Activation of mammalian Chk1 during DNA replication arrest: a role for Chk1 in the intra-S phase checkpoint monitoring replication origin firing.
Checkpoints maintain order and fidelity in the cell cycle by blocking late-occurring events when earlier events are improperly executed. Here we describe evidence for the participation of Chk1 in an intra-S phase checkpoint in mammalian cells. We show that both Chk1 and Chk2 are phosphorylated and activated in a caffeine-sensitive signaling pathway during S phase, but only in response to replication blocks, not during normal S phase progression. Replication block-induced activation of Chk1 and Chk2 occurs normally in ataxia telangiectasia (AT) cells, which are deficient in the S phase response to ionizing radiation (IR). Resumption of synthesis after removal of replication blocks correlates with the inactivation of Chk1 but not Chk2. Using a selective small molecule inhibitor, cells lacking Chk1 function show a progressive change in the global pattern of replication origin firing in the absence of any DNA replication. Thus, Chk1 is apparently necessary for an intra-S phase checkpoint, ensuring that activation of late replication origins is blocked and arrested replication fork integrity is maintained when DNA synthesis is inhibited.
Feijoo
C
C
Division of Cell Signaling, School of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland, United Kingdom.
Hall-Jackson
C
C
Wu
R
R
Jenkins
D
D
Leitch
J
J
Gilbert
D M
DM
Smythe
C
C
D000470:Alkaloids / Q000494:pharmacology; D000818:Animals; D016590:Aphidicolin / Q000494:pharmacology; D001260:Ataxia Telangiectasia / Q000378:metabolism; D002110:Caffeine / Q000494:pharmacology; D002458:Cell Fractionation; D002460:Cell Line; D002469:Cell Separation; D000071877:Checkpoint Kinase 1; D064447:Checkpoint Kinase 2; D004261:DNA Replication / Q000502:physiology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005434:Flow Cytometry; D006801:Humans; D006918:Hydroxyurea / Q000494:pharmacology; D015151:Immunoblotting; D008856:Microscopy, Fluorescence; D019384:Nucleic Acid Synthesis Inhibitors / Q000494:pharmacology; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D011494:Protein Kinases / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases; D011839:Radiation, Ionizing; D018741:Replication Origin / Q000502:physiology*; D016196:S Phase / Q000502:physiology; D019311:Staurosporine / Q000031:analogs & derivatives
D000470:Alkaloids; D004791:Enzyme Inhibitors; D019384:Nucleic Acid Synthesis Inhibitors; D010726:Phosphodiesterase Inhibitors; D016590:Aphidicolin; D002110:Caffeine; C054852:7-hydroxystaurosporine; D011494:Protein Kinases; D064447:Checkpoint Kinase 2; C000605867:CHEK1 protein, human; C578634:CHEK2 protein, human; D000071877:Checkpoint Kinase 1; D017346:Protein Serine-Threonine Kinases; D019311:Staurosporine; D006918:Hydroxyurea