title: Sensitivity of the origin decision point to specific inhibitors of cellular signaling and metabolism.
abstract: Chinese hamster ovary (CHO) cells become committed to initiate DNA replication at specific sites within the dihydrofolate reductase (DHFR) locus at a discrete point during G1 phase, the origin decision point (ODP). To better understand the requirements for passage through the ODP, we evaluated the ability of various inhibitors of G1-phase progression to prevent passage through the ODP. Of several protein kinase inhibitors tested, only inhibitors of cyclin-dependent kinase (cdk) activity (roscovitine, olomoucine) prevented passage through the ODP. Inhibitors of MAP kinase (PD98059), PKA (KT5720), PKG (KT5823), as well as inhibition of integrin-mediated signaling by preventing cell adhesion, all arrested cells in the post-ODP stages of G1 phase. Intriguingly, inhibitors of proteasome-dependent proteolysis (MG132, ALLN, lactacystin) and transcription (DRB, alpha-amanitin, actinomycin D) also inhibited passage through the ODP, whereas inhibition of protein synthesis (cycloheximide) had no effect on the ODP. Cross-checking each inhibitor for its affect on transcription revealed that the ODP could be uncoupled from transcription; MG132 and lactacystin did not inhibit transcription, and KT5720 was a potent inhibitor of transcription. Importantly, cells that were arrested upstream of the ODP with either roscovitine or lactacystin contained functional prereplication complexes (pre-RCs), supporting previous findings that pre-RC formation is not sufficient for origin specification. These results demonstrate that specification of the DHFR origin is independent of growth signaling mechanisms and does not require G1-phase synthesis of a protein regulator such as a cyclin or Dbf4/ASK1, positioning the ODP after pre-RC formation but prior to the activation of the known S-phase promoting kinases.
authors.affiliation: Department of Biochemistry and Molecular Biology, S.U.N.Y. Upstate Medical University, 750 East Adams Street, Syracuse, New York 13210, USA.
authors.forename: Susan M
authors.initials: SM
authors.lastname: Keezer
authors.forename: David M
authors.initials: DM
authors.lastname: Gilbert
mesh_terms: D000818:Animals; D015153:Blotting, Western; D001973:Bromodeoxyuridine; D016466:CHO Cells; D006224:Cricetinae; D016213:Cyclins / Q000378:metabolism; D004261:DNA Replication / Q000502:physiology*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D016193:G1 Phase / Q000187:drug effects / Q000502:physiology; D008934:Mitogens / Q000494:pharmacology; D047428:Protein Kinase Inhibitors; D018741:Replication Origin / Q000502:physiology*; D016196:S Phase / Q000187:drug effects / Q000502:physiology; D015398:Signal Transduction / Q000187:drug effects*; D013762:Tetrahydrofolate Dehydrogenase / Q000378:metabolism; D014158:Transcription, Genetic; D014981:Xenopus
chemical_list: D016213:Cyclins; D004791:Enzyme Inhibitors; D008934:Mitogens; D047428:Protein Kinase Inhibitors; D013762:Tetrahydrofolate Dehydrogenase; D001973:Bromodeoxyuridine