Recombinant Cdt1 induces rereplication of G2 nuclei in Xenopus egg extracts
Recombinant Cdt1 induces rereplication of G2 nuclei in Xenopus egg extracts
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DOI：
10.1016/j.cub.2004.12.002
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发表时间：
2005-01-26
期刊：
影响因子：
9.2
通讯作者：
Mechali, M
中科院分区：
文献类型：
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作者：
Maiorano, D;Krasinska, L;Mechali, M
维持真核生物基因组完整性的一个关键调控是限制DNA在S期的复制仅为一轮。已经提出了几个模型；其中之一，许可模型，预测核膜的形成限制了染色质进入一个正复制因子[1]。Cdt1是一种结合起源并招募MCM2-7解旋酶的因子，已被确定为非洲爪蟾和其他真核生物[2]许可系统的一个组成部分。然而，缺乏证据证明非预定的Cdt1表达在促进非法重新启动DNA合成中的直接作用。我们发现Xenopus Cdt1在G2细胞核中不存在，这表明它可能被降解或输出。重组Cdt1，在G2中加入到鸡蛋提取物中，穿过核膜，与染色质结合，并与染色质结合的Cdc6结合，重新许可染色体进行新一轮的DNA合成。其机制涉及MCM3与染色质的再结合。再起始仅在G2中被双联蛋白阻断，而Cdc6不刺激，这表明Cdt1而不是Cdc6限制了卵提取物的再起始。这些结果表明，在G2中，从染色质中去除Cdt1及其核排斥对于调节许可至关重要，并且这种控制的覆盖足以促进不合法的起源激活。
A crucial regulation for maintaining genome integrity in eukaryotes is to limit DNA replication in S phase to only one round. Several models have been proposed; one of which, the licensing model, predicted that formation of the nuclear membrane restricts access to chromatin to a positive replication factor [1]. Cdt1, a factor binding to origins and recruiting the MCM2-7 helicase, has been identified as a component of the licensing system in Xenopus and other eukaryotes [2]. Nevertheless, evidence is missing demonstrating a direct role for unscheduled Cdt1 expression in promoting illegitimate reinitiation of DNA synthesis. We show here that Xenopus Cdt1 is absent in G2 nuclei, suggesting that it might be either degraded or exported. Recombinant Cdt1, added to egg extracts in G2, crosses the nuclear membrane, binds to chromatin, and relicenses the chromosome for new rounds of DNA synthesis in combination with chromatin bound Cdc6. The mechanism involves rebinding of MCM3 to chromatin. Reinitiation is blocked by geminin only in G2 and is not stimulated by Cdc6, demonstrating that Cdt1, but not Cdc6, is limiting for reinitiation in egg extracts. These results suggest that removal of Cdt1 from chromatin and its nuclear exclusion in G2 is critical in regulating licensing and that override of this control is sufficient to promote illegitimate firing of origins.