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 G1-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 G1-phase protein kinase pathway by the addition of 2-aminopurine (2-AP) prior to the ODP arrests CHO cells in G1-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 G1-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 G1-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.
Wu
J R
JR
Department of Biochemistry and Molecular Biology, S.U.N.Y. Health Science Center, 750 East Adams Street, Syracuse, NY 13210, USA.
Keezer
S M
SM
Gilbert
D M
DM
D015075:2-Aminopurine / Q000494:pharmacology; D000818:Animals; D000963:Antimetabolites / Q000494:pharmacology; D016590:Aphidicolin / Q000494:pharmacology; D016466:CHO Cells; D002472:Cell Transformation, Viral / Q000502:physiology*; D006224:Cricetinae; D004261:DNA Replication / Q000235:genetics; D004791:Enzyme Inhibitors / Q000494:pharmacology; D016193:G1 Phase / Q000187:drug effects / Q000235:genetics*; D018741:Replication Origin / Q000502:physiology*; D016196:S Phase / Q000235:genetics; D013539:Simian virus 40 / Q000502:physiology; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics*
D000963:Antimetabolites; D004791:Enzyme Inhibitors; D016590:Aphidicolin; D015075:2-Aminopurine; D013762:Tetrahydrofolate Dehydrogenase