ORC is necessary at the interphaseto-mitosis transition to recruit cdc2 kinase and disassemble RPA foci
ORC is necessary at the interphaseto-mitosis transition to recruit cdc2 kinase and disassemble RPA foci
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DOI：
10.1016/j.cub.2006.01.059
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发表时间：
2006-03-07
期刊：
影响因子：
9.2
通讯作者：
Méchali, M
中科院分区：
文献类型：
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作者：
Cuvier, O;Lutzmann, M;Méchali, M
起源识别复合体(ORC)在确定DNA复制起源[1]和染色体分离[2，3]中起着至关重要的作用。最近对果蝇orc2突变体[4-6]和缺失orc2的人类细胞[3]的研究表明，该因子也与有丝分裂染色体组装有关。我们询问ORC是否需要独立于其在DNA复制中的功能来组装M期染色体。在M期染色体组装与DNA复制偶联或非偶联的条件下，我们对非洲爪哇卵提取液进行了耗竭分析和重组实验。我们表明，虽然ORC对于有丝分裂染色体凝聚是必不可少的，但在分裂间期转变时，正确的有丝分裂染色体组装是必要的，这一反应并不严格依赖于DNA复制。这一功能包括CDC2激酶重新聚集到染色质和间期复制蛋白A(RPA)焦点的染色质分解[7，8]。此外，我们发现RPA在cdc2激酶位点的突变阻止了RPA从染色质上解离，并在不影响DNA复制的情况下损害了有丝分裂染色体的组装。我们的结果支持这样的结论，即ORC除了在复制前复合体(Pre-RCs)的组装中发挥作用外，在G1-S的转变中，ORC也需要在有丝分裂进入时对它们进行分解。
The origin-recognition complex (ORC) has an essential role in defining DNA replication origins [1] and in chromosome segregation [2, 3]. Recent studies in Drosophila orc2 mutants [4-6], and in human cells depleted of ORC2 [3], have suggested that this factor is also implicated in mitotic chromosome assembly. We asked whether ORC was required for M phase chromosome assembly independently of its function in DNA replication. We performed depletion assays and reconstitution experiments in Xenopus egg extracts, in conditions of M phase chromosome assembly coupled or uncoupled from DNA replication. We show that, although ORC is dispensable for mitotic chromosome condensation, it is necessary at the interphasemitosis transition for proper mitotic chromosome assembly to occur in a reaction not strictly dependent on DNA replication. This function involves the recruitment to chromatin of cdc2 kinase and the chromatin disassembly of interphasic replication protein A (RPA) foci [7, 8]. Furthermore, we show that mutations of RPA at the cdc2 kinase site prevents RPA dissociation from chromatin and impairs mitotic chromosome assembly without affecting DNA replication. Our results support the conclusion that in addition to its role in the assembly of prereplication complexes (pre-RCs), at the G1-S transition, ORC is also required for their disassembly at mitotic entry.