Molecular architecture of the DNA replication origin activation checkpoint.
Perturbation of DNA replication initiation arrests human cells in G1, pointing towards an origin activation checkpoint. We used RNAi against Cdc7 kinase to inhibit replication initiation and dissect this checkpoint in fibroblasts. We show that the checkpoint response is dependent on three axes coordinated through the transcription factor FoxO3a. In arrested cells, FoxO3a activates the ARF-∣Hdm2-∣p53 → p21 pathway and mediates p15(INK4B) upregulation; p53 in turn activates expression of the Wnt/β-catenin signalling antagonist Dkk3, leading to Myc and cyclin D1 downregulation. The resulting loss of CDK activity inactivates the Rb-E2F pathway and overrides the G1-S transcriptional programme. Fibroblasts concomitantly depleted of Cdc7/FoxO3a, Cdc7/p15, Cdc7/p53 or Cdc7/Dkk3 can bypass the arrest and proceed into an abortive S phase followed by apoptosis. The lack of redundancy between the checkpoint axes and reliance on several tumour suppressor proteins commonly inactivated in human tumours provides a mechanistic basis for the cancer-cell-specific killing observed with emerging Cdc7 inhibitors.
Tudzarova
Slavica
S
Wolfson Institute for Biomedical Research, University College London, London, UK.
Trotter
Matthew W B
MW
Wollenschlaeger
Alex
A
Mulvey
Claire
C
Godovac-Zimmermann
Jasminka
J
Williams
Gareth H
GH
Stoeber
Kai
K
D048868:Adaptor Proteins, Signal Transducing; D015153:Blotting, Western; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D002458:Cell Fractionation; D002460:Cell Line; D018925:Chemokines; D050762:Cyclin-Dependent Kinase Inhibitor p15 / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics; D004261:DNA Replication / Q000235:genetics*; D005455:Fluorescent Antibody Technique; D000071316:Forkhead Box Protein O3; D051858:Forkhead Transcription Factors / Q000235:genetics / Q000378:metabolism*; D016193:G1 Phase / Q000502:physiology*; D005786:Gene Expression Regulation / Q000235:genetics*; D006801:Humans; D015151:Immunoblotting; D047468:Immunoprecipitation; D036341:Intercellular Signaling Peptides and Proteins / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D016271:Proto-Oncogene Proteins c-myc / Q000378:metabolism; D034622:RNA Interference; D018741:Replication Origin / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000235:genetics*
D048868:Adaptor Proteins, Signal Transducing; C493966:CDKN2B protein, human; D018797:Cell Cycle Proteins; D018925:Chemokines; D050762:Cyclin-Dependent Kinase Inhibitor p15; C498624:DKK3 protein, human; D017931:DNA Primers; C499517:FOXO3 protein, human; D000071316:Forkhead Box Protein O3; D051858:Forkhead Transcription Factors; D036341:Intercellular Signaling Peptides and Proteins; C489427:MYC protein, human; D016271:Proto-Oncogene Proteins c-myc; C068619:CDC7 protein, human; D017346:Protein Serine-Threonine Kinases