Cyclin E uses Cdc6 as a chromatin-associated receptor required for DNA replication.
Cyclin E uses Cdc6 as a chromatin-associated receptor required for DNA replication.
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DOI：
10.1083/jcb.152.6.1267
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发表时间：
2001-03-19
期刊：
影响因子：
--
通讯作者：
Jackson PK
中科院分区：
文献类型：
--
作者：
Furstenthal L;Kaiser BK;Swanson C;Jackson PK
通过对非洲爪哇卵提取液进行体外染色质组装实验，我们发现细胞周期蛋白E通过三个阶段与染色质特异地结合。在第一阶段，对于依赖于ATP的细胞周期蛋白E-CDK2与DNA的结合来说，起源识别复合体和CDC6复制前蛋白是必需的，而不是微染色体维持复合体。我们发现Cyclin E结合了含有Cy-Arg-X-Leu(RXL)基序的CDC6的NH2-末端区域。底物选择突变的Cyclin E蛋白(Met-Arg-Ala-Ile-Leu；MRAIL)不能与CDC6结合，不能与内源Cyclin E-CDk2竞争染色质结合，也不能挽救Cyclin E缺失提取物中的复制。具有三个共有的RXL基序突变的CDC6蛋白在数量上缺乏与细胞周期蛋白E的结合和在缺乏CDC6的提取物中拯救复制。因此，将CDK2复合体定位于染色质的Cyclin E-CDC6相互作用对DNA复制是重要的。在第二阶段，细胞周期蛋白E-CDK2在染色质上聚集，依赖于聚合酶的活性。在有丝分裂的第三阶段，细胞周期蛋白E被磷酸化，细胞周期蛋白E-CDK2复合体从染色质中被取代。在体外，丝裂原激活的蛋白激酶，尤其是细胞周期蛋白B-CDC2，而不是Polo样蛋白1，从染色质中去除细胞周期蛋白E-CDK2。过度磷酸化的细胞周期蛋白E-CDK2与间期染色质的重新结合需要去磷酸化，而CDK激酶指导的CDC14磷酸酶在体外足以完成这种去磷酸化。Cyclin E与染色质结合的这三个阶段可能促进了Cyclin E-CDK2在启动复制、阻止再复制以及允许有丝分裂后重新定位起始点方面的不同活动。
Using an in vitro chromatin assembly assay in Xenopus egg extract, we show that cyclin E binds specifically and saturably to chromatin in three phases. In the first phase, the origin recognition complex and Cdc6 prereplication proteins, but not the minichromosome maintenance complex, are necessary and biochemically sufficient for ATP-dependent binding of cyclin E–Cdk2 to DNA. We find that cyclin E binds the NH2-terminal region of Cdc6 containing Cy–Arg-X-Leu (RXL) motifs. Cyclin E proteins with mutated substrate selection (Met-Arg-Ala-Ile-Leu; MRAIL) motifs fail to bind Cdc6, fail to compete with endogenous cyclin E–Cdk2 for chromatin binding, and fail to rescue replication in cyclin E–depleted extracts. Cdc6 proteins with mutations in the three consensus RXL motifs are quantitatively deficient for cyclin E binding and for rescuing replication in Cdc6-depleted extracts. Thus, the cyclin E–Cdc6 interaction that localizes the Cdk2 complex to chromatin is important for DNA replication. During the second phase, cyclin E–Cdk2 accumulates on chromatin, dependent on polymerase activity. In the third phase, cyclin E is phosphorylated, and the cyclin E–Cdk2 complex is displaced from chromatin in mitosis. In vitro, mitogen-activated protein kinase and especially cyclin B–Cdc2, but not the polo-like kinase 1, remove cyclin E–Cdk2 from chromatin. Rebinding of hyperphosphorylated cyclin E–Cdk2 to interphase chromatin requires dephosphorylation, and the Cdk kinase–directed Cdc14 phosphatase is sufficient for this dephosphorylation in vitro. These three phases of cyclin E association with chromatin may facilitate the diverse activities of cyclin E–Cdk2 in initiating replication, blocking rereplication, and allowing resetting of origins after mitosis.
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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：
9.2
作者：
Follette, PJ;Duronio, RJ;O'Farrell, PH
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O'Farrell, PH
影响因子：
7.8
作者：
Lane, HA;Nigg, EA
通讯作者：
Nigg, EA
影响因子：
64.5
作者：
LIANG, C;WEINREICH, M;STILLMAN, B
通讯作者：
STILLMAN, B
影响因子：
64.8
作者：
MURRAY, AW;KIRSCHNER, MW
通讯作者：
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