Figure 2

Mec1 and Tel1 are required for phosphorylation of Rad9 S/TP sites after DNA damage. (A) Rad9-T474 phosphorylation after DNA damage depends on the apical checkpoint kinases Mec1 and Tel1. G1-arrested cells with indicated genotypes were treated with phleomycin, Rad9-T474 phosphorylation was visualized by immunoblotting. Strains containing the mec1Δ mutation are in sml1Δ background. Pgk1 immunoblot serves as loading control. An asterisk denotes a crossreactive band. FACS profiles in Fig. S2A. (B) Rad9-T474 phosphorylation after DNA damage is independent of checkpoint effector kinases Chk1 and Rad53. G1-arrested cells with indicated genotypes were treated with phleomycin and subjected to analysis with immunoblots as in (A). Strains containing the rad53Δ mutation are in sml1Δ background. FACS profiles in Fig. S2B. (C) Integrity of the Rad9 SCD domain is important for damage-induced Rad9 S/TP phosphorylation. Treatment and immunoblotting of WT, rad9-6AQ and rad9-S1129A strains as in (A). FACS profiles in Fig. S2C.