Rapid PIKK-dependent release of Chk1 from chromatin promotes the DNA-damage checkpoint response.
BACKGROUND
Checkpoint signaling pathways are of crucial importance for the maintenance of genomic integrity. Within these pathways, the effector kinase Chk1 plays a central role in mediating cell-cycle arrest in response to DNA damage, and it does so by phosphorylating key cell-cycle regulators.


RESULTS
By investigating the subcellular distribution of Chk1 by cell fractionation, we observed that around 20% of it localizes to chromatin during all phases of the cell cycle. Furthermore, we found that in response to DNA damage, Chk1 rapidly dissociates from the chromatin. Significantly, we observed a tight correlation between DNA-damage-induced Chk1 phosphorylation and chromatin dissociation, suggesting that phosphorylated Chk1 does not stably associate with chromatin. Consistent with these events being triggered by active checkpoint signaling, inhibition of the DNA-damage-activated kinases ATR and ATM, or siRNA-mediated downregulation of the DNA-damage mediator proteins Claspin and TopBP1, impaired DNA-damage-induced dissociation of Chk1 from chromatin. Finally, we established that Chk1 phosphorylation occurs at localized sites of DNA damage and that constitutive immobilization of Chk1 on chromatin results in a defective DNA-damage-induced checkpoint arrest.


CONCLUSIONS
Chromatin association and dissociation appears to be important for proper Chk1 regulation. We propose that in response to DNA damage, PIKK-dependent checkpoint signaling leads to phosphorylation of chromatin-bound Chk1, resulting in its rapid release from chromatin and facilitating the transmission of DNA-damage signals to downstream targets, thereby promoting efficient cell-cycle arrest.
Smits
Veronique A J
VA
The Wellcome Trust and Cancer Research UK Gurdon Institute, Tennis Court Road, Cambridge CB2 1QN, United Kingdom. v.a.j.smits@erasmusmc.nl
Reaper
Philip M
PM
Jackson
Stephen P
SP
D064007:Ataxia Telangiectasia Mutated Proteins; D018797:Cell Cycle Proteins / Q000502:physiology*; D002460:Cell Line; D000071877:Checkpoint Kinase 1; D002843:Chromatin / Q000201:enzymology*; D004249:DNA Damage; D004268:DNA-Binding Proteins / Q000502:physiology*; D006801:Humans; D008954:Models, Biological; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism / Q000502:physiology*; D011499:Protein Processing, Post-Translational; D017346:Protein Serine-Threonine Kinases / Q000502:physiology*; D015398:Signal Transduction / Q000528:radiation effects; D025521:Tumor Suppressor Proteins / Q000502:physiology*; D014466:Ultraviolet Rays
D018797:Cell Cycle Proteins; D002843:Chromatin; D004268:DNA-Binding Proteins; D025521:Tumor Suppressor Proteins; D011494:Protein Kinases; C576324:ATM protein, human; C576597:ATR protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; C000605867:CHEK1 protein, human; D000071877:Checkpoint Kinase 1; D017346:Protein Serine-Threonine Kinases