Functions of ORCI to establish replicative complex on chromatin in human

起源识别复合体（ORC）在真核生物DNA复制起始调控中起着核心作用。我们观察到ORC1的细胞周期依赖性振荡（ORC1周期），其中它在G1期积累并在S期降解，尽管其他ORC亚单位（ORCs2-5）在整个细胞周期中几乎以恒定的水平存在。我们研究了它们在人类细胞核中的行为与orc1振荡的关系。结果表明，ORCs2-5在整个细胞周期中形成复合物，并根据ORC1在G1核中的积累进一步与ORC1暂时结合。ORCs2-5存在于核酸酶不溶和可溶组分中。前者群体与ORC1的积累平行，ORC1与核酸酶抗性相关，非染色质核结构。因此，ORC1-5将通过形成ORC1-5复合物而暂时被招募到抗核酸酶结构中。事实上，在人类细胞中通过转染其siRNA人工降低ORC1水平导致ORC2向核酸酶敏感人群转移。此外，我们观察到MCM与染色质组分的关联也被这种处理阻断。这些数据表明，ORC1的振荡通过将ORCs2-5拴在细胞核结构上，积极调节人类细胞核中ORC复合物的状态。这种动态变化进一步驱动mcm在染色质上的装载。因此，我们认为人类细胞中的预复制复合体将受到G1核中ORC1的时间积累的调节。

Origin recognition complex (ORC) plays a central role in regulating initiation of DNA replication in eukaryotes. We have observed cell cycle dependent oscillation of ORC1 (the ORC1 cycle), in which it accumulates in G1 and degrades in S phase, although other ORC subunits (ORCs2-5) exist at almost constant levels throughout the cell cycle. We studied their behavior in human cell nuclei in relation with the ORC1oscilliation. The results demonstrated that ORCs2-5 form a complex throughout the cell cycle, which further associates with ORC1 temporarily according to accumulation of ORC1 in G1 nuclei. ORCs2-5 exist both in nuclease insoluble and soluble fractions. The former population appears in parallel with the accumulation of ORC1 associating with nuclease resistant, non-chromatin nuclear structures. Thus, ORCs2-5 will be temporarily recruited to the nuclease resistant structures by formation of ORC1-5 complex. Indeed, artificial reduction of ORC1 level by transfection of its siRNA in human cells resulted in a shift of ORC2 to the nuclease sensitive population. Furthermore, we observed that association of MCM to chromatin fractions is also blocked by this treatment. These data propose that oscillation of ORC1 actively regulates the status of ORC complex in human cell nuclei by tethering ORCs2-5 to the nuclear structures. This dynamic shift further drives loading of MCMs on chromatin. Thus, it is suggested that the pre-replication complex in human cells will be regulated by temporal accumulation of ORC1 in G1 nuclei.