The structure of a ring-opened proliferating cell nuclear antigen-replication factor C complex revealed by fluorescence energy transfer
The structure of a ring-opened proliferating cell nuclear antigen-replication factor C complex revealed by fluorescence energy transfer
复制标题
DOI：
10.1073/pnas.0511263103
复制
发表时间：
2006-02-21
影响因子：
11.1
通讯作者：
Benkovic, SJ
中科院分区：
文献类型：
--
作者：
Zhuang, ZH;Yoder, BL;Benkovic, SJ
已知许多在DNA代谢途径中起作用的蛋白质与增殖细胞核抗原（PCNA）相互作用。PCNA在刺激各种细胞活动中的重要功能需要其与DNA的拓扑连接。将环状PCNA装载到双链DNA上需要clamp-loader [复制因子C（RFC）]复合物的活性和来自ATP水解的能量。关于RFC复合物加载PCNA的机制和结构细节仍在发展中。特别是，一个长期假设的结构，一个开放的钳-RFC复合物作为一个中间负载的积极识别仍然难以捉摸。在这项研究中，我们捕获一个开放的酵母细胞增殖细胞核抗原钳与RFC复合物通过荧光能量转移实验。我们还遵循的拓扑结构转换的PCNA的钳加载途径的各个步骤，通过稳态和停流荧光研究。我们发现，ATP有效地驱动钳加载过程完成与DNA结合的封闭的PCNA的形成，而ATP γ S不能。从这项工作中获得的信息补充了以前的结构和机制研究，并提供了一个更完整的图片真核细胞的钳加载途径，使用酵母作为范例。
Numerous proteins that function in DNA metabolic pathways are known to interact with the proliferating cell nuclear antigen (PCNA). The important function of PCNA in stimulating various cellular activities requires its topological linkage with DNA. Loading of the circular PCNA onto duplex DNA requires the activity of a clamp-loader [replication factor C (RFC)] complex and the energy derived from ATP hydrolysis. The mechanistic and structural details regarding PCNA loading by the RFC complex are still developing. In particular, the positive identification of a long-hypothesized structure of an open clamp-RFC complex as an intermediate in loading has remained elusive. In this study, we capture an open yeast PCNA clamp in a complex with RFC through fluorescence energy transfer experiments. We also follow the topological transitions of PCNA in the various steps of the clamp-loading pathway through both steady-state and stopped-flow fluorescence studies. We find that ATP effectively drives the clamp-loading process to completion with the formation of the closed PCNA bound to DNA, whereas ATP gamma S cannot. The information derived from this work complements that obtained from previous structural and mechanistic studies and provides a more complete picture of a eukaryotic clamp-loading pathway using yeast as a paradigm.