A Cdt1-geminin complex licenses chromatin for DNA replication and prevents rereplication during S phase in Xenopus
A Cdt1-geminin complex licenses chromatin for DNA replication and prevents rereplication during S phase in Xenopus
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DOI：
10.1038/sj.emboj.7601436
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发表时间：
2006-12-13
期刊：
影响因子：
11.4
通讯作者：
Mechali, Marcel
中科院分区：
文献类型：
--
作者：
Lutzmann, Malik;Maiorano, Domenico;Mechali, Marcel
DNA合成的起始涉及通过Cdt 1（来源许可）将MCM 2 -7解旋酶加载到染色质上。Geminin被认为通过结合和抑制Cdt 1来防止再授权。在这里，我们表明，使用非洲爪蟾卵提取物，结合Cdt 1的双生蛋白是不足以阻止其活动和Cdt 1-双生蛋白复合物许可染色质，但防止再复制，作为一个分子开关在复制起点。我们证明，双生蛋白已在许可期间被招募到染色质，而批量双生蛋白在S期开始时被招募。重组Cdt 1-双生蛋白复合物结合染色质，与MCM 2 -7复合物相互作用，并在每个细胞周期许可染色质一次。因此，虽然重组Cdt 1在G1或G2诱导再复制并激活ATM/ATR依赖性检查点，但Cdt 1-双生蛋白复合物不会。我们进一步证明，Cdt 1-双生蛋白复合物的化学计量调节其活性。我们的研究结果表明，MCM 2 -7的解旋酶装载到染色质上的Cdt 1双生蛋白复合物，这是灭活后，通过结合额外的双生蛋白的起源射击模型。如果染色质上仅存在游离Cdt 1，则不会发生这种起源失活反应。
Initiation of DNA synthesis involves the loading of the MCM2-7 helicase onto chromatin by Cdt1 (origin licensing). Geminin is thought to prevent relicensing by binding and inhibiting Cdt1. Here we show, using Xenopus egg extracts, that geminin binding to Cdt1 is not sufficient to block its activity and that a Cdt1-geminin complex licenses chromatin, but prevents rereplication, working as a molecular switch at replication origins. We demonstrate that geminin is recruited to chromatin already during licensing, while bulk geminin is recruited at the onset of S phase. A recombinant Cdt1-geminin complex binds chromatin, interacts with the MCM2-7 complex and licenses chromatin once per cell cycle. Accordingly, while recombinant Cdt1 induces rereplication in G1 or G2 and activates an ATM/ATR-dependent checkpoint, the Cdt1-geminin complex does not. We further demonstrate that the stoichiometry of the Cdt1-geminin complex regulates its activity. Our results suggest a model in which the MCM2-7 helicase is loaded onto chromatin by a Cdt1 geminin complex, which is inactivated upon origin firing by binding additional geminin. This origin inactivation reaction does not occur if only free Cdt1 is present on chromatin.