title: Chromatin-bound Cdc6 persists in S and G2 phases in human cells, while soluble Cdc6 is destroyed in a cyclin A-cdk2 dependent process.
abstract: Cdc6 is essential for the initiation of DNA replication in all organisms in which it has been studied. In addition, recombinant Cdc6 can stimulate initiation in G(1) nuclei in vitro. We have analysed the behaviour of recombinant Cdc6 in mammalian cell extracts under in vitro replication conditions. We find that Cdc6 is imported into the nucleus in G(1 )phase, where it binds to chromatin and remains relatively stable. In S phase, exogenous Cdc6 is destroyed in a process that requires import into the nucleus and phosphorylation by a chromatin-bound protein kinase. Recombinant cyclin A-cdk2 can completely substitute for the nucleus in promoting destruction of soluble Xenopus and human Cdc6. Despite this regulated destruction, endogenous Cdc6 persists in the nucleus after initiation, although the amount falls. Cdc6 levels remain constant in G(2) then fall again before mitosis. We propose that cyclin A-cdk2 phosphorylation results in destruction of any Cdc6 not assembled into replication complexes, but that assembled proteins remain, in the phosphorylated state, in the nucleus. This process could contribute to the prevention of reinitiation in human cells by making free Cdc6 unavailable for re-assembly into replication complexes after G(1) phase.
authors.affiliation: Wellcome/CRC Institute, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QR and Department of Zoology, University of Cambridge, Cambridge, UK. dcl34@hermes.cam.ac.uk.
authors.forename: D
authors.initials: D
authors.lastname: Coverley
authors.forename: C
authors.initials: C
authors.lastname: Pelizon
authors.forename: S
authors.initials: S
authors.lastname: Trewick
authors.forename: R A
authors.initials: RA
authors.lastname: Laskey
mesh_terms: D016475:3T3 Cells; D000818:Animals; D042846:CDC2-CDC28 Kinases; D018797:Cell Cycle Proteins / Q000378:metabolism*; D002467:Cell Nucleus / Q000378:metabolism; D002843:Chromatin / Q000378:metabolism*; D019925:Cyclin A / Q000378:metabolism*; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D016195:G2 Phase / Q000502:physiology*; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D016196:S Phase / Q000502:physiology*; D029701:Saccharomyces cerevisiae Proteins; D014981:Xenopus; D029867:Xenopus Proteins
chemical_list: C066503:CDC6 protein, S cerevisiae; D018797:Cell Cycle Proteins; D002843:Chromatin; D019925:Cyclin A; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D029867:Xenopus Proteins; D017346:Protein Serine-Threonine Kinases; D042846:CDC2-CDC28 Kinases; C495894:CDK2 protein, human; C495891:Cdk2 protein, Xenopus; C495893:Cdk2 protein, mouse; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases