Molecular Cell Biology Laboratory, Research Institute of Pharmaceutical Sciences, Faculty of Pharmacy, Musashino University, 1-1-20 Shinmachi, Nishitokyo-shi, Tokyo 202-8585, Japan.
Biochem Biophys Res Commun. 2011 Oct 22;414(2):298-303. doi: 10.1016/j.bbrc.2011.09.037. Epub 2011 Sep 14.
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 2 days, 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.
Claspin 最初被鉴定为 Check1 (Chk1) 的相互作用蛋白。据报道，Claspin 和 Rad17 参与了 DNA 损伤诱导的 Chk1 的磷酸化，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，我们使用含有外源性 Claspin 的鸡 DT40 细胞生成了 Claspin(-/-)和 Claspin(-/-)/RAD17(-)细胞，该外源性 Claspin 可以通过添加强力霉素 (Dox) 来抑制。在 Dox 的存在下，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 延伸率显著降低，表明在没有外源性应激的情况下，Claspin 在 DNA 复制中发挥关键作用。当细胞暴露于甲基甲磺酸酯 (MMS) 等 DNA 损伤剂时，RAD17(-)细胞在细胞周期的进展和 Chk1 的磷酸化方面显示出比 Claspin(-/-)细胞更大的检查点激活缺陷。敲除 RAD17 基因在 Claspin 耗尽的细胞中对细胞死亡和 DNA 延伸率几乎没有叠加效应。
