Phosphorylation-dependent assembly and coordination of the DNA damage checkpoint apparatus by Rad4(TopBP1)

Mol Cell. 2013 Sep 26;51(6):723-736. doi: 10.1016/j.molcel.2013.08.030.


The BRCT-domain protein Rad4(TopBP1) facilitates activation of the DNA damage checkpoint in Schizosaccharomyces pombe by physically coupling the Rad9-Rad1-Hus1 clamp, the Rad3(ATR) -Rad26(ATRIP) kinase complex, and the Crb2(53BP1) mediator. We have now determined crystal structures of the BRCT repeats of Rad4(TopBP1), revealing a distinctive domain architecture, and characterized their phosphorylation-dependent interactions with Rad9 and Crb2(53BP1). We identify a cluster of phosphorylation sites in the N-terminal region of Crb2(53BP1) that mediate interaction with Rad4(TopBP1) and reveal a hierarchical phosphorylation mechanism in which phosphorylation of Crb2(53BP1) 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 Rad4(TopBP1) molecule with both Thr187 phosphorylation sites in a Crb2(53BP1) dimer is essential for establishing the DNA damage checkpoint.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Binding Sites
  • Cell Cycle Proteins / chemistry
  • Cell Cycle Proteins / genetics
  • Cell Cycle Proteins / metabolism*
  • Crystallography, X-Ray
  • DNA Damage / genetics*
  • DNA-Binding Proteins* / chemistry
  • DNA-Binding Proteins* / genetics
  • DNA-Binding Proteins* / metabolism
  • Nuclear Proteins / chemistry
  • Nuclear Proteins / genetics
  • Nuclear Proteins / metabolism*
  • Phosphorylation / genetics*
  • Protein Binding
  • Protein Structure, Tertiary
  • Schizosaccharomyces
  • Schizosaccharomyces pombe Proteins / chemistry
  • Schizosaccharomyces pombe Proteins / genetics
  • Schizosaccharomyces pombe Proteins / metabolism*
  • Transglutaminases* / chemistry
  • Transglutaminases* / genetics
  • Transglutaminases* / metabolism


  • Cell Cycle Proteins
  • Crb2 protein, S pombe
  • DNA-Binding Proteins
  • Nuclear Proteins
  • RAD4 protein, S pombe
  • Schizosaccharomyces pombe Proteins
  • rad9 protein
  • Transglutaminases

Associated data

  • PDB/4BMC
  • PDB/4BMD
  • PDB/4BU0
  • PDB/4BU1