Initiation of human DNA replication in vitro using nuclei from cells arrested at an initiation-competent state
Initiation of human DNA replication in vitro using nuclei from cells arrested at an initiation-competent state
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DOI：
10.1074/jbc.275.18.13699
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发表时间：
2000-05-05
影响因子：
4.8
通讯作者：
Krude, T
中科院分区：
文献类型：
--
作者：
Krude, T
在体外研究人类 DNA 复制的启动，使用从通过用 0.5 mM 含羞草处理 24 小时而停滞在 G(1) 期晚期的细胞中分离的启动活性细胞核 (Krude, T, (1999) Exp. Cell Res. 247, 148-159)。从含羞草碱阻滞的 HeLa 细胞中分离出的细胞核在增殖人类细胞的胞质提取物中孵育后，会启动半保守 DNA 复制。起始发生在核膜不存在和存在的情况下。细胞周期蛋白依赖性激酶 (Cdk) 抑制剂 roscovitine 和 olomoucine 抑制 DNA 复制的起始，表明起始对 Cdk 活性的依赖性。细胞分级分离显示细胞周期蛋白 A、E 和 Cdk2 与含羞草碱阻滞细胞的细胞核结合。外源添加的人细胞周期蛋白 A Cdk2 和细胞周期蛋白 E Cdk2 复合物（但不是细胞周期蛋白 B1/Cdk1 或细胞周期蛋白 D2/Cdk6）可以克服体外 roscovitine 的起始抑制。从胞浆提取物中耗尽 Cdk2 并不会阻止起始，这表明细胞周期蛋白 Cdk2 复合物在可溶性提取物中不是必需的，而是由细胞核提供的。启动进一步取决于增殖细胞的胞质提取物中存在的必需且可溶的活性，而不是来自含羞草碱阻滞细胞的胞质提取物，其与核细胞周期蛋白/Cdk2活性一起作用。
Initiation of human DNA replication is investigated in vitro, using initiation-competent nuclei isolated from cells arrested in late G(1) phase by a 24-h treatment with 0.5 mM mimosine (Krude, T, (1999) Exp. Cell Res. 247, 148-159). Nuclei isolated from mimosine-arrested HeLa cells initiate semiconservative DNA replication upon incubation in cytosolic extracts from proliferating human cells. Initiation occurs in the absence and presence of a nuclear membrane. The cyclin-dependent kinase (Cdk) inhibitors roscovitine and olomoucine inhibit initiation of DNA replication, indicating a dependence of initiation on Cdk activity. Cell fractionation shows that cyclins A, E, and Cdk2 are bound to nuclei from mimosine-arrested cells. Exogenously added human cyclin A Cdk2 and cyclin E Cdk2 complexes, but not cyclin B1/Cdk1 or cyclin D2/Cdk6, can overcome inhibition of initiation by roscovitine in vitro. Depleting Cdk2 from cytosolic extract does not prevent initiation, demonstrating that cyclin Cdk2 complexes are not required in the soluble extract, but are provided by the nuclei. Initiation depends further on an essential and soluble activity present in cytosolic extracts from proliferating cells, but not from mimosine-arrested cells, acting together with nuclear cyclin/Cdk2 activity.