(A) Isogenic ionizing radiation (IR) sensitive diploid deletion strains were grown at 30°C for two days in liquid YPD medium in 96 well plates. Serial 5-fold dilutions were made in sterile water and 2 µl aliquots were replica plated to YPD solid medium with and without the indicated doses of doxorubicin. Plates were subsequently incubated for 3 days at 30°C. Arrows indicate the direction of decreasing cell concentration. When compared to WT (row 1), defects in genes within the CCR4-NOT complex (rows 2–5) confer checkpoint adaptation functions and show intermediate sensitivity to doxorubicin. Defects in members of the RAD52 recombination repair group (rows 6–8) are required for double strand break repair and are hypersensitive to doxorubicin. (B) Diploid WT, ccr4Δ and rad51Δ cells were grown to logarithmic phase in liquid YPD and individual unbudded (G1) cells were plated in a 5×4 cell grid pattern to YPD containing doxorubicin at the indicated dose within one microscopic field of view using a Singer MSM micromanipulator. DOX-induced inhibition of cell cycle progression in G1 and G1/S phases of the cell cycle was monitored by photomicroscopy at hourly intervals. Cells were incubated at 30°C during cell cycle progression analysis. (C) Similar to panel B except individual unbudded cells were gridded onto YPD plates containing 50 µg/ml DOX. (D) Similar to panel B except individual unbudded cells were gridded onto YPD medium without doxorubicin to demonstrate normal cell cycle progression and 100% cell viability. (E) Isogenic respiratory competent and petite WT, ccr4Δ and pop2Δ strains were grown in liquid YPD and serially diluted in 96 well plates as described in panel A. Both the ccr4Δ and pop2Δ petite strains show enhanced resistance to the lethal effects of doxorubicin. (F) Similar to panel C except individual unbudded cells were from petite strains that lacked respiratory function.

(A) Haploid (1n MATα) yeast deletion strains (rows 3, 4, 7, 8, 11, 12, 15, 16) were compared to their isogenic diploid (2n) deletion counterparts (rows 1, 2, 5, 6, 9, 10, 13, 14) for enhanced hypersensitivity to doxorubicin (DOX), hydroxyurea (HU) and methyl methanesulfonate (MMS) relative to wild type (WT; rows 1, 3, 5, 7, 9, 11, 13, 15) at the indicated concentrations in YPD agar medium. Cells were grown, diluted and plated as described in Fig. 1A. Arrows indicate direction of decreasing cell concentration. Deletion of BEM1(rows 6, 8), CTK1 (rows 10, 12) or THO2 (rows 14, 16) show enhanced sensitivity to the lethal effects of doxorubicin as a diploid. Deletion of AKR1 demonstrated hypersensitivity to doxorubicin in both the diploid (row 2) and haploid (row 4) derivatives. All deletion strains with the exception of the haploid ctk1Δ (above) and hfiΔ (not shown) strains demonstrated hypersensitivity to HU and MMS when compared to WT. (B) Diploid specific hypersensitivity of ctk1Δ and hfi1Δ strains to doxorubicin-induced cytotoxicity in the diploid BY4743 background as compared to the isogenic BY4741 (MATa), BY4742 (MATa) haploid backgrounds. Dilution plating conditions were similar to that as described in Fig. 1A. (C) Some diploid-specific deletion strains demonstrate enhanced mating capability as diploids. All diploid-specific gene deletions were examined for the ability to mate to the haploid mating type tester strains147 (MATa) or 148 (MATα). WT diploid strains are non-maters. Some (ctk1Δ, hfi1Δ, nup133Δ and ctf4Δ) but not all (tho2Δ, bem1Δ, mft1Δ, thp1Δ and thp2Δ) diploid strains showed enhanced capability for mating and subsequent growth on minimal (MIN) agar medium. Representative diploid deletion strains which show enhanced mating capability (ctk1Δ) or no enhanced mating capability (tho2Δ) by growth on MIN medium (arrow *) are depicted.

(A) WT and mutant diploid deletion strains were grown to logarithmic phase in liquid YPD. Single unbudded (G1) cells were arrayed into 5×4 cell grids on YPD with and without doxorubicin (50 µg/ml). Representative photomicrographs of mutant cells arrested in G1 or at G1/S following exposure to doxorubicin have been shown following 15 or 30 hr growth at 30°C. Only WT diploid cells were capable of forming viable microcolonies when exposed to doxorubicin. Most unbudded cells (>70%) from the WT and mutant diploid strains demonstrated rapid cell cycle progression and microcolony formation in the absence of DOX (data not shown). The mean gene conversion frequency of the his3Δ1 allele to HIS⁺ was determined in WT and mutant diploid strains following transformation of a PCR fragment capable of restoring the HIS3 allele following recombination. Conversion frequencies for the WT, bem1Δ, ctf4Δ and nup133Δ strains (*) have been previously reported [40]. The HIS3 conversion frequencies for the diploid ctk1Δ, hfi1Δ and tho2Δ strains are the mean of 3–10 replica experiments±1 standard deviation. (B) Expression of BEM1 within the diploid bem1Δ strain suppresses cell cycle arrest in G1 and restores viability following exposure to doxorubicin. The diploid bem1Δ strain was transformed with either empty vector or plasmid DLB1974 expressing the WT BEM1 gene. Unbudded cells from the diploid bem1Δ strain with or without plasmid were grown as described above in liquid YPD or synthetic complete glucose containing medium lacking uracil (SC-ura) to maintain plasmid selection. Single unbudded cells were exposed to doxorubicin (50 µg/ml) in either synthetic complete glucose containing agar medium lacking uracil (SC-ura+DOX) to maintain the plasmid or YPD+DOX (for cells not containing plasmid). The bem1Δ cells exposed to DOX on YPD agar plates progress from G1 into S phase and arrest as budded cells (upper panels). Cells that harbor the BEM1 expression plasmid (bem1Δ+BEM1) do not arrest in G1 or G1/S but form viable microcolonies by 24 hrs that continue to grow in the presence of DOX (middle panels). Diploid bem1Δ cells containing vector alone, arrest in G1 when exposed to DOX on SC-ura agar medium (bottom panels). (C) Expression plasmids containing HFI1, NUP133 and CTK1 suppress doxorubicin-induced lethality in the corresponding diploid deletion strains. Galactose-inducible expression constructs for HFI1 and NUP133 cloned within the selectable (URA3) plasmid BG1805 or vector alone were transformed into hfi1Δ and nup133Δ diploid strains respectively. The selectable (HIS3) plasmid containing CTK1 and empty vector have been previously described [59]. Plasmid bearing cells were grown overnight at 30°C in either liquid SC-uracil containing galactose (for hfi1Δ and nup133Δ plasmid bearing strains) or SC-histidine glucose containing medium (for the ctk1Δ plasmid bearing strains) in 96 well dishes. Following serial 5-fold dilution, aliquots of each cell dilution were plated to the corresponding solid dropout medium with and without doxorubicin at the indicated concentration. Plates were photographed following 3 days growth at 30°C. In all cases, the expression plasmid restored resistance to DOX-induced lethality in the appropriate deletion strain. Arrows indicate direction of decreasing cell concentration.

(A) Using the 9 diploid-specific DOX resistance genes identified in this study (BEM1, CTF4, CTK1, HFI1, NUP133, MFT1, THO2, THP1 and THP2; red octagon symbols), genetic and proteomic interactions were batch downloaded from data annotated at SGD as of Nov. 2, 2008. Genetic and physical interaction data sets were retrieved and visualized using Cytoscape v2.6.1. This initial genetic interaction network map contained a total of 502 nodes (genes) and 1075 edges (interactions) and the physical interaction map contained 188 nodes with 314 edges (data not shown). These were combined and all essential genes (i.e. deletions not represented in the diploid deletion collection) were eliminated resulting in a final combined interaction map with 500 nodes and 1154 edges. Genetic and proteomic interactions are indicated with a solid or dashed line respectively. Nodes (genes) that were identified in the initial diploid screen as conferring DOX resistance are denoted as red circles. Using the interactive genetic map as a predictive tool, additional DOX-resistance genes (red squares) were subsequently identified (see panel B). Some interactive genes/proteins (green circles) did not confer resistance to DOX but did confer resistance to other DNA damaging agents (HU and/or MMS, see panels B and C). Other gene deletion strains examined (black circles) did not show sensitivity to any of the damage agent tested when compared to WT. The diploid gene deletions associated with the remaining nodes (orange circles) were not tested for enhanced sensitivity to DNA damaging agents. (B) Identification of additional damage resistance genes based on genetic interactions with diploid-specific DOX resistance genes. Fourteen diploid deletion strains predicted to be DOX sensitive based on genetic interactions (rows 2–8 and 10–16) were obtained from the diploid deletion collection and tested for enhanced sensitivity to DOX, HU and MMS when compared to WT. Cell growth, dilution and replica plating techniques were as described in Fig. 1A. Some strains showed enhanced sensitivity to DOX (ccs1Δ, row 4; get2Δ, row 7; hir1Δ, row 8; lrs4Δ, row10 and pap2Δ, row12) when compared to WT (rows 1 and 9). These strains demonstrate modest (5-fold; get2Δ and hir1Δ) to moderate (25–125 fold; ccs1Δ, lrs4Δ and pap2Δ) enhanced sensitivity to DOX as indicated (*). Some strains showed enhanced sensitivity (5–125 fold) to HU (bim1Δ, row 2 and csm1Δ, row5) or MMS (tof1Δ, row 16) without accompanying sensitivity to DOX. (C) Identification of additional damage resistance genes based on proteomic interactions with diploid-specific DOX resistance genes/proteins. Diploid deletion strains predicted to be DOX sensitive based on proteomic interaction map were obtained from the diploid deletion collection and tested for enhanced sensitivity to DOX, HU and MMS when compared to WT. Cell growth, dilution and replica plating techniques were as described in Fig. 1A. None of the deletion strains were found to show enhanced sensitivity to DOX. However, some strains (hrb1Δ, row 4; imd3Δ, row 5; pcl9Δ, row 6; tex1Δ, row7; and yck1Δ, row 8) showed enhanced sensitivity to MMS (5–625 fold) and hog1Δ (row 10) showed enhanced sensitivity to HU (5 fold) when compared to WT (rows 1 and 9) as indicated (*).