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) ce lls. ORC1, ORC4 and Cdc6 were stable (T-1/2 >2 h) and associated with a chr omatin-containing fraction throughout the cell cycle. Green fluorescent pro tein-tagged ORC1 associated with chromatin throughout mitosis in living cel ls and co-localized with ORC4 in metaphase spreads. Association of Mcm prot eins with chromatin took place during telophase, similar to 30 min after th e destruction of geminin and cyclins A and B, and was coincident with the l icensing of chromatin to replicate in geminin-supplemented Xenopus egg extr acts. Neither Mcm recruitment nor licensing required protein synthesis thro ughout mitosis. Moreover, licensing could be uncoupled from origin specific ation in geminin-supplemented extracts; site-specific initiation within the dihydrofolate reductase locus required nuclei from cells that had passed t hrough the origin decision point (ODP). These results demonstrate that mamm alian pre-RC assembly takes place during telophase, mediated by post-transl ational modifications of pre-existing proteins, and is not sufficient to se lect specific origin sites. A subsequent, as yet undefined, step selects wh ich pre-RCs will function as replication origins.