Stability, chromatin association and functional activity of mammalian pre-replication complex proteins during the cell cycle.
We have examined the behavior of pre-replication complex (pre-RC) proteins in relation to key cell cycle transitions in Chinese Hamster Ovary (CHO) cells. ORC1, ORC4 and Cdc6 were stable (T1/2 >2 h) and associated with a chromatin-containing fraction throughout the cell cycle. Green fluorescent protein-tagged ORC1 associated with chromatin throughout mitosis in living cells and co-localized with ORC4 in metaphase spreads. Association of Mcm proteins with chromatin took place during telophase, approximately 30 min after the destruction of geminin and cyclins A and B, and was coincident with the licensing of chromatin to replicate in geminin-supplemented Xenopus egg extracts. Neither Mcm recruitment nor licensing required protein synthesis throughout mitosis. Moreover, licensing could be uncoupled from origin specification in geminin-supplemented extracts; site-specific initiation within the dihydrofolate reductase locus required nuclei from cells that had passed through the origin decision point (ODP). These results demonstrate that mammalian pre-RC assembly takes place during telophase, mediated by post-translational modifications of pre-existing proteins, and is not sufficient to select specific origin sites. A subsequent, as yet undefined, step selects which pre-RCs will function as replication origins.
Okuno
Y
Y
Department of Biochemistry and Molecular Biology, SUNY Upstate Medical University, 750 East Adams Street, Syracuse, NY 13210, USA.
McNairn
A J
AJ
den Elzen
N
N
Pines
J
J
Gilbert
D M
DM
D000705:Anaphase; D000818:Animals; D016466:CHO Cells; D002453:Cell Cycle; D018797:Cell Cycle Proteins / Q000378:metabolism*; D002843:Chromatin / Q000378:metabolism*; D006224:Cricetinae; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000378:metabolism*; D016193:G1 Phase; D064248:Geminin; D006801:Humans; D008322:Mammals; D064126:Minichromosome Maintenance Complex Component 3; D008938:Mitosis; D009687:Nuclear Proteins / Q000378:metabolism*; D051738:Origin Recognition Complex; D016196:S Phase; D013692:Telophase; D029867:Xenopus Proteins; D014982:Xenopus laevis
C103706:CDC6 protein, human; D018797:Cell Cycle Proteins; D002843:Chromatin; D004268:DNA-Binding Proteins; C577998:GMNN protein, Xenopus; C577995:GMNN protein, human; D064248:Geminin; C066470:MCM3 protein, human; D009687:Nuclear Proteins; C497223:ORC1 protein, human; D051738:Origin Recognition Complex; D029867:Xenopus Proteins; D064126:Minichromosome Maintenance Complex Component 3