Chemistry, 414 Wartik Laboratory, The Pennsylvania State University, University Park, Pennsylvania 16802, USA.
Biochemistry. 2006 Jul 4;45(26):7976-89. doi: 10.1021/bi0601205.
In bacteriophage T4, a clamp loading pathway that utilizes the T4 clamp loader (gp44/62) and ATP hydrolysis initially to form a complex with the clamp (gp45) has been demonstrated, followed by interaction with DNA and closing of the clamp. However, the recent observation that gp45 exists as an opened form in solution raises the possibility of other pathways for clamp loading. In this study, an alternative clamp loading sequence is evaluated in which gp44/62 first recognizes the DNA substrate and then sequesters the clamp from solution and loads it onto DNA. This pathway differs in terms of the initial formation of a gp44/62-DNA complex that is capable of loading gp45. In this work, we demonstrate ATP-dependent DNA binding by gp44/62. Among various DNA structures that were tested, gp44/62 binds specifically to primer-template DNA but not to single-stranded DNA or blunt-end duplex DNA. By tracing the dynamic clamp closing with pre-steady-state FRET measurements, we show that the clamp loader-DNA complex is functional in clamp loading. Furthermore, pre-steady-state ATP hydrolysis experiments suggest that 1 equiv of ATP is hydrolyzed when gp44/62 binds to DNA, and additional ATP hydrolysis is associated with the completion of the clamp loading process. We also investigated the detailed kinetics of binding of MANT-nucleotide to gp44/62 through stopped-flow FRET and demonstrated a conformational change as the result of ATP, but not ADP binding. The collective kinetic data allowed us to propose and evaluate a sequence of steps describing this alternative pathway for clamp loading and holoenzyme formation.
在噬菌体T4中，已经证明了一种利用T4夹子加载器（gp44/62）和ATP水解最初与夹子（gp45）形成复合物的夹子加载途径，随后与DNA相互作用并关闭夹子。然而，最近观察到gp45在溶液中以开放形式存在，这增加了夹子加载存在其他途径的可能性。在本研究中，评估了一种替代的夹子加载序列，其中gp44/62首先识别DNA底物，然后从溶液中隔离夹子并将其加载到DNA上。该途径在能够加载gp45的gp44/62-DNA复合物的初始形成方面有所不同。在这项工作中，我们证明了gp44/62的ATP依赖性DNA结合。在测试的各种DNA结构中，gp44/62特异性结合引物-模板DNA，但不结合单链DNA或平端双链DNA。通过用预稳态荧光共振能量转移（FRET）测量追踪动态夹子关闭，我们表明夹子加载器-DNA复合物在夹子加载中起作用。此外，预稳态ATP水解实验表明，当gp44/62与DNA结合时，1当量的ATP被水解，额外的ATP水解与夹子加载过程的完成相关。我们还通过停流FRET研究了MANT-核苷酸与gp44/62结合的详细动力学，并证明了ATP结合而非ADP结合导致的构象变化。这些综合的动力学数据使我们能够提出并评估描述这种替代夹子加载和全酶形成途径的一系列步骤。
