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. 2012 Jul 5;8(7):e1002817. doi: 10.1371/journal.pgen.1002817

Figure 2. Two conserved SQ/TQ motifs in the N-terminal region of Crb2 are essential for Chk1 recruitment and activation.

Figure 2

(A) Sequence alignment of S. pombe Crb2 and its orthologs from three other fission yeast species revealed two conserved neighboring SQ/TQ motifs in the N-terminal region of Crb2. The positions of the two motifs in S. pombe Crb2 are labeled on top. (B) Mutations in Crb2 SQ/TQ cluster resulted in DNA damage hypersensitivity. Fivefold serial dilutions of cells were spotted on YES plates and incubated at 30°C. Photos were taken 2 d later for untreated, UV-treated, IR-treated and CPT-containing plates. The HU-containing plates were photographed 3 d later. Strains used were LD195, LD346, DY377, DY369, DY370 and DY371. (C) DNA damage-induced Chk1 phosphorylation is defective in Crb2 SQ/TQ cluster mutants. Cells were untreated or treated with 20 µM CPT for 2 h. Cell lysates were separated on SDS-PAGE and probed with an anti-Myc antibody by immunoblotting. Strains used were DY377, LD195, DY369, DY370 and DY371. (D) Mutations in Crb2 SQ/TQ cluster diminished Chk1 foci but not Crb2 foci. Cells expressing Chk1-GFP and CFP-Crb2 were challenged with S-phase IR treatment as in Figure 1A and examined by fluorescence microscopy. Arrows indicate dim Chk1 foci in crb2-T73A and crb2-S80A cells. Strains used were DY6503, DY6504, DY6505 and DY6506. Bar, 5 µm.