Architecture of the human origin recognition complex
Architecture of the human origin recognition complex
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DOI：
10.1074/jbc.m103078200
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发表时间：
2001-08-03
影响因子：
4.8
通讯作者：
Dutta, A
中科院分区：
文献类型：
--
作者：
Dhar, SK;Delmolino, L;Dutta, A
迄今为止，酿酒酵母起源识别复合物六个亚基的人类同源物均已报道。然而，关于人类起源识别复合体的性质和特征的报道并不多。在尝试从感染表达 HsORC 亚基的杆状病毒的昆虫细胞中纯化重组人 ORC 时，我们发现人 ORC2、-3、-4 和 -5 形成核心复合物。 HsORC1 和 HsORC6 亚基没有进入该核心复合物，表明这两个亚基与核心 ORC2-5 复合物的相互作用极其不稳定。我们发现ORC2的C端区域与ORC3的N端区域直接相互作用。然而，ORC3 的 C 末端区域是将 ORC4 和 ORC5 引入核心复合物所必需的。含有 ORC3 N 端 200 个残基的片段 (ORC3N) 竞争性抑制 ORC2-ORC3 相互作用。该片段在 U2OS 细胞中的过度表达阻断了细胞的 G(1) 状态，这提供了第一个证据，表明哺乳动物 ORC 亚基对于哺乳动物细胞中的 G(1)-S 转变很重要。
All the human homologs of the six subunits of Saccharomyces cerevisiae origin recognition complex have been reported so far. However, not much has been reported on the nature and the characteristics of the human origin recognition complex. In an attempt to purify recombinant human ORC from insect cells infected with baculoviruses expressing HsORC subunits, we found that human ORC2, -3, -4, and -5 form a core complex. HsORC1 and HsORC6 subunits did not enter into this core complex, suggesting that the interaction of these two subunits with the core ORC2-5 complex is extremely labile. We found that the C-terminal region of ORC2 interacts directly with the N-terminal region of ORC3. The C-terminal region of ORC3 was, however, necessary to bring ORC4 and ORC5 into the core complex. A fragment containing the N-terminal 200 residues of ORC3 (ORC3N) competitively inhibited the ORC2-ORC3 interaction. Overexpression of this fragment in U2OS cells blocked the cells in G(1), providing the first evidence that a mammalian ORC subunit is important for the G(1)-S transition in mammalian cells.