Guan Ling, He Peng, Yang Fang, Zhang Yuan, Hu Yunfei, Ding Jienv, Hua Yu, Zhang Yi, Ye Qiong, Hu Jiazhi, Wang Tao, Jin Changwen, Kong Daochun
From the Peking-Tsinghua Center for Life Sciences, National Laboratory of Protein and Plant Gene Research, College of Life Sciences.
the Beijing Nuclear Magnetic Resonance Center.
J Biol Chem. 2017 Apr 14;292(15):6056-6075. doi: 10.1074/jbc.M116.767806. Epub 2017 Feb 21.
A central step in the initiation of chromosomal DNA replication in eukaryotes is the assembly of pre-replicative complex (pre-RC) at late M and early G phase of the cell cycles. Since 1973, four proteins or protein complexes, including cell division control protein 6 (Cdc6)/Cdc18, minichromosome maintenance protein complex, origin recognition complex (ORC), and Cdt1, are known components of the pre-RC. Previously, we reported that a non-ORC protein binds to the essential element Δ9 of the DNA-replication origin ARS3001. In this study, we identified that the non-ORC protein is Sap1. Like ORC, Sap1 binds to DNA origins during cell growth cycles. But unlike ORC, which binds to asymmetric AT-rich sequences through its nine AT-hook motifs, Sap1 preferentially binds to a DNA sequence of 5'-(A/T) (C/G)(A/T)(G/C)(A/T) -3' ( ≥ 1). We also found that Sap1 and ORC physically interact. We further demonstrated that Sap1 is required for the assembly of the pre-RC because of its essential role in recruiting Cdc18 to DNA origins. Thus, we conclude that Sap1 is a replication-initiation factor that directly participates in the assembly of the pre-RC. DNA-replication origins in fission yeast are defined by possessing two essential elements with one bound by ORC and the other by Sap1.
真核生物中启动染色体DNA复制的核心步骤是在细胞周期的M期末期和G期早期组装前复制复合体（pre-RC）。自1973年以来，包括细胞分裂控制蛋白6（Cdc6）/Cdc18、微小染色体维持蛋白复合体、复制起点识别复合体（ORC）和Cdt1在内的四种蛋白质或蛋白质复合体是pre-RC的已知组成部分。此前，我们报道一种非ORC蛋白与DNA复制起点ARS3001的必需元件Δ9结合。在本研究中，我们鉴定出该非ORC蛋白是Sap1。与ORC一样，Sap1在细胞生长周期中与DNA起点结合。但与通过其九个AT钩基序与富含AT的不对称序列结合的ORC不同，Sap1优先与5'-(A/T) (C/G)(A/T)(G/C)(A/T) -3'（≥1）的DNA序列结合。我们还发现Sap1与ORC存在物理相互作用。我们进一步证明Sap1是pre-RC组装所必需的，因为它在将Cdc18招募到DNA起点中起关键作用。因此，我们得出结论，Sap1是一种直接参与pre-RC组装的复制起始因子。裂殖酵母中的DNA复制起点由两个必需元件定义，其中一个由ORC结合，另一个由Sap1结合。
