THE REPLICATION ORIGIN DECISION POINT IS A MITOGEN-INDEPENDENT, 2-AMINOPURINE-SENSITIVE, G(1)-PHASE EVENT THAT PRECEDES RESTRICTION POINT CONTROL

At a distinct point during G(1) phase (the origin decision point [ODP] ), Chinese hamster ovary (CHO) cell nuclei experience a transition (or igin choice) that is required for specific recognition of the dihydrof olate reductase (DHFR) origin locus by Xenopus egg extracts. We have i nvestigated the relationship between the ODP and progression of CHO ce lls through G(1) phase. Selection of the DHFR origin at the ODP was ra pidly inhibited by treatment of early G(1)-phase cells with the protei n kinase inhibitor 2-aminopurine (2-AP). Inhibition of the ODP require d administration of 2-AP at least 3 h prior to phosphorylation of the retinoblastoma tumor suppressor protein (Rb) and the restriction point (R point). Cells deprived of either serum or isoleucine from metaphas e throughout early G(1) phase acquired the capacity to replicate in Xe nopus egg extract (replication licensing) and subsequently passed thro ugh the ODP on the same schedule as cells cultured in complete growth medium, After growth arrest at the R point with hypophosphorylated Rb protein, serum- or isoleucine-deprived cells experienced a gradual los s of replication licensing. However, recognition of the DHFR origin by Xenopus egg cytosol remained stable in growth-arrested cells until th e point at which all nuclei had lost the capacity to initiate replicat ion. These results provide evidence that the ODP requires a mitogen-in dependent protein kinase that is activated after replication licensing and prior to R-point control.