Regulation of checkpoint kinases through dynamic interaction with Crb2.
ATR/Rad3-like kinases promote the DNA damage checkpoint through regulating Chk1 that restrains the activation of cyclin-dependent kinases. In fission yeast, Crb2, a BRCT-domain protein that is similar to vertebrate 53BP1, plays a crucial role in establishing this checkpoint. We report here that Crb2 regulates DNA damage checkpoint through temporal and dynamic interactions with Rad3, Chk1 and replication factor Cut5. The active complex formation between Chk1 and Crb2 is regulated by Rad3 and became maximal during the checkpoint arrest. Chk1 activation seems to need two steps of interaction changes: the loss of Rad3-Chk1 and Rad3-Crb2 interactions, and the association between hyperphosphorylated forms of Chk1 and Crb2. Chk1 is the major checkpoint kinase for the arrest of DNA polymerase mutants. The in vitro assay of Chk1 showed that its activation requires the presence of Crb2 BRCT. Hyperphosphorylation of Crb2 is also dependent on its intact BRCT. Finally, we show direct interaction between Rad3 and Crb2, which is inhibitory to Rad3 activity. Hence, Crb2 is the first to interact with both Rad3 and Chk1 kinases.
Mochida
Satoru
S
Graduate School of Biostudies, Kyoto University, Kitashirakawa-Oiwakecho, Sakyo-ku, Kyoto, Japan.
Esashi
Fumiko
F
Aono
Nobuki
N
Tamai
Katsuyuki
K
O'Connell
Matthew J
MJ
Yanagida
Mitsuhiro
M
D002453:Cell Cycle; D018797:Cell Cycle Proteins / Q000737:chemistry / Q000378:metabolism* / Q000502:physiology; D000071877:Checkpoint Kinase 1; D064447:Checkpoint Kinase 2; D004249:DNA Damage; D004259:DNA-Directed DNA Polymerase / Q000235:genetics; D046911:Macromolecular Substances; D009154:Mutation; D009687:Nuclear Proteins / Q000737:chemistry / Q000378:metabolism* / Q000502:physiology; D011494:Protein Kinases / Q000378:metabolism* / Q000502:physiology; D017434:Protein Structure, Tertiary; D012568:Schizosaccharomyces / Q000201:enzymology / Q000378:metabolism* / Q000528:radiation effects; D029702:Schizosaccharomyces pombe Proteins / Q000737:chemistry / Q000378:metabolism* / Q000502:physiology; D014466:Ultraviolet Rays
D018797:Cell Cycle Proteins; C109867:Crb2 protein, S pombe; D046911:Macromolecular Substances; D009687:Nuclear Proteins; D029702:Schizosaccharomyces pombe Proteins; D011494:Protein Kinases; D064447:Checkpoint Kinase 2; D000071877:Checkpoint Kinase 1; C000605873:Chk1 protein, S pombe; C456840:rad3 protein, S pombe; D004259:DNA-Directed DNA Polymerase