Chromatin association of human origin recognition complex, Cdc6, and minichromosome maintenance proteins during the cell cycle:: Assembly of prereplication complexes in late mitosis
Chromatin association of human origin recognition complex, Cdc6, and minichromosome maintenance proteins during the cell cycle:: Assembly of prereplication complexes in late mitosis
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DOI：
10.1128/mcb.20.22.8602-8612.2000
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发表时间：
2000-11-01
影响因子：
5.3
通讯作者：
Stillman, B
中科院分区：
文献类型：
--
作者：
Méndez, J;Stillman, B
从酵母和非洲爪蟾的研究中获得的证据表明，DNA复制的启动是一个多步骤的过程。起点识别复合物（ORC），Cdc6p和微小染色体维持（MCM）蛋白是建立复制前复合物所必需的，在复制前复合物的启动由细胞周期蛋白依赖性激酶和Dbf4p依赖性激酶Cdc7p的激活触发。这些复制蛋白的人类同源物的鉴定允许在哺乳动物细胞中的S期调节的调查。使用几种人类细胞系的离心淘洗，我们证明，虽然人类Orc2（hOrc2p）和hMcm蛋白存在于整个细胞周期，hCdc6p水平变化，在早期G（1）非常低，并积累，直到细胞进入有丝分裂。hCdc6p在体内可被多聚泛素化，并可被蛋白酶体抑制剂稳定。与hOrc2p的情况类似，hCdc6p的显著部分存在于整个细胞周期的染色质上，而hMcm蛋白在可溶性和染色质结合形式之间交替。在有丝分裂晚期，细胞周期蛋白B被破坏，hMcm蛋白被加载到染色质上，这表明有丝分裂激酶活性抑制了人类细胞中复制前复合物的形成。
Evidence obtained from studies with yeast and Xenopus indicate that the initiation of DNA replication is a multistep process. The origin recognition complex (ORC), Cdc6p, and minichromosome maintenance (MCM) proteins are required for establishing prereplication complexes, upon which initiation is triggered by the activation of cyclin-dependent kinases and the Dbf4p-dependent kinase Cdc7p. The identification of human homologues of these replication proteins allows investigation of S-phase regulation in mammalian cells. Using centrifugal elutriation of several human cell lines, we demonstrate that whereas human Orc2 (hOrc2p) and hMcm proteins are present throughout the cell cycle, hCdc6p levels vary, being very low in early G(1) and accumulating until cells enter mitosis. hCdc6p can be polyubiquitinated in vivo, and it is stabilized by proteasome inhibitors. Similar to the case for hOrc2p, a significant fraction of hCdc6p is present on chromatin throughout the cell cycle, whereas hMcm proteins alternate between soluble and chromatin-bound forms. Loading of hMcm proteins onto chromatin occurs in late mitosis concomitant with the destruction of cyclin B, indicating that the mitotic kinase activity inhibits prereplication complex formation in human cells.