THE NUCLEAR-ENVELOPE PREVENTS REINITIATION OF REPLICATION BY REGULATING THE BINDING OF MCM3 TO CHROMATIN IN XENOPUS EGG EXTRACTS
THE NUCLEAR-ENVELOPE PREVENTS REINITIATION OF REPLICATION BY REGULATING THE BINDING OF MCM3 TO CHROMATIN IN XENOPUS EGG EXTRACTS
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DOI：
10.1016/s0960-9822(95)00253-3
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发表时间：
1995-11-01
期刊：
影响因子：
9.2
通讯作者：
LASKEY, RA
中科院分区：
文献类型：
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作者：
MADINE, MA;KHOO, CY;LASKEY, RA
背景：MCM蛋白复合物通过“复制许可”确保DNA在每个细胞周期中只复制一次。核膜也参与复制许可，但MCM蛋白与核膜之间的关系尚不清楚。在这里，我们研究了XMCM3 （Xenopus MCM复合物的一个组成部分）、核膜通透性和每个细胞周期DNA复制起始之间的关系。结果：我们的研究结果表明，核膜不阻止XMCM3进入细胞核，但它确实阻止XMCM3与染色质结合。我们还鉴定了爪蟾MCM复合体的另一个组成部分，作为裂糖菌pombe蛋白Cdc21的同源物。XMCM3不优先与DNA复制位点共定位。相反，它几乎均匀地分布在染色质上，并在复制过程中突然丢失，XMCM3穿过g2期HeLa细胞的完整核膜，但随后不能与染色质结合。核膜的渗透性允许XMCM3与g2期染色质结合。因此，我们将复制许可分为两个阶段。第一种方法需要核膜排除的细胞质“装载因子”的进入；随后，MCM3可以在核膜存在或不存在的情况下与染色质结合，但前提是装载因子在没有核膜的情况下进入。结论：非洲爪蟾MCM复合物含有酵母MCM2、MCM3、MCM5和Cdc21蛋白的同源物。在复制过程中，XMCM3从染色质中移出。核膜允许XMCM3进入细胞核，但调节其与染色质的结合；结合需要一个不能穿过核膜的装载因子。基于这些结果，我们提出了复制许可的两阶段模型。
Background: A complex of MCM proteins is implicated in ensuring that DNA replicates only once in each cell cycle, by 'replication licensing'. The nuclear membrane is also implicated in replication licensing, but the relationship between the MCM proteins and the nuclear membrane is unclear. Here, we investigate the relationship between XMCM3 (a component of the Xenopus MCM complex), nuclear envelope permeability and the initiation of DNA replication once per cell cycle.Results: Our results show that the nuclear envelope does not prevent the entry of XMCM3 into the nucleus, but that it does prevent the binding of XMCM3 to chromatin. We have also identified another component of the Xenopus MCM complex as a homologue of the Schizosaccharomyces pombe protein Cdc21. XMCM3 does not preferentially co-localize with sites of DNA replication. Instead, it is almost uniformly distributed on chromatin and is suddenly lost during replication, XMCM3 crosses intact nuclear membranes of G2-phase HeLa cells but cannot then bind to chromatin. Permeabilization of the nuclear envelope allows the binding of XMCM3 to G2-phase chromatin. We have therefore resolved replication licensing into two stages. The first requires the entry of a cytosolic 'loading factor' that is excluded by the nuclear membrane; subsequently, MCM3 can bind to chromatin in the presence or absence of a nuclear membrane, but only if the loading factor has gained access in the absence of the membrane.Conclusions: The Xenopus MCM complex contains homologues of yeast MCM2, MCM3, MCM5 and Cdc21 proteins. XMCM3 is displaced from chromatin during replication. The nuclear envelope allows entry of XMCM3 into the nucleus, but regulates its binding to chromatin; binding requires a loading factor which cannot cross the nuclear envelope. Based on these results, we present a two-stage model for replication licensing.