Anti-Sml1 | Suppressor of Mec1 lethality

Deletion of IXR1 leads to increased Rnr3 and Rnr4 levels and decreased Sml1 levels.(A) Western blot analysis of Rnr3-HA and Rnr4 levels in wild-type (AC447-2A), ixr1-S366F (TOY619), and ixr1? (TOY621) strains before and after 2 hours treatment with 0.2 mg/L 4-nitroquinoline 1-oxide (4-NQO), 200 mM HU, or 0.02% methyl methanesulfonate (MMS). Rnr3 and Rnr4 levels were quantified in relative units (RU, levels of Rnr3 or Rnr4 divided by the levels of tubulin in corresponding sample) as described in Materials and Methods. ND – not detected. (B) Western blot analysis of Rnr2 levels in wild-type (AC447-2A) and ixr1? (TOY621) strains before treatment and after 2 hours treatment with 0.2 mg/L 4-NQO, 0.02% MMS, or 200 mM HU. (C) Western blot analysis of Sml1 levels in wild-type (W1588-4C) and ixr1? (TOY736) strains before treatment and after 2 hours treatment with 0.02% MMS. (D) Western blot analysis of Rad53 phosphorylation status in wild-type (W1588-4C) and ixr1? (TOY736) strains before treatment and after 2 hours treatment with 0.02% MMS. (E) Deletion of RAD53 but not of SML1 or DUN1 abolishes the upregulation of Rnr3 and Rnr4 levels in ixr1?. Western blot analysis of Rnr3-HA and Rnr4 levels. The following strains were analyzed: wt (AC447-2A), ixr1? (TOY732), ixr1? sml1? (TOY778), ixr1? dun1? sml1? (TOY772), and ixr1? rad53? sml1? (TOY781).

Curcumin induces iron starvation and Sml1p degradation.A & B) RT-PCR Analysis; Logarithmically grown wild-type (1588-4C) cells in standard SC liquid media were treated with either DMSO or curcumin (400 ?M) for 3 hr. Total RNA were extracted and reverse transcribed to cDNA. Semi-quantitative PCR analysis was performed to assess the levels of FRE1, FET3, ACO1, RNR1/2/3/4, HUG1 and ACT1 transcripts. C) Wild-type (1588-4C) cells were cultured up to log phase and treated with either DMSO or curcumin (400 ?M) for 3 hr in triplicate. Whole cell extracts were prepared by TCA extraction method and samples were subjected to western blot anlaysis using antibodies against Sml1, Rad53, Tbp and Rap1. D) Wild-type (1588-4C) cells were grown up to log phase, cultures of cells were divided equally and pre-incubated with either 1 mM PMSF or 100 mM MG132 for 90 min and then treated with curcumin (400 ?M) for 3 hr. In addition, some cells were treated with iron (100 ?M) and rapamycin (50 ng/ml) in presence of curcumin (400 ?M) for 3 hr. Sml1 protein levels were analyzed by western blotting and cellular levels of Rap1, Tbp and Gapdh were used as loading controls.

Curcumin induces iron starvation and Sml1p degradation.A & B) RT-PCR Analysis; Logarithmically grown wild-type (1588-4C) cells in standard SC liquid media were treated with either DMSO or curcumin (400 ?M) for 3 hr. Total RNA were extracted and reverse transcribed to cDNA. Semi-quantitative PCR analysis was performed to assess the levels of FRE1, FET3, ACO1, RNR1/2/3/4, HUG1 and ACT1 transcripts. C) Wild-type (1588-4C) cells were cultured up to log phase and treated with either DMSO or curcumin (400 ?M) for 3 hr in triplicate. Whole cell extracts were prepared by TCA extraction method and samples were subjected to western blot anlaysis using antibodies against Sml1, Rad53, Tbp and Rap1. D) Wild-type (1588-4C) cells were grown up to log phase, cultures of cells were divided equally and pre-incubated with either 1 mM PMSF or 100 mM MG132 for 90 min and then treated with curcumin (400 ?M) for 3 hr. In addition, some cells were treated with iron (100 ?M) and rapamycin (50 ng/ml) in presence of curcumin (400 ?M) for 3 hr. Sml1 protein levels were analyzed by western blotting and cellular levels of Rap1, Tbp and Gapdh were used as loading controls.

Curcumin induces iron starvation and Sml1p degradation.A & B) RT-PCR Analysis; Logarithmically grown wild-type (1588-4C) cells in standard SC liquid media were treated with either DMSO or curcumin (400 ?M) for 3 hr. Total RNA were extracted and reverse transcribed to cDNA. Semi-quantitative PCR analysis was performed to assess the levels of FRE1, FET3, ACO1, RNR1/2/3/4, HUG1 and ACT1 transcripts. C) Wild-type (1588-4C) cells were cultured up to log phase and treated with either DMSO or curcumin (400 ?M) for 3 hr in triplicate. Whole cell extracts were prepared by TCA extraction method and samples were subjected to western blot anlaysis using antibodies against Sml1, Rad53, Tbp and Rap1. D) Wild-type (1588-4C) cells were grown up to log phase, cultures of cells were divided equally and pre-incubated with either 1 mM PMSF or 100 mM MG132 for 90 min and then treated with curcumin (400 ?M) for 3 hr. In addition, some cells were treated with iron (100 ?M) and rapamycin (50 ng/ml) in presence of curcumin (400 ?M) for 3 hr. Sml1 protein levels were analyzed by western blotting and cellular levels of Rap1, Tbp and Gapdh were used as loading controls.

Sen1 is required to maintain basal levels of RNR1 through transcription.A) Untreated WT, Sen1-1 and Sen1-2 cells were grown in liquid YPD to mid-log and used for making whole cell protein extracts by TCA precipitation. Samples were analyzed by western blotting with Rnr1, Rnr2, Sml1, Rap1 and Gapdh antibodies. CBBR stands for Coomassie Brilliant Blue R.B) Semi-quantitative analysis of RNR1, HUG1 genes; logarithmically grown Sen1 strains (WT, sen1-1, Sen1-2) in YPD were left untreated for 3 hr. Total RNA was extracted and reverse transcribed to cDNA. Semi-quantitative PCR analysis was performed to assess the levels of RNR1, HUG1 and ACT1 transcripts. The expected sizes of the PCR product for RNR1, HUG1 and ACT1 are 219, 190, and 520 bp respectively. Two repeats of RT-PCR amplifications are shown here.

Mutations in Sen1p cause sensitivity to DNA damaging agents.A) Growth Assay; Sen1-TAP (Wild-type) and the different mutants of Sen1 [(Sen1(G1747D), Sen1-2(?1-975), Sen1(K128E), Sen1(R302W)] were grown up to log-phase. 3 µl of each undiluted and 10-fold serially diluted cultures were spotted on control YPDA or YPDA plates containing 0.02% MMS, 100 mM HU, 2 mM H2O2 and one set exposed to UV of 100 J/m2. All plates were incubated at 30°C for 3 days and photographed. B) Growth of Sen1 strains was recorded in the presence of different agents like MMS (0.03%), Hydroxy Urea (0.2 M), H2O2 (2 mM) and analyzed in comparison to untreated (control) cells. The average cell density (OD600) of two independent isolates of Sen1 strains with error bars was plotted against different time points (0, 2, 4, 8 and 12 hr). C) Viability Assay; Sen1-TAP (Wt), Sen1-1 and sen1-2 strains were cultured up to mid log phase and treated with 0.03% MMS for 12 hr and cells were stained with 0.3% methylene blue for checking viability. Untreated and heat killed cells were taken as negative and positive controls respectively; viability was observed under light microscope (40X) and photographed. D) MMS (0.03%) treated and untreated Sen1-TAP (WT), Sen1-1, Sen1-2, Sen1(K128E) and Sen1(R302W) cells were used for making whole cell protein extracts by TCA precipitation, samples were analyzed by western blotting. Rnr1, Rnr2, Rnr3 antibodies were used to examine the expression of ribonucleotide reductase proteins and Sml1. E) Quantification of Rnr proteins (Rnr1, 2 & 3) level in Sen1 strains from Fig.4A before and after 3 h of MMS (0.03%) treatment. ‘Lamin A’ was used as the internal control (refer materials and methods). Error bars represent standard error of the mean (SEM).

The Crt1/Dun1 pathway of the replication stress checkpoint is activated in dpb2-103 cells under MMS or HU treatment.(A) Western-Blot detection of Sml1 degradation. Extracts from WT or dpb2-103 yeast cells treated with 0,05% MMS were analyzed. (B) Quantitative RT-PCR analysis of RNR3 and HUG1 transcripts in yeast cells released from G1-arrest in YNBD (green) or YNBD with 200 mM HU (red). Transcript levels were analyzed after 120 and 240 minutes of growth and normnalized to wild-type G1-synchronized cells.