Study of the cell cycle-dependent assembly of the DNA pre-replication centres in Xenopus egg extracts.
RPA is a cellular, three-subunit, single-stranded (ss) DNA binding protein, which assists T-antigen in the assembly of the pre-priming complex in the SV40 replication system. By immunodepletion and complementation, we have identified RPA as an essential factor for cellular DNA replication in Xenopus extracts. RPA assembles post-mitotically on the decondensing chromosomes into numerous subnuclear pre-replication centres (preRCs) which serve, upon formation of the nuclear membrane, as RCs for the initiation of DNA synthesis. By a variety of experiments including the use of isolated components, we demonstrate that an inactive cdc2-cyclin B kinase complex is essential to allow post-mitotic assembly of the preRCs. In contrast, the active cdk2-cyclin A kinase does not impede or facilitate the assembly of preRCs. Digestion analysis using the single-strand-specific P1 nuclease as well as competition experiments with ssDNA, reveal that replication-associated unwinding of the DNA, assisted by RPA, requires the formation of the nuclear membrane. The p21 cdk-interacting protein Cip1 appears to inhibit DNA replication prior to the unwinding DNA step, but after assembly of preRC and nuclear reconstruction.
Adachi
Y
Y
Department of Biochemistry, University of Geneva, Switzerland.
Laemmli
U K
UK
D000818:Animals; D001667:Binding, Competitive; D016203:CDC2 Protein Kinase / Q000378:metabolism; D042846:CDC2-CDC28 Kinases; D002453:Cell Cycle / Q000502:physiology*; D002467:Cell Nucleus / Q000502:physiology; D051357:Cyclin-Dependent Kinase 2; D050759:Cyclin-Dependent Kinase Inhibitor p21; D018844:Cyclin-Dependent Kinases; D016213:Cyclins / Q000378:metabolism; D004265:DNA Helicases / Q000302:isolation & purification / Q000378:metabolism*; D004261:DNA Replication; D004277:DNA, Single-Stranded / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D005260:Female; D008297:Male; D008938:Mitosis / Q000502:physiology; D009685:Nuclear Envelope / Q000378:metabolism; D009690:Nucleic Acid Conformation; D010063:Ovum / Q000502:physiology*; D047428:Protein Kinase Inhibitors; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D013094:Spermatozoa / Q000502:physiology; D029867:Xenopus Proteins; D014982:Xenopus laevis
D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D047428:Protein Kinase Inhibitors; D029867:Xenopus Proteins; C076713:P70 protein, Xenopus; D017346:Protein Serine-Threonine Kinases; D016203:CDC2 Protein Kinase; D042846:CDC2-CDC28 Kinases; C495891:Cdk2 protein, Xenopus; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases; D004265:DNA Helicases