Chen Yueyue, Qian Jie, You Li, Zhang Xiufeng, Jiao Jinxia, Liu Yang, Zhao Jie
State Key Laboratory of Hybrid Rice, College of Life Sciences, Wuhan University, Wuhan, China.
Front Plant Sci. 2018 Jun 19;9:779. doi: 10.3389/fpls.2018.00779. eCollection 2018.
Replication factor C (RFC) is a multisubunit complex that opens the sliding clamp and loads it onto the DNA chain in an ATP-dependent manner and is thus critical for high-speed DNA synthesis. In yeast () and humans, biochemical studies and structural analysis revealed interaction patterns between the subunits and architectures of the clamp loaders. Mutations of lead to loss of cell viability and defective replication. However, the functions of RFC subunits in higher plants are unclear, except for , and the interaction and arrangement of the subunits have not been studied. Here, we identified /+, /+, and /+ mutants in , and found that embryos and endosperm arrested at the 2/4-celled embryo proper stage and 6-8 nuclei stages, respectively. Subcellular localization analysis revealed that AtRFC1 and OsRFC1/4/5 proteins were localized in the nucleus, while AtRFC2/3/4/5 and OsRFC2/3 proteins were present both in the nucleus and cytoplasm. By using yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) techniques, we demonstrated the interactions of and rice () RFC subunits, and proposed arrangements of the five subunits within the RFC complex, which were AtRFC5-AtRFC4-AtRFC3/2-AtRFC2/3-AtRFC1 and OsRFC5-OsRFC2-OsRFC3-OsRFC4-OsRFC1, respectively. In addition, AtRFC1 could interact with AtRFC2/3/4/5 in the presence of other subunits, while OsRFC1 directly interacted with the other four subunits. To further characterize the regions required for complex formation, truncated RFC proteins of the subunits were created. The results showed that C-termini of the RFC subunits are required for complex formation. Our studies indicate that the localization and interactions of RFCs in and rice are distinctly discrepant.
复制因子C（RFC）是一种多亚基复合体，它能打开滑动夹并以ATP依赖的方式将其加载到DNA链上，因此对高速DNA合成至关重要。在酵母（）和人类中，生化研究和结构分析揭示了亚基之间的相互作用模式以及夹加载器的结构。的突变导致细胞活力丧失和复制缺陷。然而，除了之外，RFC亚基在高等植物中的功能尚不清楚，并且亚基之间的相互作用和排列尚未得到研究。在这里，我们在中鉴定出 /+、 /+ 和 /+ 突变体，发现胚胎和胚乳分别在2/4细胞期的胚体阶段和6 - 8核阶段停滞。亚细胞定位分析表明，AtRFC1和OsRFC1/4/5蛋白定位于细胞核，而AtRFC2/3/4/5和OsRFC2/3蛋白在细胞核和细胞质中均有存在。通过使用酵母双杂交（Y2H）和双分子荧光互补（BiFC）技术，我们证明了和水稻（）RFC亚基之间的相互作用，并提出了RFC复合体中五个亚基的排列方式，分别为AtRFC5 - AtRFC4 - AtRFC3/2 - AtRFC2/3 - AtRFC1和OsRFC5 - OsRFC2 - OsRFC3 - OsRFC4 - OsRFC1。此外，在其他亚基存在的情况下，AtRFC1可以与AtRFC2/3/4/5相互作用，而OsRFC1直接与其他四个亚基相互作用。为了进一步表征复合体形成所需的区域，构建了亚基的截短RFC蛋白。结果表明，RFC亚基的C末端是复合体形成所必需的。我们的研究表明，RFC在和水稻中的定位和相互作用明显不同。
