Structural and functional analysis of the Crb2-BRCT2 domain reveals distinct roles in checkpoint signaling and DNA damage repair.
Schizosaccharomyces pombe Crb2 is a checkpoint mediator required for the cellular response to DNA damage. Like human 53BP1 and Saccharomyces cerevisiae Rad9 it contains Tudor(2) and BRCT(2) domains. Crb2-Tudor(2) domain interacts with methylated H4K20 and is required for recruitment to DNA dsDNA breaks. The BRCT(2) domain is required for dimerization, but its precise role in DNA damage repair and checkpoint signaling is unclear. The crystal structure of the Crb2-BRCT(2) domain, alone and in complex with a phosphorylated H2A.1 peptide, reveals the structural basis for dimerization and direct interaction with gamma-H2A.1 in ionizing radiation-induced foci (IRIF). Mutational analysis in vitro confirms the functional role of key residues and allows the generation of mutants in which dimerization and phosphopeptide binding are separately disrupted. Phenotypic analysis of these in vivo reveals distinct roles in the DNA damage response. Dimerization mutants are genotoxin sensitive and defective in checkpoint signaling, Chk1 phosphorylation, and Crb2 IRIF formation, while phosphopeptide-binding mutants are only slightly sensitive to IR, have extended checkpoint delays, phosphorylate Chk1, and form Crb2 IRIF. However, disrupting phosphopeptide binding slows formation of ssDNA-binding protein (Rpa1/Rad11) foci and reduces levels of Rad22(Rad52) recombination foci, indicating a DNA repair defect.
Kilkenny
Mairi L
ML
CR-UK DNA Repair Enzymes Group, Section of Structural Biology, The Institute of Cancer Research, Chelsea, London SW3 6JB, United Kingdon.
Doré
Andrew S
AS
Roe
S Mark
SM
Nestoras
Konstantinos
K
Ho
Jenny C Y
JC
Watts
Felicity Z
FZ
Pearl
Laurence H
LH
D000595:Amino Acid Sequence; D002166:Camptothecin / Q000494:pharmacology; D002453:Cell Cycle / Q000235:genetics / Q000502:physiology*; D018797:Cell Cycle Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D018360:Crystallography, X-Ray; D004249:DNA Damage / Q000187:drug effects / Q000528:radiation effects; D004260:DNA Repair; D019281:Dimerization; D004307:Dose-Response Relationship, Radiation; D006639:Histidine / Q000378:metabolism; D006918:Hydroxyurea / Q000494:pharmacology; D007259:Infrared Rays; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D009687:Nuclear Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D012568:Schizosaccharomyces / Q000235:genetics* / Q000378:metabolism*; D029702:Schizosaccharomyces pombe Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction / Q000502:physiology; D014466:Ultraviolet Rays
D018797:Cell Cycle Proteins; C109867:Crb2 protein, S pombe; D009687:Nuclear Proteins; D029702:Schizosaccharomyces pombe Proteins; D006639:Histidine; D006918:Hydroxyurea; D002166:Camptothecin