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. 2015 Feb 4;43(4):2138–2151. doi: 10.1093/nar/gkv082

Figure 3.

Figure 3.

Down-regulation of Ku genes triggers the DNA damage response. (A) Cell images of UMP111 (wt), UCS43 (uku70nar1) and UCS42 (uku80nar1) strains carrying a Chk1-3GFP fusion grown for 8 h in permissive (MMD) and restrictive (YPD) conditions. All cells were shown at the same magnification (Bar: 15 μm). (B) Quantification of the cellular response to DNA damage as the percentage of cells carrying a clear nuclear GFP fluorescence signal. (C) In vivo phosphorylation of Chk1 in response to Ku70 protein depletion. UMP124 (wt) and UMP231 (uku70nar1) cells carrying an endogenous Chk1-3MYC allele were incubated in YPD (restrictive conditions) for the indicated time. Protein extracts were immunoprecipitated with a commercial anti-MYC antibody and the immunoprecipitates were subjected to SDS-PAGE and immunoblotting using anti-MYC antibody. Phosphorylated Chk1 migrated as a smear (brackets) most likely because the presence of multiple species of phosphorylated protein. (D) Levels of Cdk1 inhibitory phosphorylation upon Ku70 depletion. Protein extracts from the indicated strains (FB1, wt; UCS33, uku70nar1; UCS35, uku70nar1 chk1Δ; UMP218, uku70nar1 mre11Δ) that were grown in restrictive conditions (YPD) for 8 h were separated by SDS-PAGE. Immunoblots were probed successively with an antibody that recognizes phosphorylated Cdk1 (α-Cdc2-Y15P) and anti-PSTAIRE. (E) Levels of Cdk1 phosphorylation were determined by quantifying the level of antibody signal using a ChemiDoc unit (Bio-Rad). Signal from the phosphopeptide-specific antibodies was normalized to the amount of phosphorylation in the control strain (FB1). Differences in loading of samples were corrected by dividing each phosphopeptide-specific antibody signal by the Cdk1 (α-PSTAIRE) antibody signal. Two independent experiments were used to calculate the mean and s.d. (F) Growth of conditional strains in solid medium. Serial 10-fold dilutions of UCS33 (uku70nar1), UMP221 (uku70nar1 Pscp:cdk1), UMP222 (uku70nar1 Pscp:cdk1AF) cultures were applied to solid rich medium (YPD) and minimal medium with nitrate (MMD). YPD plates were incubated for 2 days at 28°C.