Cdk2 Activity Is Dispensable for the Onset of DNA Replication during the First Mitotic Cycles of the Sea Urchin Early Embryo

Abstract

Earlier work reported the important role of Cdk2 as a regulator of DNA replication in somatic cells and inXenopusextracts. In the present report we analyzein vivothe involvement of Cdk2 in DNA replication during early embryogenesis using the first mitotic cycles of sea urchin embryos. UnfertilizedSphaerechinus granulariseggs are arrested after the second meiotic cytokinesis. Fertilization resumes the block and induces DNA replication after a short lag period, making sea urchin early embryo a good model for studyingin vivothe onset of DNA replication. We show that Cdk2 as well as its potential partner cyclin A are present in the nucleus in G1 and S phase and therefore available for DNA replication. In accordance with data obtained inXenopusegg extracts we observed that Cdk2 kinase activity is low and stable during the entire cycle. However, in contrast with thisin vitrosystem in which Cdk2 activity is required for the onset of DNA replication, the specific inhibition of Cdk2 kinase by microinjection of the catalytically inactive Cdk2-K33R or the inhibitor p21Cip1does not prevent DNA replication. Because olomoucine, DMAP, and emetine treatments did not preclude DNA synthesis, neither cyclin A/Cdk1 nor cyclin B/Cdk1 kinase activities are necessary to replace the absence of Cdk2 kinase in promoting DNA replication. These data suggest that during early embryogenesis Cdks activities, in particular Cdk2, are dispensablein vivofor the initiation step of DNA replication. However, the specific localization of Cdk2 in the nucleus from the beginning of M phase to the end of S phase suggests its involvement in other mechanisms regulating DNA replication such as inhibition of DNA re-replication and/or that its regulating role is achieved through a pathway independent of the kinase activity. We further demonstrate that even after inhibition of Cdk activities, the permeabilization of the nuclear membrane is required to allow a second round of DNA replication. However, in contrast toXenopusegg extracts, re-replication can take place in the absence of DMAP-sensitive kinase.