Transformation abrogates an early G1-phase arrest point required for specification of the Chinese hamster DHFR replication origin

The origin decision point (ODP) was originally identified as a distinct point during G 1 ‐phase when Chinese hamster ovary (CHO) cell nuclei experience a transition that is required for specific recognition of the dihydrofolate reductase (DHFR) origin locus by Xenopus egg extracts. Passage of cells through the ODP requires a mitogen‐independent protein kinase that is activated prior to restriction point control. Here we show that inhibition of an early G 1 ‐phase protein kinase pathway by the addition of 2‐aminopurine (2‐AP) prior to the ODP arrests CHO cells in G 1 ‐phase. Transformation with simian virus 40 (SV40) abrogated this arrest point, resulting in the entry of cultured cells into S‐phase in the presence of 2‐AP and a disruption of the normal pattern of initiation sites at the DHFR locus. Cells treated with 2‐AP after the ODP initiated replication specifically within the DHFR origin locus. Transient exposure of transformed cells to 2‐AP during the ODP transition also disrupted origin choice, whereas non‐transformed cells arrested in G 1 ‐phase and then passed through a delayed ODP after removal of 2‐AP from the medium. We conclude that mammalian cells have many potential sites at which they can initiate replication. Normally, events occurring during the early G 1 ‐phase ODP transition determine which of these sites will be the preferred initiation site. However, if chromatin is exposed to S‐phase‐promoting factors prior to this transition, mammalian cells, like Xenopus and Drosophila embryos, can initiate replication without origin specification.