ctk mutants are sensitive to HU and UV irradiation. Wild-type (WT), ctk1Δ, ctk1Δ/YCp22CTK1-HA, ctk2Δ, ctk2Δ/YCp22CTK2-HA, ctk3Δ, and ctk3Δ/YCp22CTK3-Myc YPD diluted fivefold and spotted onto YPD plates. The first plate was left untreated, the second plate contained 0.1 M HU, and the third plate was irradiated with 40 J of UV-B light per m². All plates were subsequently incubated for 3 days at 30°C and photographed with a Kodak digital camera.

HU treatment increases CTD phosphorylation on Ser2. (A) Wild-type (WT) cells were treated with 0.1 M HU for the indicated times. The resulting protein extracts were resolved on SDS-polyacrylamide gels and immunoblotted with monoclonal antibodies specific for phospho-Ser2 (H5), phospho-Ser5 (H14), and unphosphorylated RNA polymerase II (8WG16). Proteins were detected by chemiluminescence. (B) Wild-type and isogenic ctk1Δ cells were treated with 0.1 M HU for the indicated times. Proteins were extracted and immunoblotted as described above.

Ctk1p is required for induction of RNR genes by HU and MMS. Wild-type (WT), ctk1Δ, and ctk1Δ/YCp22CTK1-HA cells were either left untreated (−) (lanes 1 to 3), treated with 0.1 M HU for 60 min (lanes 4 to 6), or treated with 0.03% MMS for 60 min (lanes 7 to 9). Total RNA was isolated, resolved on a formaldehyde-agarose gel, and transferred to a nylon membrane. The membrane was sequentially hybridized with ³²P-labeled probes corresponding to RNR1, RNR2, RNR3, and RPS3. The results of hybridization were visualized by autoradiography. The level of ribosomal RPS3 mRNA served as a loading control.

Transcriptional responses of wild-type (WT) and ctk1Δ cells to HU. (A) Results of five experiments (numbered on the right) are shown. Experiments 1 and 2 compared mRNA levels in wild-type cells treated with HU for 30 and 60 min, respectively, to those in untreated wild-type cells. Experiment 3 compared mRNA levels in untreated ctk1Δ cells to those in untreated wild-type cells. Experiments 4 and 5 compared mRNA levels in HU-treated ctk1Δ cells to those in HU-treated wild-type cells after 30 and 60 min of treatment, respectively. Only the 187 genes whose expression changed at least threefold in at least one experiment were included in the K-mean clustering. The color scale at the bottom represents the fold change in transcript abundance; red indicates gene induction by HU, and green indicates gene repression. (B) Genes activated by HU in wild-type cells but not in ctk1Δ cells (cluster III). Only the subset of genes with known functions is shown. Annotations for gene functions are from the Yeast Proteome Database. The full set of 187 genes and their fold changes are available from the authors.

Activation of DNA damage response genes is compromised in ctk mutants. (A) Wild-type (WT), ctk1Δ, ctk2Δ, and ctk3Δ cells were either grown under normal conditions (−) (lanes 1, 4, 7, and 10), treated with 0.03% MMS for 60 min (lanes 2, 5, 8, and 11), or treated with 0.1 M HU for 60 min (lanes 3, 6, 9, and 12). Total RNA was isolated, resolved on a formaldehyde-agarose gel, and transferred to a nylon membrane. The membrane was hybridized sequentially with ³²P-labeled probes corresponding to FLR1, GTT2, YNL134C, and RPS3. Results of hybridizations were visualized by autoradiography. (B) Wild-type and ctk1Δ mutant cells were incubated with 0.1 M HU for 0, 1, 2, 4, or 6 h. Total RNA was isolated and analyzed by Northern blot hybridization with probes corresponding to FLR1, GTT2, YNL134C, and ADH1, which served as a loading control.

HU increases phosphorylation of RNA polymerase II associated with induced genes. Wild-type (WT) and ctk1Δ cells were either left untreated (−) (lanes 1, 3, 6, and 8) or treated with 0.1 M HU for 60 min (lanes 2, 4, 7, and 9). Protein-DNA complexes were cross-linked with formaldehyde, and RNA polymerase II was immunoprecipitated from cell extracts with antibodies against phospho-Ser2 (H5) and phospho-Ser5 (H14). DNA fragments associated with RNA polymerase II were PCR amplified and labeled with ³²P. The resulting PCR products were resolved on 8% polyacrylamide gels and visualized by autoradiography. PCR mixtures contained two pairs of primers; the first pair corresponded to the transcription start site of the indicated gene, and the second pair amplified an intergenic region on chromosome V, which was a control for nonspecific binding (indicated by asterisks). The input control contained DNA isolated from whole-cell extracts.

Transcriptional response of wild-type (WT) and ctk1Δ cells to amino acid starvation. (A) Results of five experiments (numbered on the right) are shown. Experiments 1 and 2 compared mRNA levels in wild-type cells depleted of amino acids for 60 or 120 min, respectively, to those in wild-type cells maintained in a rich medium. Experiment 3 compared mRNA levels in ctk1Δ cells and wild-type cells in a rich medium. Experiments 4 and 5 compared mRNA levels in amino acid-depleted ctk1Δ cells to those in wild-type cells depleted of amino acids for 60 and 120 min, respectively. Only the 328 genes whose expression levels changed at least threefold in at least one experiment were included in the K-mean clustering. Roman numerals indicate five major gene clusters with similar expression patterns. Colors indicate fold changes in transcript abundance (see the color scale at bottom right). (B) Genes activated by amino acid starvation in wild-type cells but not in ctk1Δ cells (cluster III). Only the subset of genes strongly dependent on Ctk1p is shown. Annotations for gene functions are from the Yeast Proteome Database. The full set of 328 genes and their fold changes are available from the authors.