Genotoxin-induced Rad9-Hus1-Rad1 (9-1-1) chromatin association is an early checkpoint signaling event.
Rad17, Rad1, Hus1, and Rad9 are key participants in checkpoint signaling pathways that block cell cycle progression in response to genotoxins. Biochemical and molecular modeling data predict that Rad9, Hus1, and Rad1 form a heterotrimeric complex, dubbed 9-1-1, which is loaded onto chromatin by a complex of Rad17 and the four small replication factor C (RFC) subunits (Rad17-RFC) in response to DNA damage. It is unclear what checkpoint proteins or checkpoint signaling events regulate the association of the 9-1-1 complex with DNA. Here we show that genotoxin-induced chromatin binding of 9-1-1 does not require the Rad9-inducible phosphorylation site (Ser-272). Although we found that Rad9 undergoes an additional phosphatidylinositol 3-kinase-related kinase (PIKK)-dependent posttranslational modification, we also show that genotoxin-triggered 9-1-1 chromatin binding does not depend on the catalytic activity of the PIKKs ataxia telangiectasia-mutated (ATM), ataxia telangiectasia and Rad3-related (ATR), or DNA-PK. Additionally, 9-1-1 chromatin binding does not require DNA replication, suggesting that the complex can be loaded onto DNA in response to DNA structures other than stalled DNA replication forks. Collectively, these studies demonstrate that 9-1-1 chromatin binding is a proximal event in the checkpoint signaling cascade.
Roos-Mattjus
Pia
P
Department of Biochemistry and Molecular Biology, Mayo Graduate School, Rochester, Minnesota 55905, USA.
Vroman
Benjamin T
BT
Burtelow
Matthew A
MA
Rauen
Matthew
M
Eapen
Alex K
AK
Karnitz
Larry M
LM
D018797:Cell Cycle Proteins / Q000737:chemistry / Q000378:metabolism*; D002843:Chromatin / Q000378:metabolism*; D004247:DNA / Q000378:metabolism; D004249:DNA Damage; D018076:DNA, Complementary / Q000378:metabolism; D004268:DNA-Binding Proteins; D004720:Endonucleases / Q000737:chemistry / Q000378:metabolism*; D016193:G1 Phase; D006801:Humans; D015151:Immunoblotting; D020014:K562 Cells; D009154:Mutation; D010766:Phosphorylation; D011485:Protein Binding; D029702:Schizosaccharomyces pombe Proteins; D015398:Signal Transduction; D014162:Transfection; D014407:Tumor Cells, Cultured
D018797:Cell Cycle Proteins; D002843:Chromatin; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D029702:Schizosaccharomyces pombe Proteins; C106332:hus1 protein, S pombe; C069930:rad9 protein; D004247:DNA; D004720:Endonucleases