Characterization of a novel ATR-dependent, Chk1-independent, intra-S-phase checkpoint that suppresses initiation of replication in Xenopus.
In most eukaryotes, replication origins fire asynchronously throughout S-phase according to a precise timing programme. When replication fork progression is inhibited, an intra-S-phase checkpoint is activated that blocks further origin firing and stabilizes existing replication forks to prevent them undergoing irreversible collapse. We show that chromatin incubated in Xenopus egg extracts displays a replication-timing programme in which firing of new replication origins during S phase depends on the continued activity of S-phase-inducing cyclin-dependent kinases. We also show that low concentrations of the DNA-polymerase inhibitor aphidicolin, which only slightly slows replication-fork progression, strongly suppress further initiation events. This intra-S-phase checkpoint can be overcome by caffeine, an inhibitor of the ATM/ATR checkpoint kinases, or by neutralizing antibodies to ATR. However, depletion or inhibition of Chk1 did not abolish the checkpoint. We could detect no significant effect on fork stability when this intra-S-phase checkpoint was inhibited. Interestingly, although caffeine could prevent the checkpoint from being activated, it could not rescue replication if added after the timing programme would normally have been executed. This suggests that special mechanisms might be necessary to reverse the effects of the intra-S-phase checkpoint once it has acted on particular origins.
Luciani
M Gloria
MG
Wellcome Trust Biocentre, University of Dundee, Dow Street, Dundee, DD1 5EH, UK.
Oehlmann
Maren
M
Blow
J Julian
JJ
D000818:Animals; D016590:Aphidicolin / Q000378:metabolism / Q000494:pharmacology; D064007:Ataxia Telangiectasia Mutated Proteins; D002110:Caffeine / Q000378:metabolism / Q000494:pharmacology; D018797:Cell Cycle Proteins / Q000378:metabolism / Q000502:physiology*; D002467:Cell Nucleus / Q000378:metabolism; D000071877:Checkpoint Kinase 1; D002843:Chromatin / Q000378:metabolism; D019925:Cyclin A / Q000494:pharmacology; D004261:DNA Replication; D004587:Electrophoresis, Agar Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007700:Kinetics; D008297:Male; D019384:Nucleic Acid Synthesis Inhibitors; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism; D011494:Protein Kinases / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000502:physiology*; D011687:Purines / Q000494:pharmacology; D000077546:Roscovitine; D016196:S Phase; D013094:Spermatozoa / Q000378:metabolism; D013997:Time Factors; D014981:Xenopus; D029867:Xenopus Proteins / Q000378:metabolism / Q000502:physiology*
C502211:Cdc45 protein, Xenopus; C491876:Cdc45 protein, mouse; D018797:Cell Cycle Proteins; D002843:Chromatin; D019925:Cyclin A; D004791:Enzyme Inhibitors; D019384:Nucleic Acid Synthesis Inhibitors; D018809:Proliferating Cell Nuclear Antigen; D011687:Purines; D029867:Xenopus Proteins; D000077546:Roscovitine; D016590:Aphidicolin; D002110:Caffeine; D011494:Protein Kinases; C576898:Atr protein, Xenopus; D064007:Ataxia Telangiectasia Mutated Proteins; D000071877:Checkpoint Kinase 1; C000605875:Chek1 protein, Xenopus; C000605869:Chek1 protein, mouse; D017346:Protein Serine-Threonine Kinases