Chk1 activation requires Rad9 S/TQ-site phosphorylation to promote association with C-terminal BRCT domains of Rad4TOPBP1.
To gain insight into the function and organization of proteins assembled on the DNA in response to genotoxic insult we investigated the phosphorylation of the Schizosaccharomyces pombe PCNA-like checkpoint protein Rad9. C-terminal T412/S423 phosphorylation of Rad9 by Rad3(ATR) occurs in S phase without replication stress. Rad3(ATR) and Tel1(ATM) phosphorylate these same residues, plus additional ones, in response to DNA damage. In S phase and after damage, only Rad9 phosphorylated on T412/S423, but not unphosphorylated Rad9, associates with a two-BRCT-domain region of the essential Rad4(TOPBP1) protein. Rad9-Rad4(TOPBP1) interaction is required to activate the Chk1 damage checkpoint but not the Cds1 replication checkpoint. When the Rad9-T412/S423 are phosphorylated, Rad4(TOPBP1) coprecipitates with Rad3(ATR), suggesting that phosphorylation coordinates formation of an active checkpoint complex.
Furuya
Kanji
K
Genome Damage and Stability Centre, University of Sussex, Brighton, BN1 9RQ, UK.
Poitelea
Marius
M
Guo
Liandi
L
Caspari
Thomas
T
Carr
Antony M
AM
D001665:Binding Sites / Q000235:genetics; D018797:Cell Cycle Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000071877:Checkpoint Kinase 1; D064447:Checkpoint Kinase 2; D004249:DNA Damage; D004261:DNA Replication; D004271:DNA, Fungal / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004789:Enzyme Activation; D008954:Models, Biological; D009154:Mutation; D010766:Phosphorylation; D011494:Protein Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016196:S Phase; D012568:Schizosaccharomyces / Q000166:cytology / Q000235:genetics / Q000378:metabolism; D029702:Schizosaccharomyces pombe Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011503:Transglutaminases / Q000378:metabolism*
D018797:Cell Cycle Proteins; D004271:DNA, Fungal; D004268:DNA-Binding Proteins; C082463:RAD4 protein, S pombe; D029702:Schizosaccharomyces pombe Proteins; C069930:rad9 protein; D011503:Transglutaminases; D011494:Protein Kinases; D064447:Checkpoint Kinase 2; D000071877:Checkpoint Kinase 1; C000605873:Chk1 protein, S pombe; C456840:rad3 protein, S pombe