Determination of initiation of DNA replication before and after nuclear formation in Xenopus egg cell free extracts.
Xenopus egg extracts prepared before and after egg activation retain M- and S-phase specific activity, respectively. Staurosporine, a potent inhibitor of protein kinase, converted M-phase extracts into interphase-like extracts that were capable of forming nuclei upon the addition of sperm DNA. The nuclei formed in the staurosporine treated M-phase extract were incapable of replicating DNA, and they were unable to initiate replication upon the addition of S-phase extracts. Furthermore, replication was inhibited when the staurosporine-treated M-phase extract was added in excess to the staurosporine-treated S-phase extract before the addition of DNA. The membrane-depleted S-phase extract supported neither nuclear formation nor replication; however, preincubation of sperm DNA with these extracts allowed them to form replication-competent nuclei upon the addition of excess staurosporine-treated M-phase extract. These results demonstrate that positive factors in the S-phase extracts determined the initiation of DNA replication before nuclear formation, although these factors were unable to initiate replication after nuclear formation.
Kubota
Y
Y
Department of Biology, Faculty of Science, Osaka University, Japan.
Takisawa
H
H
D000470:Alkaloids / Q000494:pharmacology; D000818:Animals; D002453:Cell Cycle / Q000187:drug effects*; D002467:Cell Nucleus / Q000378:metabolism* / Q000648:ultrastructure; D002474:Cell-Free System; D004261:DNA Replication; D066298:In Vitro Techniques; D008938:Mitosis; D009865:Oocytes; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D016196:S Phase; D019311:Staurosporine; D014982:Xenopus laevis
D000470:Alkaloids; D011493:Protein Kinase C; D019311:Staurosporine