Nuclear organization of DNA replication initiation proteins in mammalian cells
Nuclear organization of DNA replication initiation proteins in mammalian cells
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DOI：
10.1074/jbc.m111398200
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发表时间：
2002-03-22
影响因子：
4.8
通讯作者：
Tsurumi, T
中科院分区：
文献类型：
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作者：
Fujita, M;Ishimi, Y;Tsurumi, T
起源识别复合物（ORC）、CDC6和MCM蛋白依次组装形成复制前染色质。然而，它们的组织在哺乳动物细胞中仍然很不清楚。在这里，我们表明，ORC1蛋白与非染色质核结构和组装在哺乳动物细胞的核灶中使用体内化学交联方法。还发现CDC6蛋白在非染色质核结构上的核灶中组装，尽管它们与ORC I的物理关联尚未检测到。与酵母细胞中的情况相反，发现CDC6即使在细胞进入S期后仍与非染色质核结构相关联。相反，ORC1蛋白被发现在S期期间通过蛋白酶体依赖性途径降解。我们还发现，一些ORC2蛋白与非染色质核结构如ORC1相关，尽管其余蛋白与核酸酶敏感的染色质结合。进一步的分析表明，ORC2与ORC1在非染色质核结构上物理相互作用。另一方面，我们的研究结果表明，虽然一小部分的MCM复合物加载到染色质区域附近的ORC焦点，他们中的大多数是更广泛的分布。这种组织的复制前染色质和复杂的起源规范在高等真核细胞之间的可能关系进行了讨论。
Origin recognition complex (ORC), CDC6, and MCM proteins assemble sequentially to form prereplication chromatin. However, their organization remains largely unclear in mammalian cells. Here we show that ORC1 proteins are associated with non-chromatin nuclear structures and assemble in nuclear foci in mammalian cells using an in vivo chemical cross-linking method. CDC6 proteins were also found to assemble in nuclear foci on non-chromatin nuclear structures, although their physical association with ORC I has been undetectable. In contrast to the situation in yeast cells, CDC6 was found to remain associated with non-chromatin nuclear structures even after cells entered into S phase. Instead, ORC1 proteins were found to be degraded by a proteasome-dependent pathway during S phase. We also found that some ORC2 proteins are associated with non-chromatin nuclear structures like ORC1, although the remainder binds to nuclease-sensitive chromatin. Further analyses indicate that ORC2 physically interacts with ORC1 on non-chromatin nuclear structures. On the other hand, our results suggest that although a small proportion of MCM complexes are loaded onto chromatin regions near ORC foci, most of them are more widely distributed. Possible relations between such organization of prereplication chromatin and complicated origin specification in higher eukaryotic cells are discussed.