Multiple phosphorylation of Rad9 by CDK is required for DNA damage checkpoint activation.
The DNA damage checkpoint controls cell cycle arrest in response to DNA damage, and activation of this checkpoint is in turn cell cycle-regulated. Rad9, the ortholog of mammalian 53BP1, is essential for this checkpoint response and is phosphorylated by the cyclin-dependent kinase (CDK) in the yeast Saccharomyces cerevisiae. Previous studies suggested that the CDK consensus sites of Rad9 are important for its checkpoint activity. However, the precise CDK sites of Rad9 involved have not been determined. Here we show that CDK consensus sites of Rad9 function in parallel to its BRCT domain toward checkpoint activation, analogous to its fission yeast ortholog Crb2. Unlike Crb2, however, mutation of multiple rather than any individual CDK site of Rad9 is required to completely eliminate its checkpoint activity in vivo. Although Dpb11 interacts with CDK-phosphorylated Rad9, we provide evidence showing that elimination of this interaction does not affect DNA damage checkpoint activation in vivo, suggesting that additional pathway(s) exist. Taken together, these findings suggest that the regulation of Rad9 by CDK and the role of Dpb11 in DNA damage checkpoint activation are more complex than previously suggested. We propose that multiple phosphorylation of Rad9 by CDK may provide a more robust system to allow Rad9 to control cell cycle-dependent DNA damage checkpoint activation.
Ludwig Institute for Cancer Research, University of California at San Diego, La Jolla, CA, USA.
Guoliang
G
Wang
Xiangyan
X
Tong
Stephanie
S
Weng
Huilin
H
Zhou
D000595:Amino Acid Sequence; D059447:Cell Cycle Checkpoints / Q000235:genetics*; D018797:Cell Cycle Proteins / Q000235:genetics* / Q000378:metabolism*; D018844:Cyclin-Dependent Kinases / Q000235:genetics / Q000378:metabolism*; D004249:DNA Damage; D015966:Gene Expression Regulation, Fungal; D008969:Molecular Sequence Data; D009154:Mutation; D010766:Phosphorylation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D012568:Schizosaccharomyces / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction
D018797:Cell Cycle Proteins; C097185:DPB11 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; C069930:rad9 protein; D018844:Cyclin-Dependent Kinases