Functional relationship between Claspin and Rad17.

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.