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Functions of ORCI to establish replicative complex on chromatin in human

ORCI在人类染色质上建立复制复合体的功能

基本信息

批准号：
13680786
负责人：
OBUSE Chikasi
金额：
$ 2.3万
依托单位：
NARA INSTITUTE OF SCIENCE AND TECHNOLOGY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2002
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13680786/
关键词：
ORC heterochromatin chromosome mass spectrography functional regulation affinity putification replication proteomics 複合体抗体

项目摘要

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.

起源识别复合物 (ORC) 在调节真核生物 DNA 复制的起始过程中发挥着核心作用。我们观察到 ORC1（ORC1 周期）的细胞周期依赖性振荡，其中它在 G1 期积累并在 S 期降解，尽管其他 ORC 亚基 (ORCs2-5) 在整个细胞周期中以几乎恒定的水平存在。我们研究了它们在人类细胞核中与 ORC1 振荡相关的行为。结果表明，ORCs2-5在整个细胞周期中形成复合物，根据ORC1在G1细胞核中的积累，进一步与ORC1暂时结合。 ORCs2-5 存在于核酸酶不溶性和可溶性级分中。前一个群体的出现与 ORC1 的积累同时出现，ORC1 与核酸酶抗性、非染色质核结构相关。因此，ORCs2-5将通过形成ORC1-5复合物而暂时招募到核酸酶抗性结构中。事实上，通过在人类细胞中转染 ORC1 的 siRNA 来人为降低 ORC1 水平，导致 ORC2 转变为核酸酶敏感群体。此外，我们观察到这种处理也阻断了 MCM 与染色质组分的关联。这些数据表明，ORC1 的振荡通过将 ORCs2-5 束缚到核结构来主动调节人类细胞核中 ORC 复合物的状态。这种动态变化进一步驱动了染色质上 MCM 的负载。因此，表明人类细胞中的预复制复合体将受到ORC1在G1核中的时间积累的调节。