Cyclin A-dependent kinase activity affects chromatin binding of ORC, Cdc6, and MCM in egg extracts of Xenopus laevis
Cyclin A-dependent kinase activity affects chromatin binding of ORC, Cdc6, and MCM in egg extracts of Xenopus laevis
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DOI：
10.1046/j.1432-1327.1999.00613.x
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发表时间：
1999-09-01
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影响因子：
--
通讯作者：
Strausfeld, U
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文献类型：
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作者：
Findeisen, M;El-Denary, M;Strausfeld, U
真核生物DNA复制的起始需要将起源识别复合体（ORC）、Cdc6和小染色体维持（MCM）蛋白装载到染色质上形成起始前复合体。在爪蟾卵提取物中，Orc1、Orc2、Cdc6和Mcm4蛋白在间期被低磷酸化，在中期被高磷酸化。我们发现ORC、Cdc6和MCM蛋白的染色质结合不需要细胞周期蛋白依赖的激酶活性。高细胞周期蛋白a依赖性激酶活性抑制并促进爪蟾ORC、Cdc6和MCM从精子染色质上的释放，但对对照蛋白的染色质结合没有影响。在DNA复制假核中，细胞周期蛋白A与ORC、Cdc6和MCM蛋白结合在精子染色质上。相比之下，高周期蛋白E/cdk2未在染色质上检测到，但在核质中发现可溶解。高周期蛋白E激酶活性允许爪蟾ORC和Cdc6结合到精子染色质上，而不是MCM，即使该激酶不直接使MCM磷酸化。我们得出结论，染色质结合的细胞周期蛋白A激酶通过蛋白磷酸化和Cdc6和MCM的染色质释放来控制DNA复制，而可溶性细胞周期蛋白E激酶通过抑制过早的MCM染色质结合来防止细胞周期中的rereplication。
The initiation of DNA replication in eukaryotes requires the loading of the origin recognition complex (ORC), Cdc6, and minichromosome maintenance (MCM) proteins onto chromatin to form the preinitiation complex. In Xenopus egg extract, the proteins Orc1, Orc2, Cdc6, and Mcm4 are underphosphorylated in interphase and hyperphosphorylated in metaphase extract. We find that chromatin binding of ORC, Cdc6, and MCM proteins does not require cyclin-dependent kinase activities. High cyclin A-dependent kinase activity inhibits the binding and promotes the release of Xenopus ORC, Cdc6, and MCM from sperm chromatin, but has no effect on chromatin binding of control proteins. Cyclin A together with ORC, Cdc6 and MCM proteins is bound to sperm chromatin in DNA replicating pseudonuclei. In contrast, high cyclin E/cdk2 was not detected on chromatin, but was found soluble in the nucleoplasm. High cyclin E kinase activity allows the binding of Xenopus ORC and Cdc6, but not MCM, to sperm chromatin, even though the kinase does not phosphorylate MCM directly. We conclude that chromatin-bound cyclin A kinase controls DNA replication by protein phosphorylation and chromatin release of Cdc6 and MCM, whereas soluble cyclin E kinase prevents rereplication during the cell cycle by the inhibition of premature MCM chromatin association.