Venclovas Ceslovas, Colvin Michael E, Thelen Michael P
Computational and Systems Biology Division, Biology and Biotechnology Research Program, Lawrence Livermore National Laboratory, California 94550, USA.
Protein Sci. 2002 Oct;11(10):2403-16. doi: 10.1110/ps.0214302.
Replication and related processes in eukaryotic cells require replication factor C (RFC) to load a molecular clamp for DNA polymerase in an ATP-driven process, involving multiple molecular interactions. The detailed understanding of this mechanism is hindered by the lack of data regarding structure, mutual arrangement, and dynamics of the players involved. In this study, we analyzed interactions that take place during loading onto DNA of either the PCNA clamp or the Rad9-Rad1-Hus1 checkpoint complex, using computationally derived molecular models. Combining the modeled structures for each RFC subunit with known structural, biochemical, and genetic data, we propose detailed models of how two of the RFC subunits, RFC1 and RFC3, interact with the C-terminal regions of PCNA. RFC1 is predicted to bind PCNA similarly to the p21-PCNA interaction, while the RFC3-PCNA binding is proposed to be similar to the E. coli delta-beta interaction. Additional sequence and structure analysis, supported by experimental data, suggests that RFC5 might be the third clamp loader subunit to bind the equivalent PCNA region. We discuss functional implications stemming from the proposed model of the RFC1-PCNA interaction and compare putative clamp-interacting regions in RFC1 and its paralogs, Rad17 and Ctf18. Based on the individual intermolecular interactions, we propose RFC and PCNA arrangement that places three RFC subunits in association with each of the three C-terminal regions in PCNA. The two other RFC subunits are positioned at the two PCNA interfaces, with the third PCNA interface left unobstructed. In addition, we map interactions at the level of individual subunits between the alternative clamp loader/clamp system, Rad17-RFC(2-5)/Rad9-Rad1-Hus1. The proposed models of interaction between two clamp/clamp loader pairs provide both structural framework for interpretation of existing experimental data and a number of specific findings that can be subjected to direct experimental testing.
真核细胞中的复制及相关过程需要复制因子C（RFC）在由ATP驱动的过程中为DNA聚合酶加载分子夹子，这涉及多种分子相互作用。由于缺乏有关所涉及分子的结构、相互排列和动力学的数据，对该机制的详细理解受到阻碍。在本研究中，我们使用计算推导的分子模型分析了在将增殖细胞核抗原（PCNA）夹子或Rad9-Rad1-Hus1检查点复合物加载到DNA过程中发生的相互作用。将每个RFC亚基的建模结构与已知的结构、生化和遗传数据相结合，我们提出了RFC的两个亚基RFC1和RFC3如何与PCNA的C末端区域相互作用的详细模型。预计RFC1与PCNA的结合类似于p21与PCNA的相互作用，而RFC3与PCNA的结合被认为类似于大肠杆菌的δ-β相互作用。由实验数据支持的额外序列和结构分析表明，RFC5可能是结合等效PCNA区域的第三个夹子加载器亚基。我们讨论了从所提出的RFC1与PCNA相互作用模型得出的功能含义，并比较了RFC1及其旁系同源物Rad17和Ctf18中假定的与夹子相互作用的区域。基于个体分子间相互作用，我们提出了RFC和PCNA的排列方式，即三个RFC亚基与PCNA的三个C末端区域中的每一个相关联。另外两个RFC亚基位于PCNA的两个界面处，而第三个PCNA界面保持畅通。此外，我们绘制了替代夹子加载器/夹子系统Rad17-RFC(2-5)/Rad9-Rad1-Hus1中各个亚基之间的相互作用。所提出的两个夹子/夹子加载器对之间的相互作用模型既为解释现有实验数据提供了结构框架，也提供了许多可直接进行实验测试的具体发现。
