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.