Replication origins are already licensed in G1 arrested unfertilized sea urchin eggs.
Fertilization relieves the oocyte from a cell cycle arrest, inducing progression towards mitotic cycles. While the signalling pathways involved in oocyte to embryo transition have been widely investigated, how they specifically trigger DNA replication is still unclear. We used sea urchin eggs whose oocytes are arrested in G1 to investigate in vivo the molecular mechanisms regulating initiation of replication after fertilization. Unexpectedly, we found that CDC6, Cdt1 and MCM3, components of the pre-replication complexes (pre-RC) which license origins for replication, were already loaded on female chromatin before fertilization. This is the first demonstration of a cell cycle arrest in metazoan in which chromatin is already licensed for replication. In contrast pre-RC assemble on chromatin post-fertilization as in other organisms. These differences in the timing of pre-RC assembly are accompanied by differences in Cdk2 requirement for DNA replication initiation between female and male chromatin post-fertilization. Finally, we demonstrated that a concomitant inhibition of MAP kinase and ATM/ATR pathways releases the block to DNA synthesis. Our findings provide new insight into the mechanisms contributing to the release of G1 arrest and the control of S-phase entry at fertilization.
Université Pierre et Marie Curie-Paris6, Laboratoire Arago, Banyuls-sur-mer, BP44, Avenue du Fontaulé, F-66651, France.
Antoine
A
Aze
Claire
C
Fayet
Laure
L
Lapasset
A M
AM
Genevière
D000818:Animals; D001483:Base Sequence; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism; D002843:Chromatin / Q000378:metabolism; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D005260:Female; D005306:Fertilization / Q000502:physiology; D016650:Fluorescein-5-isothiocyanate / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D005456:Fluorescent Dyes / Q000378:metabolism; D016193:G1 Phase / Q000502:physiology; D049452:Green Fluorescent Proteins / Q000378:metabolism; D007150:Immunohistochemistry; D007211:Indoles / Q000378:metabolism; D008969:Molecular Sequence Data; D010063:Ovum / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D018741:Replication Origin; D012617:Sea Urchins / Q000235:genetics / Q000378:metabolism*
D018797:Cell Cycle Proteins; D002843:Chromatin; D005456:Fluorescent Dyes; D007211:Indoles; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; C007293:DAPI; D048049:Extracellular Signal-Regulated MAP Kinases; D016650:Fluorescein-5-isothiocyanate