Volume 11, Issue 3, Pages 827-835 (March 2003) PP2C Phosphatases Ptc2 and Ptc3 Are Required for DNA Checkpoint Inactivation after a Double-Strand Break  Christophe Leroy, Sang Eun Lee, Moreshwar B. Vaze, Françoise Ochsenbien, Raphaël Guerois, James E. Haber, Marie-Claude Marsolier-Kergoat  Molecular Cell  Volume 11, Issue 3, Pages 827-835 (March 2003) DOI: 10.1016/S1097-2765(03)00058-3 Copyright © 2003 Cell Press Terms and Conditions

Figure 1 Ptc2 Affects Cell Cycle Resumption and the Phosphorylation of Rad53 and Rad9 after Formation of HO Cuts (A and B) Strains Lev348 (wild-type), MCM302 (ptc2Δ), Lev370 (yku80Δ), and YSL85 (cdc5-ad) with or without the pGAL-PTC2 plasmid were tested for their proficiency at adaptation with the microcolony assay. (C and D) Lev348 (wild-type) and MCM302 (ptc2Δ) cells were grown overnight in raffinose medium, and HO expression was induced by galactose at t = 0 hr. Aliquots were taken at the indicated times after addition of galactose and analyzed by FACS and by Western blotting. (E) Lev358 (RAD9-13myc::kanMX) cells harboring a deletion of PTC2 or the pGAL-PTC2 plasmid were treated as described above, and Rad9 phosphorylation was analyzed by Western blotting. (F) Deletion of PTC2 does not affect 5′ to 3′ DNA degradation. The kinetics of 5′ to 3′ resection after HO cleavage was analyzed in wild-type (Lev348 and MCM302, squares) and yku80Δ (Lev370 and MCM304, circles) cells harboring either the wild-type PTC2 allele (closed symbols) or a deletion of PTC2 (open symbols). Molecular Cell 2003 11, 827-835DOI: (10.1016/S1097-2765(03)00058-3) Copyright © 2003 Cell Press Terms and Conditions

Figure 2 Ptc3 Affects Adaptation to HO-Induced DSB (A and B) Strains Lev348 (wild-type), MCM302 (ptc2Δ), MCM296 (ptc3Δ), and MCM298 (ptc2Δ ptc3Δ) were grown on raffinose medium overnight, and HO expression was induced by galactose at t = 0 hr. Four hours after HO induction, a cell aliquot was sonicated and plated to galactose medium for microcolony assay (A). Aliquots were also retrieved at the indicated times after HO induction and analyzed by FACS (B). (C) Ptc2 phosphatase activity is required for adaptation to HO-induced DSB. MCM298 cells (ptc2Δ ptc3Δ) transformed with plasmids carrying PTC2, ptc2D234A, or ptc2E37A/D38A under the control of a galactose-inducible promoter were treated as described above for the microcolony assay. Molecular Cell 2003 11, 827-835DOI: (10.1016/S1097-2765(03)00058-3) Copyright © 2003 Cell Press Terms and Conditions

Figure 3 Ptc2 and Ptc3 Are Required for Recovery after HO-Induced DSB YMV2 cells deleted for the PTC2 and/or PTC3 genes (YMV56, MCM390, and MCM392) were grown on raffinose medium overnight, and HO expression was induced by the addition of galactose at t = 0 hr. At t = 4 hr, a cell aliquot was sonicated, plated to galactose medium, and incubated overnight at 30°C to monitor cell viability (A). Aliquots were taken at the indicated times and analyzed by FACS (B) and Western blots (C). The efficiency of repair by single-strand annealing is shown in Southern blots (D), as in Vaze et al. (2002). Molecular Cell 2003 11, 827-835DOI: (10.1016/S1097-2765(03)00058-3) Copyright © 2003 Cell Press Terms and Conditions

Figure 4 Ptc2 Interacts Specifically with Rad53 FHA1 Domain (A and B) pGBT9/PTC2 and pPC97 RAD9-FLAG expressing the Gal4BD-Ptc2 and Gal4BD-Rad9 fusion proteins were introduced into the Y190 tester strain along with the pACTIIst/RAD53(1-821), pACTIIst/RAD53(1-164), pACTIIst/RAD53(156-573), and pACTIIst/RAD53(564-821) plasmids harboring the sequence of Gal4 activating domain fused to the sequences encoding full-length Rad53, or encompassing Rad53 FHA1, kinase (KD), or FHA2 domains, respectively. In (B), the pACTIIst/RAD53 constructs contain the sequence encoding Rad53(7-164). The two-hybrid interaction is revealed by growth on plates lacking histidine (−His) complemented with 25 mM 3-amino-triazole (3AT) and by X-gal staining. (C) Ptc2 phosphorylation is required for its interaction with Rad53. Extracts (1 mg protein) from E. coli cells expressing a histidine-tagged, full-length version of Rad53 (lanes 1 and 3) or from yeast MCM298 (ptc2Δ ptc3Δ) cells (lanes 2 and 4) were incubated with equal amounts (100 μg) of immobilized GST-Ptc2, either mock-treated or treated with casein kinase II (CKII), as indicated. GST-Ptc2 bound proteins were analyzed by Western blotting. (D) Phosphorylated Ptc2 binds to Rad53 irrespective of its phosphorylation state. Lev348 cells were grown on raffinose medium overnight, and HO expression was induced by the addition of galactose at t = 0 hr. Extracts (0.5 mg protein) prepared from aliquots taken at the indicated times were treated as described in (C). Molecular Cell 2003 11, 827-835DOI: (10.1016/S1097-2765(03)00058-3) Copyright © 2003 Cell Press Terms and Conditions