Fig 2: Mec1-Rad53-Dun1–dependent regulation of S. cerevisiae ribonucleotide reductase.The activated Dun1 kinase relieves inhibition of RNR by targeting the transcriptional repressor Crt1(Rfx1) and protein inhibitors Sml1 and Dif1.

Fig 3: 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(Δ1-975), 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).

Fig 4: Depletion of Rnr1 in BY4741 background mildly triggers the S-phase checkpoint, greatly reduces dNTP levels and significantly prolongs S-phase. (A) Cartoon depicting the gene RNR1 under the control of the inducible PGAL1/10 promoter. The promoter is induced in galactose plus sucrose (gal + suc) media and inhibited in glucose (glu) media. Rnr1 is epitope-tagged with 3x hemagglutinin (3HA) at its N-terminus. (B) Rnr3 protein is mildly expressed in single mutant PGAL:3HA-RNR1 depleted of Rnr1. Strains PGAL:3HA-RNR1 and PGAL:3HA-RNR1 crt1? were grown at 30°C in liquid minimal medium lacking histidine with 2% galactose and 2% sucrose. To trigger Rnr1 depletion, cells at OD600 ~0.2 were transferred to liquid minimal medium lacking histidine with 2% glucose. Cells were harvested before transfer (0 h) and 2, 4 and 6 h after transfer to glucose-containing medium (see also Materials and Methods). Strain WT was grown in rich YEPD (2% glucose) medium at 30°C, in absence of drugs (i.e. unperturbed conditions), or for 3 h in presence of either 200 mM HU (labeled +HU) or 0.03% MMS (labeled +MMS) (see also Materials and Methods). Strain crt1? was grown in rich YEPD (2% glucose) medium at 30°C. Note that all cell samples were harvested in exponential phase. Total proteins were separated on a 4–20% SDS-polyacrylamide gel and then electro-blotted. The filter was stained with Ponceau Red (bottom sub-panel). The same filter was probed separately with antibodies against HA tag (3HA-Rnr1), Rnr3, PGK1 and Sml1. Relevant protein molecular weights (KDa) are indicated at the left-hand. For the ease of comparison, each well is allocated a unique Latin letter, which is repeated in each sub-panel. The length of Rnr1 depletion in hours (hr) is indicated above the wells a-d and e-h. One representative experiment is shown of at least three independent ones. (C) dNTP levels, particularly dGTPs, are greatly decreased in single mutant PGAL:3HA-RNR1 depleted of Rnr1. The WT strain was cultured in rich YEPD medium (2% glucose) at 30°C and harvested at OD600 ~0.4. The single mutant PGAL:3HA-RNR1 was cultured as explained in (B) and harvested 6 h after transfer to glucose-containing media at OD600 ~0.4. dNTP levels were normalized to rNTP levels and values were adjusted to the total number of cells used for the preparation. Error bars reflect S.E.M. of 2 independent repeats. Symbols on the organigram: + and – indicate that Rnr1 is present or absent, respectively. For the ease of comparison, WT strain and single mutant PGAL:3HA-RNR1 are also represented in Supplementary Figure S3A (see also Supplementary Table S4). (D) Rad53 is mildly phosphorylated in single mutant PGAL:3HA-RNR1 depleted of Rnr1. Strains and growth conditions are as in (B). Total proteins were separated on a 6.5% SDS-polyacrylamide gel. The Filter was probed with antibody against Rad53 (P-Rad53 represents the phosphorylated form of Rad53). For other details, see (B). (E) Cells depleted of Rnr1 accumulate significantly in S-phase in the presence of Crt1. Flow cytometry (FACS) histograms of cells from strains PGAL:3HA-RNR1 and PGAL:3HA-RNR1 crt1?, before (0 h), and 2, 4 and 6 h after transfer to glucose medium. One representative experiment is shown of at least three independent ones. Note that cell samples from the same cultures were used for FACS, western-blotting in (B) and (D), and RT-qPCR in Supplementary Figure S2. (F) Cells depleted of Rnr1 grow much slower than the WT strain, but their growth is fully restored in the absence of Crt1. Drop test growth assays of strain WT, strains deleted of the gene CRT1, SML1, or DUN1, and strains carrying PGAL:3HA-RNR1 without gene deletion (labeled “none"), or with deletion of the gene CRT1, SML1 or DUN1. Cells were pre-grown in YPGS (2% galactose and 1% sucrose) liquid medium overnight. Serial dilutions were plated on YEPD (2% glucose) and YPGS solid media. Plates were incubated at 25°C. Photographs were taken at the indicated number of days (d). For the ease of comparison, a unique Latin alphabet letter is allocated for each row. ‘Glu’ stands for glucose and ‘Gal + Suc’ stands for galactose plus sucrose. The horizontal line across the images is included for clarity. See Supplementary Table S1 for the list of strains. One representative experiment is shown of at least three independent ones.

Fig 5: NSNM exposure leads to degradation of Sml1 without activating RNR genes or Rad52 foci formation.(A) Whole cell extracts were prepared by TCA extraction method and samples were subjected to western blot analysis with indicated antibodies. Blotting with antibodies against Tbp and Rap1 or Ponceau S staining of representative blot were used as loading controls. (B) Sml1-YFP tagged strain were treated with NSNM (100 μM) for 3 h. For control same strains were treated with MMS (0.03%), images were taken as described in materials and methods. (C and D) Growth Assay; wild type and different mutant yeast strains were spotted onto control SCA (DMSO) plates or SCA plates containing 100, 200 or 300 μM NSNM. All plates were incubated at 30°C for 72 h and photographed. (E) NSNM exposure does not lead to Rad52 foci formation. Rad52-YFP tagged yeast strain was treated with 100 μM NSNM for 3 h, same strain was treated with 0.03% MMS (control) prior to visualization of foci by confocal microscopy.