Phosphorylation-dependent interactions between Crb2 and Chk1 are essential for DNA damage checkpoint.
In response to DNA damage, the eukaryotic genome surveillance system activates a checkpoint kinase cascade. In the fission yeast Schizosaccharomyces pombe, checkpoint protein Crb2 is essential for DNA damage-induced activation of downstream effector kinase Chk1. The mechanism by which Crb2 mediates Chk1 activation is unknown. Here, we show that Crb2 recruits Chk1 to double-strand breaks (DSBs) through a direct physical interaction. A pair of conserved SQ/TQ motifs in Crb2, which are consensus phosphorylation sites of upstream kinase Rad3, is required for Chk1 recruitment and activation. Mutating both of these motifs renders Crb2 defective in activating Chk1. Tethering Crb2 and Chk1 together can rescue the SQ/TQ mutations, suggesting that the main function of these phosphorylation sites is promoting interactions between Crb2 and Chk1. A 19-amino-acid peptide containing these SQ/TQ motifs is sufficient for Chk1 binding in vitro when one of the motifs is phosphorylated. Remarkably, the same peptide, when tethered to DSBs by fusing with either recombination protein Rad22/Rad52 or multi-functional scaffolding protein Rad4/Cut5, can rescue the checkpoint defect of crb2Δ. The Rad22 fusion can even bypass the need for Rad9-Rad1-Hus1 (9-1-1) complex in checkpoint activation. These results suggest that the main role of Crb2 and 9-1-1 in DNA damage checkpoint signaling is recruiting Chk1 to sites of DNA lesions.
College of Biological Sciences, China Agricultural University, Beijing, China.
Meng
M
Qu
Bing
B
Yang
Li
L
Tao
John R
JR
Yates
Paul
P
Russell
Meng-Qiu
MQ
Dong
Li-Lin
LL
Du
D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism; D000071877:Checkpoint Kinase 1; D064447:Checkpoint Kinase 2; D053903:DNA Breaks, Double-Stranded; D004249:DNA Damage / Q000235:genetics; D004268:DNA-Binding Proteins; D018816:Genes, cdc; D009154:Mutation; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D054730:Protein Interaction Domains and Motifs / Q000235:genetics; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary; D012568:Schizosaccharomyces / Q000235:genetics*; D029702:Schizosaccharomyces pombe Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction
D018797:Cell Cycle Proteins; C109867:Crb2 protein, S pombe; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D029702:Schizosaccharomyces pombe Proteins; D011494:Protein Kinases; D064447:Checkpoint Kinase 2; D000071877:Checkpoint Kinase 1; C000605873:Chk1 protein, S pombe; C456840:rad3 protein, S pombe