ATP-dependent assembly of the human origin recognition complex
ATP-dependent assembly of the human origin recognition complex
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DOI：
10.1074/jbc.m705905200
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发表时间：
2007-11-02
影响因子：
4.8
通讯作者：
Stillman, Bruce
中科院分区：
文献类型：
--
作者：
Siddiqui, Khalid;Stillman, Bruce
起源识别复合物 (ORC) 最初是在芽殖酵母提取物中发现的，它是一种蛋白质复合物，它以 ATP 依赖性方式与自主复制序列高亲和力结合。我们已经克隆了 ORC 亚基的人类同源物并将其表达为重组蛋白。与迄今为止研究的其他真核起始子相反，人 ORC 的体外组装依赖于 ATP 结合。 Orc4 或 Orc5 ATP 结合位点的突变会损害复合物的组装，而 Orc1 ATP 结合不是必需的。使用抗 Orc3 抗体对人类细胞进行免疫荧光染色，证明细胞周期依赖性与核结构的关联。免疫沉淀实验表明，随着细胞进入 S 期，ORC 会分解。 Orc6 蛋白直接与 Orc3 亚基结合，并作为 ORC 的一部分在体内相互作用。这些数据表明，人类细胞中 ORC 的组装和分解受到独特的调节，可能有助于将 DNA 复制限制在每个细胞分裂周期一次。
The origin recognition complex (ORC) was initially discovered in budding yeast extracts as a protein complex that binds with high affinity to autonomously replicating sequences in an ATP-dependent manner. We have cloned and expressed the human homologs of the ORC subunits as recombinant proteins. In contrast to other eukaryotic initiators examined thus far, assembly of human ORC in vitro is dependent on ATP binding. Mutations in the ATP-binding sites of Orc4 or Orc5 impair complex assembly, whereas Orc1 ATP binding is not required. Immunofluorescence staining of human cells with anti-Orc3 antibodies demonstrate cell cycle-dependent association with a nuclear structure. Immunoprecipitation experiments show that ORC disassembles as cells progress through S phase. The Orc6 protein binds directly to the Orc3 subunit and interacts as part of ORC in vivo. These data suggest that the assembly and disassembly of ORC in human cells is uniquely regulated and may contribute to restricting DNA replication to once in every cell division cycle.