Multi-step Loading of Human Minichromosome Maintenance Proteins in Live Human Cells
Multi-step Loading of Human Minichromosome Maintenance Proteins in Live Human Cells
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DOI：
10.1074/jbc.m113.474825
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发表时间：
2013-12-13
影响因子：
4.8
通讯作者：
Lygerou, Zoi
中科院分区：
文献类型：
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作者：
Symeonidou, Ioanna-Eleni;Kotsantis, Panagiotis;Lygerou, Zoi
背景：将MCM2-7装载到染色质上许可复制起始物。结果：MCMS在有丝分裂晚期与染色质短暂相互作用，在G(1)期结合稳定，在G(1)期负荷增加。结论：MCM2-7对染色质负荷的多水平调节发生在有丝分裂过程中，发生在G(1)/S相变之前。意义：人类活细胞内DNA许可系统的动态揭示了多个控制点。每个细胞周期一次的复制是通过组装到染色质上的多亚单位蛋白质复合体来调节的，这些复合体允许DNA进行下一轮复制。许可包括在G(1)阶段将六聚体MCM2-7复合体加载到染色质上，并取决于许可因子CDT1。体外实验表明，微染色体维持(MCM)蛋白有两步结合模式，短暂的初始相互作用转化为稳定的染色质负载。在这里，我们使用体内许可实验评估活的人类细胞中的MCM载量，该方法基于GFP标记的MCM蛋白亚单位在整个细胞周期中的光漂白后的荧光恢复。我们发现，在末期，MCM2和MCM4与染色质保持瞬时相互作用，表现出类似于CDT1的动力学。这些相互作用从早期的G(1)相转变为稳定的相互作用。在G(1)期，MCM2和MCM4的不活动部分增加，提示反复许可。在G(1)期晚期，很大一部分MCM蛋白被装载到染色质上，在S期开始之前观察到最大的许可。光漂白实验中的荧光损失表明，随着G(1)期的进展，MCM-染色质相互作用的亚核浓度不同，并且不与S相的DNA合成位点共存。
Background: MCM2-7 loading onto chromatin licenses origins for replication. Results: MCMs exhibit transient interactions with chromatin in late mitosis, stable binding in G(1) phase and increased loading in late G(1) phase. Conclusion: Multilevel regulation of MCM2-7 loading to chromatin occurs during mitosis and preceding the G(1)/S phase transition. Significance: The dynamics of the DNA licensing system within live human cells reveal multiple control points.Once-per-cell cycle replication is regulated through the assembly onto chromatin of multisubunit protein complexes that license DNA for a further round of replication. Licensing consists of the loading of the hexameric MCM2-7 complex onto chromatin during G(1) phase and is dependent on the licensing factor Cdt1. In vitro experiments have suggested a two-step binding mode for minichromosome maintenance (MCM) proteins, with transient initial interactions converted to stable chromatin loading. Here, we assess MCM loading in live human cells using an in vivo licensing assay on the basis of fluorescence recovery after photobleaching of GFP-tagged MCM protein subunits through the cell cycle. We show that, in telophase, MCM2 and MCM4 maintain transient interactions with chromatin, exhibiting kinetics similar to Cdt1. These are converted to stable interactions from early G(1) phase. The immobile fraction of MCM2 and MCM4 increases during G(1) phase, suggestive of reiterative licensing. In late G(1) phase, a large fraction of MCM proteins are loaded onto chromatin, with maximal licensing observed just prior to S phase onset. Fluorescence loss in photobleaching experiments show subnuclear concentrations of MCM-chromatin interactions that differ as G(1) phase progresses and do not colocalize with sites of DNA synthesis in S phase.