Figure 5. Fusing the Crb2(67–85) peptide with homologous recombination protein Rad22 can bypass Crb2 and Rad9 for Chk1 recruitment and activation.

(A) Rad22-Crb2(67–85) can recruit Chk1 to DSBs in the absence of Crb2 and Rad9, but not when the SQ/TQ motifs are mutated, or in the absence of Rad3. Cells were treated with 80 Gy IR and examined by fluorescence microscopy after 1.5 h. Strains used were DY6536, DY6534, DY6535, DY6989 and DY6538. Bar, 5 µm. (B and C) The Crb2(67–85) peptide fused with Rad22 is phosphorylated by Rad3 upon DNA damage treatment. Cells expressing Flag-tagged Rad22 or Flag-tagged Rad22-Crb2(67–85) fusion protein were untreated or treated with 20 µM CPT for 2 h. Rad22 was immunoprecipitated using anti-Flag beads, eluted by boiling in SDS loading buffer, and immunoblotted with anti-Flag or anti-phospho-SQ/TQ antibody. Strains used were DY6561, DY6562 and DY6565. (D) The Crb2(67–85) peptide fused with Rad22 interacts with Chk1 in a manner dependent on Rad3 kinase and the SQ/TQ motifs. Cells expressing Myc-tagged Chk1 were treated with 320 Gy IR. Rad22 was immunoprecipitated using anti-Flag beads, and Chk1 was detected by immunoblotting with anti-Myc antibody. Strains used were DY6561, DY6562, DY6563 and DY6565. (E) DNA damage sensitivity of crb2Δ can be partially rescued by expressing a Rad22-Crb2(67–85) fusion protein. Spot assay was performed as in Figure 2B. Strains used were DY6539, DY6540, DY6541, DY6543, DY6536, DY6534 and DY6535. (F) Rad22-Crb2(67–85) can rescue the checkpoint defect of crb2Δ in a manner dependent on the SQ/TQ motifs and Chk1, but not Rad9. The indicated mutants in a cdc25-22 background were synchronized at late G2 phase by incubating at 35.5°C for 2.5 h. Following 80 Gy IR treatment, cultures were returned to the permissive temperature of 25°C. Mitosis was monitored by staining cells with Hoechst and Calcofluor dyes. Strains used were DY6551, DY6550, DY6552, DY6553, DY6555 and DY6554.