Sensitivity of the origin decision point to specific inhibitors of cellular signaling and metabolism.
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.
Keezer
Susan M
SM
Department of Biochemistry and Molecular Biology, S.U.N.Y. Upstate Medical University, 750 East Adams Street, Syracuse, New York 13210, USA.
Gilbert
David M
DM
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
D016213:Cyclins; D004791:Enzyme Inhibitors; D008934:Mitogens; D047428:Protein Kinase Inhibitors; D013762:Tetrahydrofolate Dehydrogenase; D001973:Bromodeoxyuridine