Recruitment of ORC or CDC6 to DNA is sufficient to create an artificial origin of replication in mammalian cells
Recruitment of ORC or CDC6 to DNA is sufficient to create an artificial origin of replication in mammalian cells
复制标题
DOI：
10.1101/gad.1369805
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发表时间：
2005-12-01
影响因子：
10.5
通讯作者：
Dutta, A
中科院分区：
文献类型：
--
作者：
Takeda, DY;Shibata, Y;Dutta, A
在真核生物中，复制起点被认为可以募集起始蛋白，如起点识别复合物（origin recognition complex，ORC）和Cdc 6，并为DNA解旋提供平台。在这里，我们测试是否本地化的起始蛋白到DNA是足够的起源功能。ORC复合物和Cdc 6的不同组分刺激前复制复合物（前RC）的形成和复制起始时，融合到GAL 4 DNA结合域和招聘到质粒DNA含有GAL 4结合位点的串联阵列。每个细胞周期发生一次复制，并被Geminin抑制，表明质粒在细胞周期中得到适当许可。GAL 4融合蛋白募集ORC-Cdc 6复合物的其他多肽，并且新生链丰度在GAL 4结合位点附近最高。因此，人工起点重现了生理起点的许多调控特征，对哺乳动物细胞复制起始的研究具有重要价值。我们证明了该系统的实用性，通过显示人Cdc 6和Orc 1的ATP酶结构域的功能的重要性和Orc 1和Orc 2的N-末端片段在本试验中的可分配性。将真核细胞复制起始因子人工募集到DNA序列中可以产生功能性复制起点，从而为这些因子提供稳健的遗传测定和产生用于基因治疗的附加型载体的新方法。
Origins of replication are expected to recruit initiation proteins like origin recognition complex (ORC) and Cdc6 in eukaryotes and provide a platform for unwinding DNA. Here we test whether localization of initiation proteins onto DNA is sufficient for origin function. Different components of the ORC complex and Cdc6 stimulated prereplicative complex (pre-RC) formation and replication initiation when fused to the GAL4 DNA-binding domain and recruited to plasmid DNA containing a tandem array of GAL4-binding sites. Replication occurred once per cell cycle and was inhibited by Geminin, indicating that the plasmid was properly licensed during the cell cycle. The GAL4 fusion protein recruits other polypeptides of the ORC-Cdc6 complex, and nascent strand abundance was highest near the GAL4-binding sites. Therefore, the artificial origin recapitulates many of the regulatory features of physiological origins and is valuable for studies on replication initiation in mammalian cells. We demonstrated the utility of this system by showing the functional importance of the ATPase domains of human Cdc6 and Orc1 and the dispensability of the N-terminal segments of Orc1 and Orc2 in this assay. Artificial recruitment of a eukaryotic cellular replication initiation factor to a DNA sequence can create a functional origin of replication, providing a robust genetic assay for these factors and a novel approach to generating episomal vectors for gene therapy.