ATR and ATM regulate the timing of DNA replication origin firing.
Timing of DNA replication initiation is dependent on S-phase-promoting kinase (SPK) activity at discrete origins and the simultaneous function of many replicons. DNA damage prevents origin firing through the ATM- and ATR-dependent inhibition of Cdk2 and Cdc7 SPKs. Here, we establish that modulation of ATM- and ATR-signalling pathways controls origin firing in the absence of DNA damage. Inhibition of ATM and ATR with caffeine or specific neutralizing antibodies, or upregulation of Cdk2 or Cdc7, promoted rapid and synchronous origin firing; conversely, inhibition of Cdc25A slowed DNA replication. Cdk2 was in equilibrium between active and inactive states, and the concentration of replication protein A (RPA)-bound single-stranded DNA (ssDNA) correlated with Chk1 activation and inhibition of origin firing. Furthermore, ATM was transiently activated during ongoing replication. We propose that ATR and ATM regulate SPK activity through a feedback mechanism originating at active replicons. Our observations establish that ATM- and ATR-signalling pathways operate during an unperturbed cell cycle to regulate initiation and progression of DNA synthesis, and are therefore poised to halt replication in the presence of DNA damage.
Shechter
David
D
Integrated Program in Cellular, Molecular, and Biophysical Studies, and Department of Genetics and Development, Hammer Health Sciences Center, Columbia University College of Physicians and Surgeons, 701 West 168th Street, New York, NY 10032, USA.
Costanzo
Vincenzo
V
Gautier
Jean
J
D000818:Animals; D000906:Antibodies / Q000494:pharmacology; D064007:Ataxia Telangiectasia Mutated Proteins; D042846:CDC2-CDC28 Kinases / Q000235:genetics / Q000378:metabolism; D002110:Caffeine / Q000494:pharmacology; D018797:Cell Cycle Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D002457:Cell Extracts; D002474:Cell-Free System; D000071877:Checkpoint Kinase 1; D051357:Cyclin-Dependent Kinase 2; D004249:DNA Damage / Q000235:genetics; D042522:DNA Replication Timing / Q000187:drug effects / Q000235:genetics*; D004277:DNA, Single-Stranded / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D025461:Feedback, Physiological / Q000235:genetics*; D005260:Female; D006801:Humans; D009865:Oocytes; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D051716:Replication Protein A; D015398:Signal Transduction / Q000235:genetics*; D025521:Tumor Suppressor Proteins; D029867:Xenopus Proteins; D014982:Xenopus laevis; D020687:cdc25 Phosphatases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism
D000906:Antibodies; D018797:Cell Cycle Proteins; D002457:Cell Extracts; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; C497167:RPA1 protein, human; D051716:Replication Protein A; D025521:Tumor Suppressor Proteins; D029867:Xenopus Proteins; D002110:Caffeine; D011494:Protein Kinases; C576898:Atr protein, Xenopus; C489405:CDC7 protein, Xenopus; C068619:CDC7 protein, human; C576324:ATM protein, human; C576597:ATR protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; C000605867:CHEK1 protein, human; D000071877:Checkpoint Kinase 1; C000605875:Chek1 protein, Xenopus; D017346:Protein Serine-Threonine Kinases; D042846:CDC2-CDC28 Kinases; C495894:CDK2 protein, human; C495891:Cdk2 protein, Xenopus; D051357:Cyclin-Dependent Kinase 2; C517004:CDC25A protein, human; D020687:cdc25 Phosphatases