Dpb11 function is required for the Dot1-independent checkpoint activation pathway in response to UV irradiation. (A) UV survival assay. Strains K699 (WT), YMIC4E8 (rad9Δ), YFP20 (dpb11-1), YFL234 (dot1Δ), and YMAG6 (dot1Δ dpb11-1) were grown overnight to stationary phase and then diluted and plated on YPD plates, which were irradiated with the indicated UV doses. Survival was assayed by determining the number of colonies that formed on the plates after 3 days. (B) UV checkpoint assay. Yeast strains K699 (WT), YFP20 (dpb11-1), YFL234 (dot1Δ), YMAG6 (dot1Δ dpb11-1), and YMIC4F6 (mec3Δ rad9Δ) were synchronized in M phase with nocodazole, UV irradiated at 40 J/m², and released in YPD plus α-factor. Every 15 min, samples were taken and scored for the presence of binucleated cells. (C) Analysis of the phosphorylation of checkpoint factors. WT and dpb11-1, dot1Δ, and dot1Δ dpb11-1 mutant cells carrying Ddc2-HA and Rad9-myc were arrested with nocodazole and either mock or UV irradiated (75 J/m²); 30 min after irradiation, Ddc2, Rad9, and Rad53 phosphorylations were analyzed by SDS-PAGE and Western blotting. (D) Strain K699 (WT), YFP20 (dpb11-1), YFL234 (dot1Δ), and YMAG6 (dot1Δ dpb11-1) cells were cultured to mid-log phase, arrested in G₁ with 20 μg/ml α-factor, and either mock or UV irradiated (75 J/m²); 30 min after irradiation, Rad53 phosphorylation was analyzed by SDS-PAGE and Western blotting.

Dpb11 and Dot1 cooperate in the checkpoint activation pathway in response to DSB-inducing agents. (A) DSB survival assay. Strains K699 (WT), YFP20 (dpb11-1), YFL234 (dot1Δ), and YMAG6 (dot1Δ dpb11-1) were grown to mid-log phase and then treated for 30 min with Zeocin at the indicated concentrations. Serial dilutions were then spotted onto YPD plates and incubated for 3 days. (B) Analysis of checkpoint activation after treatment with DSB-inducing agents. Strains K699 (WT), YFP20 (dpb11-1), YFL234 (dot1Δ), and YMAG6 (dot1Δ dpb11-1) were cultured to mid-log phase and either mock treated or treated with 100 μg/ml Zeocin. At 15 and 45 min after drug addition, samples were taken and processed for the analysis of Rad53 phosphorylation.

UV-induced Ddc1 phosphorylation depends upon the presence of Mec1 kinase consensus sites. (A) Outline of the Cdc28 (gray) and Mec1 (black) putative phosphorylation target sites in Ddc1. Cdc28 and Mec1 target sites were mutated to alanine in ddc1-M3 and ddc1-M8 mutant strains, respectively. The ddc1-M11 mutant strain contains a combination of all of these mutations. (B) Strains YLDN25 (WT), YLDN17 (ddc1-M3), YLDN23 (ddc1-M8), and YLDN24 (ddc1-M11) were arrested with nocodazole and either UV irradiated (75 J/m²) or mock treated. Protein extracts prepared immediately after UV treatment were separated by SDS-PAGE and analyzed with anti-Ddc1 antibodies.

Phosphorylation of Ddc1 and DOT1 are required for the establishment of an effective UV response. (A) Strains YLDN25 (WT), YLDN17 (ddc1-M3), YLDN23 (ddc1-M8), YLDN24 (ddc1-M11) YFP27 (dot1Δ), YFP28 (dot1Δ ddc1-M3), YFP29 (dot1Δddc1-M8), and YFP30 (dot1Δ ddc1-M11) were arrested with nocodazole and either UV irradiated (75 J/m²) or mock treated. Rad9 and Rad53 phosphorylations were analyzed 30 min after irradiation. A protein extract from YMIC4E8 (rad9Δ) was loaded onto the same gel in order to identify the anti-Rad9 cross-reacting band, indicated by an asterisk. (B) In order to measure sensitivity to UV irradiation, 10-fold serial dilutions of overnight cultures of the strains from panel A and strain YFP152 (ddc1Δ) were spotted onto plates, which were then either mock or UV irradiated. Images of the plates were taken after 3 days to assess cell survival.

Phosphorylation of Ddc1 T602 is required for Rad53 and Rad9 phosphorylation in the absence of DOT1. (A) Strains YLDN25 (WT), YLDN9 (ddc1-T602A), YFP37 (dot1Δ ddc1-T602A), and YFP29 (dot1Δ ddc1-M8) were arrested with nocodazole and subjected to UV irradiation (75 J/m²) or mock treated. Rad53 phosphorylation was analyzed 30 min after UV treatment. A protein extract from strain YMIC4E8 (rad9Δ) was loaded onto the same gel in order to identify the anti-Rad9 cross-reacting band, indicated by an asterisk. (B) Strains YFP37 (dot1Δ ddc1-T602A), YLDN25 (WT), YFP148 (ddc1-T602S), YFP27(dot1Δ), and YFP149 (dot1Δ ddc1-T602S) were arrested in M phase with nocodazole and either UV irradiated (75 J/m²) or mock treated. Rad53 phosphorylation was analyzed 30 min after treatment.

ddc1-T602A and dpb11-1 mutations are epistatic for UV sensitivity and effect on Rad53 phosphorylation. (A) Strain YFP63 (WT), YFP64 (ddc1-T602A), YFP65 (dpb11-1), and YFP66 (dpb11-1 ddc1-T602A) cells were arrested with nocodazole and UV irradiated. Rad53 phosphorylation was assayed 30 min after treatment. (B) Strains in panel A, YFP27 (dot1Δ), YFP142 (dot1Δ dpb11-1), YFP37 (dot1Δ ddc1-T602A), and YFP144 (dot1Δ ddc1-T602A dpb11-1) were grown overnight to stationary phase, and then 10-fold dilutions were spotted onto appropriate plates and either mock treated or UV irradiated with the indicated dosages. Images were taken after 3 days to measure cell survival.

UV-induced Dpb11 phosphorylation is mediated by Ddc1-T602 and requires Mec1 kinase. (A) Strains YFP38 (WT), YFP48/3a (mec1-1), YFP49/1d (rad53Δ), YFP55/6c (ddc1Δ), YFP63 (DDC1), and YFP64 (ddc1-T602A), all expressing a myc-tagged Dpb11 protein, were blocked in nocodazole and UV irradiated (75 J/m²). Dpb11 phosphorylation was assessed 30 min after UV irradiation by SDS-PAGE and Western blotting. (B) The indicated strains were arrested in either α-factor (G₁) or nocodazole (M) and UV treated. Protein extracts prepared immediately (T0) or 30 min (T30) after UV irradiation were separated on Phos tag-conjugated acrylamide gels as described in Materials and Methods. (C) Overexposure of the T30 samples from panel B.

The interaction between Dpb11 and Ddc1 requires a functional Mec1 kinase. Plasmids pFP1 (pJG4-5-DPB11) and pFP2 (pEG202-DDC1) were cotransformed with pSH18-34, a β-galactosidase reporter plasmid, in either MEC1 or mec1-1 mutant yeast cells. A similar strategy was adopted for pFP4 (pEG202-ddc1-C), which carries only the C-terminal fragment (nucleotides 309 to 612) of DDC1, containing the 11 putative Mec1 phosphorylation target sites and for pFP10 (pEG202-ddc1M8). To assess two-hybrid interaction, these strains were patched onto 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-Gal) plates containing either raffinose (Raf; Dpb11 prey repressed) or galactose (Gal; Dpb11 prey expressed) as a carbon source. After 3 days, the plates were analyzed. The strains in panel A are YFP50 (MEC1, top), YFP52 (MEC1, bottom), YFP113 (mec1-1, top), and YFP114 (mec1-1, bottom). A positive control in the mec1-1 mutant strain (p53 versus large T antigen [TAg]) was also used. The strains in panel B are, from left to right, YFP50, YFP86, YFP54, and YFP153.

Dpb11 is required for the full activation of Mec1. (A) Protein extracts from strain YMAG78/4B (dpb11^td) were prepared under different conditions to assess the presence of the Dbp11-degron protein. Cells were cultured at 28°C in YP plus raffinose (Mock Unind.) and then shifted to the degradation medium at 37°C (Mock Induced), UV irradiated, and shifted back to 25°C (T0, sample taken immediately; T30, sample taken 30 min later). The presence of Dpb11-degron was assessed by using anti-HA antibodies. (B) The experiment was repeated under the same conditions in parallel with strains YMAG82/15a (DPB11) and YMAG78/4B (dpb11^td). Ddc2 hyperphosphorylation was monitored by SDS-PAGE and Western blotting. CTRL, control.

Possible model of Dpb11 function in the UV-induced DNA damage checkpoint. Dpb11 is recruited to sites of DNA damage through the interaction of its C-terminal BRCTs with the 9-1-1 subunit Ddc1, phosphorylated by Mec1 on T602. Once recruited, it plays a double role in checkpoint activation. First, it enhances Mec1 kinase activity, and second, in G₂/M, it participates with H3-K79 in Rad9 recruitment, likely through an interaction of its N-terminal BRCTs with a Cdc28-phosphorylated site on Rad9. Full Mec1 activity and tight Rad9 recruitment allow rapid and full phosphorylation of Rad53, which correlates with checkpoint activation.