Figure 1

A CDK-independent, DNA damage-dependent mode of Rad9-S462 and -T474 phosphorylation and interaction with Dpb11. (A) DNA damage stimulates the Rad9-Dpb11 interaction in cell extracts. GST pulldown experiment with ^GSTDpb11-N (contains BRCT1 + 2, which is the Rad9 interaction site) and extracts from Rad9^9myc-expressing cells arrested in G1 (α-factor arrest) or M phase (nocodazole arrest) and treated with phleomycin or mock treated. FACS profiles in Fig. S1B. (B,C) Phosphorylation of Rad9-S462 and -T474 is stimulated by DNA damage in G1. (B) Rad9^3FLAG was purified from cells treated as in (A) by FLAG-IP. Phosphorylation of Rad9 S/TP sites was determined using Rad9-S462p and Rad9-T474p phosphorylation-specific antibodies. FACS profiles in Fig. S1C. (C) Cells treated as in (A) were used to prepare whole cell extract, which was probed with the Rad9-T474p phosphorylation-specific antibody. The rad9-AA strain (harbouring the S462A and T474A mutations) was used as specificity control. Pgk1 immunoblot serves as loading control. FACS profiles in Fig. S1D. (D,E) CDK inhibition does not affect damage-induced Rad9 S/TP phosphorylation. (D) 1-NM-PP1 was used to inhibit CDK in G1-arrested cdc28-as1 cells, but this did not affect Rad9-T474 phosphorylation after DNA damage. FACS profiles in Fig. S1E. (E) As in (D), but with M phase-arrested cells. 1-NM-PP1 treatment abolished T474 phosphorylation in undamaged cdc28-as1 cells, demonstrating that CDK is inhibited under these conditions. In contrast T474 is efficiently phosphorylated after phleomycin treatment, even after CDK inhibition. Pgk1 immunoblot serves as loading control. The asterisk denotes a crossreactive band. FACS profiles in Fig. S1F.