Phosphorylation of Claspin is triggered by the nucleocytoplasmic ratio at the Xenopus laevis midblastula transition

At the Xenopus midblastula transition (MBT), cell cycles lengthen, and checkpoints that respond to damaged or unreplicated DNA are established. The MBT is triggered by a critical nucleocytoplasmic (N/C) ratio; however, the molecular basis for its initiation remains unknown. In egg extracts, activation of Chk1 checkpoint kinase requires the adaptor protein Claspin, which recruits Chk1 for phosphorylation by ATR. At the MBT in embryos, Chk1 is transiently activated to lengthen the cell cycle. We show that Xenopus Claspin is phosphorylated at the MBT at both DNA replication checkpoint-dependent and -independent sites. Further, in egg extracts, Claspin phosphorylation depends on a threshold N/C ratio, but occurs even when ATR is inhibited. Not all phosphorylation that occurs at the MBT is reproduced in egg extracts. Our results identify Claspin as the most upstream molecule in the signaling pathway that responds to the N/C ratio and indicate that Claspin may also respond to an independent timer to trigger the MBT and activation of cell cycle checkpoints.