Phosphorylation-Dependent Assembly and Coordination of the DNA Damage Checkpoint Apparatus by Rad4TopBP1

The BRCT-domain protein Rad4TopBP1 facilitates activation of the DNA damage checkpoint in Schizosaccharomyces pombe by physically coupling the Rad9-Rad1-Hus1 clamp, the Rad3ATR -Rad26ATRIP kinase complex, and the Crb253BP1 mediator. We have now determined crystal structures of the BRCT repeats of Rad4TopBP1, revealing a distinctive domain architecture, and characterized their phosphorylation-dependent interactions with Rad9 and Crb253BP1. We identify a cluster of phosphorylation sites in the N-terminal region of Crb253BP1 that mediate interaction with Rad4TopBP1 and reveal a hierarchical phosphorylation mechanism in which phosphorylation of Crb253BP1 residues Thr215 and Thr235 promotes phosphorylation of the noncanonical Thr187 site by scaffolding cyclin-dependent kinase (CDK) recruitment. Finally, we show that the simultaneous interaction of a single Rad4TopBP1 molecule with both Thr187 phosphorylation sites in a Crb253BP1 dimer is essential for establishing the DNA damage checkpoint.