Cdk2 activity is dispensable for the onset of DNA replication during the first mitotic cycles of the sea urchin early embryo.
Earlier work reported the important role of Cdk2 as a regulator of DNA replication in somatic cells and in Xenopus extracts. In the present report we analyze in vivo the involvement of Cdk2 in DNA replication during early embryogenesis using the first mitotic cycles of sea urchin embryos. Unfertilized Sphaerechinus granularis eggs 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 studying in vivo the 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 in Xenopus egg extracts we observed that Cdk2 kinase activity is low and stable during the entire cycle. However, in contrast with this in vitro system 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 p21(Cip1) does 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 dispensable in vivo for 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 to Xenopus egg extracts, re-replication can take place in the absence of DMAP-sensitive kinase.
Laboratoire Arago, URA 2156, Banyuls-sur Mer, F66650, France.
J L
JL
Moreau
F
F
Marques
A
A
Barakat
P
P
Schatt
J C
JC
Lozano
G
G
Peaucellier
A
A
Picard
A M
AM
Genevière
D000225:Adenine / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D016203:CDC2 Protein Kinase / Q000737:chemistry / Q000502:physiology; D042846:CDC2-CDC28 Kinases; D002453:Cell Cycle / Q000502:physiology; D003001:Cloning, Molecular; D019925:Cyclin A / Q000378:metabolism; D051357:Cyclin-Dependent Kinase 2; D050759:Cyclin-Dependent Kinase Inhibitor p21; D018844:Cyclin-Dependent Kinases / Q000737:chemistry* / Q000502:physiology; D016213:Cyclins / Q000494:pharmacology; D004261:DNA Replication / Q000235:genetics*; D004640:Emetine / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005306:Fertilization / Q000502:physiology; D005455:Fluorescent Antibody Technique; D007701:Kinetin; D008540:Meiosis / Q000502:physiology; D008845:Microinjections; D008938:Mitosis / Q000502:physiology*; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry* / Q000502:physiology; D011687:Purines / Q000494:pharmacology; D012617:Sea Urchins / Q000196:embryology*; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA
D019925:Cyclin A; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D004791:Enzyme Inhibitors; D011687:Purines; C001020:N(6),N(6)-dimethyladenine; C090046:olomoucine; D017346:Protein Serine-Threonine Kinases; D016203:CDC2 Protein Kinase; D042846:CDC2-CDC28 Kinases; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases; D000225:Adenine; D007701:Kinetin; D004640:Emetine