Indicate dim Chk1 foci in crb2-T73A and crb2-S80A cells. Strains utilized had been DY6503, DY6504, DY6505 and DY6506. Bar, 5 mm. doi:10.1371/journal.pgen.1002817.gsame cells, we utilized strains Fenobucarb In Vitro expressing CFP-tagged Crb2 as the only version of Crb2. Upon IR treatment of S-phase cells, Chk1-GFP formed distinct nuclear foci in cells expressing wild-type Flufenoxuron web CFP-Crb2, and these foci absolutely overlapped with Crb2 foci (Figure 2D). The frequencies of detecting Chk1 foci dramatically decreased in cells expressing Crb2-T73A or Crb2-S80A, and only inside a compact minority of these cells (about 3 ) could we see pretty faint Chk1 foci, which had been also colocalized with Crb2 foci. No Chk1 foci may be detected in cells expressing Crb2-2AQ. In contrast for the robust reduction of Chk1 concentrate formation, the 3 mutant forms of Crb2 themselves showed robust concentrate formation like wild-type Crb2 (Figure 2D). To rule out the possibility that an effect on Crb2 recruitment was masked by the redundancy in between the two Crb2 recruitment pathways, we examined the localization of Crb2(1358)-LZ and discovered that its concentrate formation was also unaffected by the 2AQ mutations (Figure S5). As a result, the Crb2 SQ/TQ cluster will not be critical for the relocalization of Crb2 itself, but rather specifically controls the accumulation of Chk1 at DSBs. In agreement using the checkpoint defect detected by the cdc2522 block-and-release assay and also the inability to help Chk1 phosphorylation, crb2-2AQ cells didn’t elongate immediately after the S-phase IR treatment and displayed the “cut” (cell untimely torn) phenotype (Figure 2D, Figure S4B and S4C), indicating a serious defect in G2 arrest. In contrast, cells expressing Crb2-T73A or Crb2-S80A became substantially elongated, constant with their partial proficiency in Chk1 phosphorylation. We speculate that Chk1 molecules have been recruited to DSBs in crb2-T73A or crb2-S80A cells at a level high sufficient for partial checkpoint activation butPLoS Genetics | plosgenetics.orgtoo low to become clearly distinguished in the diffuse nucleoplasmic Chk1-GFP signal.Crb2 SQ/TQ cluster is phosphorylated in vivoCrb2 is known to undergo DNA damage-induced hyperphosphorylation, which manifests as mobility shift on SDS-PAGE [11,18,26]. To assess irrespective of whether the SQ/TQ cluster contributes to Crb2 phosphorylation, we examined the DNA damage-induced mobility shift of Crb2. The 2AQ mutations considerably reduced but didn’t abolish the mobility shift of Crb2 induced by IR or camptothecin (CPT) (Figure 3A). We hypothesized that other SQ/TQ motifs could contribute for the residual shift in 2AQ mutant, as you’ll find a total of 11 SQ/TQ motifs in Crb2 (Figure 3B). Hence, we mutated all remaining SQ/TQ motifs except S666, which plays a critical structural function in the Crb2 dimer interface and is unlikely to become a phosphorylation site [19,26]. The resulting 8AQ mutant didn’t show any DNA damage sensitivity (Figure S6), suggesting that T73 and S80 will be the only functionally essential ATM/ATR consensus sites. Compared to wild-type Crb2, the 8AQ mutant displayed a less pronounced IR-induced shift, which may be additional reduced by the mutations at T73 and/or S80 (Figure 3C). Residual mobility shift was still observed using the 10AQ (8AQ+2AQ) mutant, indicating that DNA damage-induced phosphorylation may well also happen on non-SQ/TQ websites. The contribution of T73 and S80 to Crb2 mobility shift suggests that they are phosphorylated in vivo just after DNA damage. Since Crb2 mobility shift is dependent onPhosphorylated Crb2.
