NBS1 directly activates ATR independently of MRE11 and TOPBP1.
NBS1 plays unique and essential roles in ATM activation in response to DNA double-strand breaks. We found that CHK1 phosphorylation and FANCD2 ubiquitination induced by various DNA replication-stalling agents were abrogated in Nbs1 knockout DT40 cells but not in conditional Mre11 knockout cells, indicating an MRE11-independent role for NBS1 in ATR activation. The results of in vitro ATR kinase assay indicated that the N-terminal region of NBS1 directly activates ATR independently of TOPBP1, consistent with the findings that this region of NBS1 directly interacts with ATR. This conclusion was furthermore supported by the results of in vivo experiments; the expression of the N-terminal region of NBS1 fused to PCNA induces ATR activation in Rad17 knockout cells, and the expression of the ATR activation domain of TOPBP1 fused to PCNA induces ATR activation in Nbs1 knockout cells. These results therefore indicate that NBS1 and TOPBP1 have the potential to activate ATR independently, although both are required for functional activation of ATR in vivo.
Department of Molecular Pathology, Cancer Research Institute, Kanazawa University, Kanazawa, Ishikawa, 920-1192, Japan. mkoba@staff.kanazawa-u.ac.jp
Masahiko
M
Kobayashi
Naoyuki
N
Hayashi
Minoru
M
Takata
Ken-Ichi
K
Yamamoto
D000818:Animals; D064007:Ataxia Telangiectasia Mutated Proteins; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D000071877:Checkpoint Kinase 1; D002645:Chickens; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D052236:Fanconi Anemia Complementation Group D2 Protein / Q000378:metabolism; D006801:Humans; D000076228:MRE11 Homologue Protein; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D011494:Protein Kinases / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D054875:Ubiquitination
D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D052236:Fanconi Anemia Complementation Group D2 Protein; C121766:MRE11 protein, human; C112156:NBN protein, human; D009687:Nuclear Proteins; C110513:TOPBP1 protein, human; D011494:Protein Kinases; C576597:ATR protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; C000605867:CHEK1 protein, human; D000071877:Checkpoint Kinase 1; D017346:Protein Serine-Threonine Kinases; D000076228:MRE11 Homologue Protein