Functional relationship between Claspin and Rad17
Functional relationship between Claspin and Rad17
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Claspin 和 Rad17 之间的功能关系
DOI：
10.1016/j.bbrc.2011.09.037
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发表时间：
2011
期刊：
影响因子：
--
通讯作者：
T.
中科院分区：
文献类型：
--
作者：
Yoshimura;A.;Akita;M.;Hosono;Y.;Abe;T.;Kobayashi;M.;Yamamoto;K.I.;Tada;S.;Seki;M.;Enomoto;T.
Claspin 最初被鉴定为 Check1 (Chk1) 相互作用蛋白。据报道，Claspin 和 Rad17 参与 DNA 损伤诱导的 Chk1 磷酸化，这是检查点激活的标志。为了了解 Claspin 的细胞功能以及 Claspin 和 Rad17 之间的功能关系，我们使用鸡 DT40 细胞生成了 Claspin−/− 和 Claspin−/−/RAD17− 细胞，其中含有外源引入的 Claspin，可通过添加强力霉素 (Dox) 来抑制。在存在 Dox 的情况下，Claspin−/− 细胞在 2 天内停止生长，导致细胞死亡。此外，在 Claspin 耗尽的细胞中观察到 DNA 延伸率显着降低，这表明在没有外源应激的情况下，Claspin 在 DNA 复制中发挥着关键作用。当细胞暴露于甲磺酸甲酯（MMS）（一种DNA损伤剂）时，通过细胞周期进展和Chk1磷酸化监测，RAD17细胞在检查点激活方面表现出比Claspin细胞更大的缺陷。在 Claspin 耗尽的细胞中，敲除 RAD17 基因对细胞死亡和 DNA 延伸率几乎没有附加影响。
Claspin was originally identified as a Check1 (Chk1)-interacting protein. Claspin and Rad17 are reportedly involved in the DNA damage-induced phosphorylation of Chk1, a hallmark of checkpoint activation. To understand the cellular functions of Claspin and the functional relationship between Claspin and Rad17, we generated Claspin−/−and Claspin−/−/RAD17−cells using chicken DT40 cells, which contain an exogenously introduced Claspin that can be suppressed by the addition of doxycycline (Dox). In the presence of Dox, Claspin−/−cells ceased growth within 2days, leading to cell death. In addition, a remarkable reduction in the rate of DNA elongation was observed in Claspin-depleted cells, suggesting that Claspin plays a critical role in DNA replication in the absence of exogenous stress. When cells were exposed to methyl methanesulfonate (MMS), a DNA damaging agent, RAD17−cells showed a greater defect in checkpoint activation than Claspin−/−cells as monitored by progression of cell cycle and phosphorylation of Chk1. Knocking out RAD17 gene showed almost no additive effects on cell death and DNA elongation rates in Claspin-depleted cells.