Stability, chromatin association and functional activity of mammalian pre-replication complex proteins during the cell cycle

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Abstract
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, ∼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.