The DNA damage-dependent intra-S phase checkpoint is regulated by parallel pathways.
To preserve genetic integrity, mammalian cells exposed to ionizing radiation activate the ATM kinase, which initiates a complex response-including the S-phase checkpoint pathways-to delay DNA replication. Defects in ATM or its substrates Nbs1 or Chk2 (ref. 3), the Nbs1-interacting Mre11 protein, or the Chk2-regulated Cdc25A-Cdk2 cascade all cause radio-resistant DNA synthesis (RDS). It is unknown, however, whether these proteins operate in a common signaling cascade. Here we show that experimental blockade of either the Nbs1-Mre11 function or the Chk2-triggered events leads to a partial RDS phenotype in human cells. In contrast, concomitant interference with Nbs1-Mre11 and the Chk2-Cdc25A-Cdk2 pathways entirely abolishes inhibition of DNA synthesis induced by ionizing radiation, resulting in complete RDS analogous to that caused by defective ATM. In addition, Cdk2-dependent loading of Cdc45 onto replication origins, a prerequisite for recruitment of DNA polymerase, was prevented upon irradiation of normal or Nbs1/Mre11-defective cells but not cells with defective ATM. We conclude that in response to ionizing radiation, phosphorylations of Nbs1 and Chk2 by ATM trigger two parallel branches of the DNA damage-dependent S-phase checkpoint that cooperate by inhibiting distinct steps of DNA replication.
Falck
Jacob
J
Institute of Cancer Biology, Danish Cancer Society, Strandboulevarden 49, DK-2100 Copenhagen, Denmark.
Petrini
John H J
JH
Williams
Bret R
BR
Lukas
Jiri
J
Bartek
Jiri
J
D064007:Ataxia Telangiectasia Mutated Proteins; D042846:CDC2-CDC28 Kinases; D018797:Cell Cycle Proteins; D002460:Cell Line; D064447:Checkpoint Kinase 2; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases / Q000378:metabolism; D004249:DNA Damage; D004268:DNA-Binding Proteins; D006801:Humans; D009687:Nuclear Proteins / Q000378:metabolism; D011494:Protein Kinases / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011839:Radiation, Ionizing; D016196:S Phase / Q000502:physiology*; D025521:Tumor Suppressor Proteins; D020687:cdc25 Phosphatases / Q000378:metabolism
D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D025521:Tumor Suppressor Proteins; D011494:Protein Kinases; D064447:Checkpoint Kinase 2; C576324:ATM protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; C578634:CHEK2 protein, human; D017346:Protein Serine-Threonine Kinases; D042846:CDC2-CDC28 Kinases; C495894:CDK2 protein, human; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases; C517004:CDC25A protein, human; D020687:cdc25 Phosphatases