Phosphorylation-dependent assembly and coordination of the DNA damage checkpoint apparatus by Rad4(TopBP1).
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.
National Institute of Biological Sciences, 7 Science Park Road, ZGC Life Science Park, Beijing 102206, China.
Meng
M
Qu
Cancer Research UK DNA Repair Enzymes Group, Genome Damage and Stability Centre, School of Life Sciences, University of Sussex, Falmer, BN1 9RQ, UK.
Mathieu
M
Rappas
MRC Genome Damage and Stability Centre, School of Life Sciences, University of Sussex, Falmer, BN1 9RQ, UK.
Christopher P
CP
Wardlaw
MRC Genome Damage and Stability Centre, School of Life Sciences, University of Sussex, Falmer, BN1 9RQ, UK.
Valerie
V
Garcia
National Institute of Biological Sciences, 7 Science Park Road, ZGC Life Science Park, Beijing 102206, China.
Jing-Yi
JY
Ren
Cancer Research UK DNA Repair Enzymes Group, Genome Damage and Stability Centre, School of Life Sciences, University of Sussex, Falmer, BN1 9RQ, UK.
Matthew
M
Day
MRC Genome Damage and Stability Centre, School of Life Sciences, University of Sussex, Falmer, BN1 9RQ, UK.
Antony M
AM
Carr
Cancer Research UK DNA Repair Enzymes Group, Genome Damage and Stability Centre, School of Life Sciences, University of Sussex, Falmer, BN1 9RQ, UK.
Antony W
AW
Oliver
National Institute of Biological Sciences, 7 Science Park Road, ZGC Life Science Park, Beijing 102206, China.
Li-Lin
LL
Du
Cancer Research UK DNA Repair Enzymes Group, Genome Damage and Stability Centre, School of Life Sciences, University of Sussex, Falmer, BN1 9RQ, UK.
Laurence H
LH
Pearl
D001665:Binding Sites; D018797:Cell Cycle Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018360:Crystallography, X-Ray; D004249:DNA Damage / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation / Q000235:genetics*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012568:Schizosaccharomyces; D029702:Schizosaccharomyces pombe Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011503:Transglutaminases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D018797:Cell Cycle Proteins; C109867:Crb2 protein, S pombe; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; C082463:RAD4 protein, S pombe; D029702:Schizosaccharomyces pombe Proteins; C069930:rad9 protein; D011503:Transglutaminases