Requirement for NBS1 in the S phase checkpoint response to DNA methylation combined with PARP inhibition.
Treatment of PARP-1-expressing cells with the combination of a DNA methylating agent (MMS) and the PARP inhibitor 4-amino-1,8-naphthalimide (4-AN) leads to an ATR/Chk1-dependent S phase checkpoint and cell death by apoptosis. Activation of ATM/Chk2 is involved in sustaining the S phase checkpoint, and double strand break (DSB) accumulation was demonstrated. NBS1, part of the MRN complex that responds to DSBs, is known to modulate ATR- and ATM-dependent checkpoint responses to UV and IR, but a role in the response to PARP inhibition has not been addressed. Here we show that the S phase checkpoint observed 4-8h after MMS+4-AN treatment was absent in cells deficient in NBS1, but was present in NBS1-complemented (i.e., functionally wild-type) cells, indicating a critical role for NBS1 in this checkpoint response. NBS1 was phosphorylated in response to MMS+4-AN treatment, and this was partially ATR- and ATM-dependent, suggesting involvement of both upstream kinases. NBS1 expression had little effect on ATR-mediated phosphorylation of Chk1 and ATM-mediated phosphorylation of Chk2 in response to MMS+4-AN. Phosphorylation of SMC1 was also observed in response to MMS+4-AN treatment. In the absence of ATM and NBS1, phosphorylation of SMC1 was weak, especially at early times after MMS+4-AN treatment. In the absence of ATR activation, reduced SMC1 phosphorylation was seen over a 24h time course. These results suggested that both ATR and ATM phosphorylate SMC1 in response to MMS+4-AN and that this phosphorylation is enhanced by phospho-NBS1. The loss of the MMS+4-AN-induced S phase checkpoint in NBS1-deficient cells may be due to a reduced cellular level of the critical downstream effector, phospho-SMC1.
Laboratory of Structural Biology, NIEHS, National Institutes of Health, Research Triangle Park, NC 27709, USA.
Julie K
JK
Horton
Donna F
DF
Stefanick
Jennifer Y
JY
Zeng
Michael J
MJ
Carrozza
Samuel H
SH
Wilson
D015057:1-Naphthylamine / Q000031:analogs & derivatives* / Q000494:pharmacology; D064007:Ataxia Telangiectasia Mutated Proteins; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D002460:Cell Line; D000071877:Checkpoint Kinase 1; D064447:Checkpoint Kinase 2; D002868:Chromosomal Proteins, Non-Histone / Q000235:genetics / Q000378:metabolism; D053903:DNA Breaks, Double-Stranded; D019175:DNA Methylation; D004268:DNA-Binding Proteins / Q000235:genetics / Q000502:physiology; D005434:Flow Cytometry; D018816:Genes, cdc; D006801:Humans; D047468:Immunoprecipitation; D008741:Methyl Methanesulfonate / Q000494:pharmacology*; D053644:Naphthalimides / Q000494:pharmacology*; D009687:Nuclear Proteins / Q000235:genetics / Q000502:physiology*; D010766:Phosphorylation / Q000187:drug effects / Q000235:genetics / Q000502:physiology; D000071137:Poly (ADP-Ribose) Polymerase-1; D000067856:Poly(ADP-ribose) Polymerase Inhibitors; D011065:Poly(ADP-ribose) Polymerases / Q000235:genetics / Q000502:physiology; D011494:Protein Kinases / Q000235:genetics / Q000502:physiology; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000502:physiology; D015363:Quinolones / Q000494:pharmacology*; D016196:S Phase / Q000187:drug effects*; D025521:Tumor Suppressor Proteins / Q000235:genetics / Q000502:physiology; D000098550:Structural Maintenance of Chromosome Protein 1
D018797:Cell Cycle Proteins; D002868:Chromosomal Proteins, Non-Histone; D004268:DNA-Binding Proteins; C112156:NBN protein, human; D053644:Naphthalimides; D009687:Nuclear Proteins; D000067856:Poly(ADP-ribose) Polymerase Inhibitors; D015363:Quinolones; D025521:Tumor Suppressor Proteins; D000098550:Structural Maintenance of Chromosome Protein 1; C086538:4-amino-1,8-naphthalimide; D015057:1-Naphthylamine; D008741:Methyl Methanesulfonate; C499320:PARP1 protein, human; D000071137:Poly (ADP-Ribose) Polymerase-1; D011065:Poly(ADP-ribose) Polymerases; D011494:Protein Kinases; D064447:Checkpoint Kinase 2; C576324:ATM protein, human; C576597:ATR protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; C000605867:CHEK1 protein, human; C578634:CHEK2 protein, human; D000071877:Checkpoint Kinase 1; D017346:Protein Serine-Threonine Kinases