Regulation of checkpoint kinases through dynamic interaction with Crb2

Abstract

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 BRCTdomain 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.

The Phosphorylation Network for Efficient Activation of the DNA Replication Checkpoint in Fission Yeast

“…S3C). This suggests that Rad9-Thr 225 and Hus1-Thr 47 may not be true phosphorylated residues but are required for the clamp structure or other functions (39). Consistent with this, mutation of the two sites to the phosphomimic residue glutamic acid made the cells even more sensitive (supplemental Fig.…”

“…Although the possibility of Thr 412 -dependent phosphorylation of Thr 225 in Rad9 cannot be completely ruled out by this experiment (25), we found no convincing evidence for crucial Rad3 phosphorylation in the PCNA-like domain of Rad9 and probably all PCNA-like domains in the 9-1-1 complex. We propose that the two residues Rad9-Thr 225 and Hus1-Thr 47 are structurally important for the 9-1-1 clamp complex.…”

“…We also mutated three other serines in the SQ cluster to cysteines. However, unlike Rad9-Thr 225 and Hus1-Thr 47 , cysteine substitutions of the serines in the SQ cluster in various combinations did not make the cells significantly more resistant (supplemental Fig. S5B).…”

“…We found no evidence for redundant Rad3 phosphorylation in the sensor proteins. 47 in Hus1. Phosphorylation of Rad9 has been studied before for Chk1 activation and regulation of the choice of DNA repair pathways (22,25,39).…”

“…Phosphorylation of Rad9, which recruits Cut5 (15,22,47), is not required for the phosphorylation of Mrc1 by Rad3, suggesting that activation of the sensor kinase Rad3 is not dependent on the recruitment of Cut5 and may involve an unknown mechanism. However, as we show below, Rad9 phosphorylation (and probably the recruitment of Cut5 by phosphorylated Rad9) is important for activation of the effector kinase Cds1.…”