A role for Cdk2 kinase in negatively regulating DNA replication during S phase of the cell cycle.
A role for Cdk2 kinase in negatively regulating DNA replication during S phase of the cell cycle.
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DOI：
10.1083/jcb.137.1.183
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发表时间：
1997-04-07
期刊：
影响因子：
--
通讯作者：
Newport J
中科院分区：
文献类型：
--
作者：
Hua XH;Yan H;Newport J
使用无细胞提取物从非洲爪蟾卵，我们表明，cdk2-细胞周期蛋白E和A激酶在负调控DNA复制中发挥重要作用。具体来说，我们证明，cdk2激酶浓度周围的染色质提取物增加200倍，一旦染色质组装成细胞核。此外，我们发现，如果cdk2-细胞周期蛋白E或A的浓度在卵细胞质中增加16倍之前，添加精子染色质，染色质未能启动DNA复制一旦组装成核。这表明cdk2-cyclin E或A可以负调控DNA复制。关于这种负调控是如何发生的，我们表明，高水平的cdk2-细胞周期蛋白E不会阻止蛋白复合物ORC与精子染色质的关联，但会阻止复制所必需的蛋白MCM3的关联。更重要的是，我们发现当cdk2-cyclin E水平增加时，与染色质预结合的MCM3不会解离。综上所述，我们的研究结果强烈表明，在胚胎细胞周期中，低浓度的cdk2-细胞周期蛋白E存在于有丝分裂后的细胞质和核形成之前，允许蛋白质的增强DNA复制结合到染色质，并在细胞核内的高浓度的cdk2-细胞周期蛋白E防止MCM复制后与染色质重新关联。这种情况可能会部分地限制DNA复制到每个细胞周期的一轮。
Using cell-free extracts made from Xenopus eggs, we show that cdk2-cyclin E and A kinases play an important role in negatively regulating DNA replication. Specifically, we demonstrate that the cdk2 kinase concentration surrounding chromatin in extracts increases 200-fold once the chromatin is assembled into nuclei. Further, we find that if the cdk2–cyclin E or A concentration in egg cytosol is increased 16-fold before the addition of sperm chromatin, the chromatin fails to initiate DNA replication once assembled into nuclei. This demonstrates that cdk2–cyclin E or A can negatively regulate DNA replication. With respect to how this negative regulation occurs, we show that high levels of cdk2–cyclin E do not block the association of the protein complex ORC with sperm chromatin but do prevent association of MCM3, a protein essential for replication. Importantly, we find that MCM3 that is prebound to chromatin does not dissociate when cdk2– cyclin E levels are increased. Taken together our results strongly suggest that during the embryonic cell cycle, the low concentrations of cdk2–cyclin E present in the cytosol after mitosis and before nuclear formation allow proteins essential for potentiating DNA replication to bind to chromatin, and that the high concentration of cdk2–cyclin E within nuclei prevents MCM from reassociating with chromatin after replication. This situation could serve, in part, to limit DNA replication to a single round per cell cycle.
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影响因子：
7.8
作者：
JACKSON, PK;CHEVALIER, S;PHILIPPE, M;KIRSCHNER, MW
通讯作者：
KIRSCHNER, MW
影响因子：
56.9
作者：
KOFF, A;GIORDANO, A;ROBERTS, JM
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ROBERTS, JM
影响因子：
64.5
作者：
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通讯作者：
NURSE, P
DOI：
10.1083/jcb.123.6.1321
发表时间：
1993-12
期刊：
The Journal of cell biology
影响因子：
--
作者：
Kubota Y;Takisawa H
通讯作者：
Takisawa H
影响因子：
64.5
作者：
LIANG, C;WEINREICH, M;STILLMAN, B
通讯作者：
STILLMAN, B