Fig 1.
Hierarchical clustering of toxin-sensitive mutants.
Mutants identified as toxin-sensitive in at least one functional profiling screen were subjected to two-dimensional hierarchical clustering analysis using their GI scores. Toxin concentrations for each screen are indicated at the top of each column. GI values were obtained from the chromium screens reported in this study or published screens using the same functional profiling platform [26, 27]. Zoom view (right panel, upper half) indicates mutants shared in cadmium and arsenic screens but not chromium. Right panel, lower half indicates Cr(VI)-sensitive mutants shared with genotoxin-sensitive screens (CPT, HU and UV) but not cadmium or arsenic. Thiabendazole (TBZ) is a microtubule-depolymerizing drug.
Table 1.
GO Biological processes enriched for Cr(VI)-sensitive mutants.
Table 2.
Summary of annotated phenotypes enriched for Cr(VI)-sensitive mutants.
Fig 2.
DNA replication stress and DNA damage checkpoint genes are required for chromium-6 resistance.
Flow cytometry-based competitive growth assays determined mutant sensitivities to the indicated concentrations of toxicants. (A) Mutants lacking subunits of the Rad3/ATR checkpoint kinase, Rad9-Hus1-Rad1 checkpoint clamp or its Rad17-RFC clamp loader are sensitive to Cr(VI), CPT, MMS and bleomycin (Bleo), but not cadmium. (B) Mutants lacking the replication checkpoint kinase Cds1, the Mrc1 mediator required for Cds1 activation, the Swi1-Swi3 fork protection complex, or the multi-BRCT protein Brc1 are sensitive to Cr(VI), CPT, and HU (except brc1Δ), but not bleomycin. (C) A mutant lacking the DNA damage checkpoint kinase Chk1 is sensitive to Cr(VI), CPT and bleomycin, but not HU. The assays with chk1Δ cells used higher concentrations of Cr(VI) and CPT because S. pombe has a long G2 phase, hence chk1Δ cells are relatively less sensitive to genotoxins that collapse DNA replication forks. Statistically significant differences as determined by a two-tailed Student’s t-test are indicated. Bars represent standard deviation from three independent experiments.
Fig 3.
Hexavalent chromium activates Cds1 and Chk1 checkpoint kinases.
(A) Exposure to Cr(VI), but not As(III) or Cd(II), triggers Cds1 phosphorylation, as detected by reduced mobility of HA-tagged Cds1 in SDS-PAGE. HU was used as a positive control. The absence of Cds1 phosphorylation in HU-treated mrc1Δ cells further validates the assay. Cells were incubated with toxicants for 2 or 4 hours at 30°C. Toxicant concentrations were 20 mM HU, 200 μM K2Cr2O7, 200 μM CdSO4 or 200 μM NaAsO2. Tubulin (anti-tub) was used as the loading control. (B) Exposure to Cr(VI), but not As(III) or Cd(II), triggers Chk1 phosphorylation, as detected by reduced mobility of HA-tagged Chk1 in SDS-PAGE. CPT was used as a positive control. Cells were incubated with toxicants for 2 or 4 hours at 30°C. Toxicant concentrations were 30 μM CPT, 200 μM K2Cr2O7, 200 μM CdSO4 or 200 μM NaAsO2. (C) Cr(VI)-treated cells activate Chk1 upon transit through S-phase. Temperature sensitive cdc25-22 cells were synchronized in late G2 by incubation at 35°C for 3 hours, followed by an additional 2-hour incubation at 35°C in the presence of 100 μM Cr(VI). At time point 0, cells were released from the cell cycle block by lowering the temperature to 25°C. Activation of HA-tagged Chk1 was monitored by immunoblotting. Chk1 phosphorylation coincides with the rise in septation index (upper panel), which correlates with passage through S-phase. No Chk1 phosphorylation was observed in Cr(VI)-treated cells maintained in late G2 by incubation for an additional 120 minutes at 35°C (rightmost lane). The leftmost lane is the untreated control.
Fig 4.
Increased RPA foci are observed following chromate exposure.
(A) Increased number of RPA-GFP foci are detected in cells treated with 50 or 100 μM Cr(VI) for 2 hours at 25°C. Mega-foci, which are potentially clusters of RPA-GFP foci, are indicated by arrows. (B) Increased number of RPA-GFP foci are detected in cells treated with CPT. There was no change in RPA-GFP foci in cadmium-treated compared to untreated cells. Cells were treated with toxicants for 2 hours at 25°C. (C) Most Cr(VI)-treated cells with RPA-GFP foci are in S-phase or early G2 phase. Cell cycle stage was estimated from cell and nuclear morphology. Bars represent standard deviation from 3 independent experiments.
Fig 5.
HR repair is crucial for chromium-6 resistance.
(A) Flow cytometry-based competitive growth assays reveal that the Mre11 subunit of MRN DNA-end processing endonuclease, Rad51 and Rad57 recombinases, and Mus81 resolvase, are essential for resistance to Cr(VI). Note that all mutants were significantly sensitive to the indicated concentrations of Cr(VI), CPT and MMS, whereas only rad51Δ and rad57Δ were sensitive to HU and bleomycin. (B) RecQ DNA helicase is important for growth in the presence of Cr(VI). The sensitivity of rqh1Δ cells is comparable to mus81Δ. Bars represent standard deviation from 3 independent experiments. Statistically significant differences as determined by a two-tailed Student’s t-test are indicated.
Fig 6.
Increased Rad52 foci in Cr(VI)-treated cds1Δ and rad1Δ cells.
(A) In comparison to untreated cells, there is an ~ 3-fold increase in Rad52-YFP nuclear foci in WT cells treated with chromium. Cells defective for intra S-checkpoint signaling and stabilization of replication fork show a 2-fold increase in Rad52 foci formation even in the absence genotoxins. The cds1Δ and rad1Δ mutants showed Rad52 foci formation in 70% of the cells, as compared to 30% in WT. (B) Quantification according to cell cycle stage indicated maximum Rad52 foci in S and early G2 phase cells in wild type, cds1Δ and rad1Δ mutants. (C) Co-localization of Rad11-GFP and Rad52-RFP foci on treatment with Cr(VI) indicates the spatial and temporal overlap of replication stress events with recombination. Arrows indicate co-localization. All foci were scored in live cells at 25°C after treatment with 100 μM Cr(VI) in EMM for 3h. Cells were washed with fresh media before scoring. Error bars are indicative of standard deviation from three independent experiments.
Fig 7.
NER-independent activity of Rad16-Swi10 endonuclease contributes to chromium resistance.
(A) Competitive growth assays were performed with the indicated concentrations of chromate, the UV mimetic 4-NQO, the DNA crosslinker cisplatin, and CPT. Cells lacking the NER proteins Rhp14/XPA, Rad13/XPG, Swi10/ERCC1 or Rad16/XPF are all highly sensitive to 4-NQO and cisplatin, but insensitive to CPT. Only cells lacking the Swi10 or Rad16 subunits of the 3’ flap endonuclease are clearly sensitive to chromate, whereas those lacking the Rad13 3’ incision endonuclease appear to have weak sensitivity to chromate. Cells lacking Rhp14 are insensitive to chromate. The graph also shows that Pso2 endonuclease is required for resistance to cisplatin but not chromate, CPT or 4-NQO. (B) Cells lacking Rev3 TLS DNA polymerase zeta are sensitive to 10μM cisplatin, but not 10μM chromate or 1μM CPT. Cells lacking Mus81 or Rad51 are sensitive to CPT and chromate resistance, but only cells lacking Rad51 are sensitive to cisplatin. Bars indicate standard deviation from 3 independent experiments. Statistically significant differences as determined by a two-tailed Student’s t-test are indicated.
Fig 8.
Post-replication repair mutants are insensitive to chromium.
Competitive growth assays were performed with the indicated concentrations of chromate, CPT, 4-NQO, and cisplatin. Cells lacking the PRR proteins Rhp18/RAD18 or Ubc13 are sensitive to 4-NQO and cisplatin but not chromate or CPT. Elimination or Rhp14/XPA further increases 4-NQO and cisplatin sensitivities in cells lacking Ubc13. Cells lacking Hrq1/RECQL4 DNA helicase are only sensitive to cisplatin, whilst even co-elimination of Hrq1 and Ubc13 fails to cause any chromate or CPT sensitivity. Bars indicate standard deviation from 3 independent experiments. Statistically significant differences as determined by a two-tailed Student’s t-test are indicated.
Fig 9.
Tdp1 and Pnk1 repair Cr-induced DNA lesions.
Competitive growth assays were performed with the indicated concentrations of chromate or MMS. Cells lacking Tdp1 are only sensitive to chromate, whereas cells lacking the BER proteins Apn2 or Nth1 are only sensitive to MMS. There appear to be no genetic interactions of tdp1Δ with apn2Δ or nth1Δ; for example, the chromate sensitivity of the tdp1Δ apn2Δ strain is similar to tdp1Δ. Cells lacking Pnk1 are sensitive to both chromate and MMS, but these sensitivities are partially rescued by tdp1Δ. Bars indicate standard deviation from 3 independent experiments. Statistically significant differences as determined by a two-tailed Student’s t-test are indicated.
Fig 10.
Tdp1 and Rad16-Swi10 provide alternative pathways for repairing Top1-independent Cr-induced DNA lesions.
Competitive growth assays were performed with the indicated concentrations of chromate or CPT. (A) Elimination of Top1 suppresses tdp1Δ CPT sensitivity but not chromate sensitivity. Elimination of Top1, which suppresses tdp1Δ swi10Δ synthetic lethality, suppresses CPT sensitivity but not chromate sensitivity. Partial inactivation of Top1 caused by attachment of an HA epitope tag (top1-HA) suppresses the tdp1Δ swi10Δ synthetic lethality but these cells remain acutely sensitive to chromate. (B) Loss of Tdp1 enhances chromate and CPT sensitivities in mu81Δ and rhp57Δ backgrounds. Bars indicate standard deviation from 3 independent experiments. Statistically significant differences as determined by a two-tailed Student’s t-test are indicated.
Fig 11.
Model for repair of Cr-DNA lesions by Tdp1-Pnk1 and Rad16-Swi10 (XPF-ERCC1) pathways.
(A) Tdp1 processes Cr-DNA SSBs to generate SSBs with a 3’-PO4 terminus that is a substrate for Pnk1. (B) Alternatively, these Cr-DNA lesions may be processed by Rad16-Swi10 (XPF-ERCC1). In the model shown, this cleavage occurs at an seDSB that is formed by replication fork collapse. After removal of Cr-DNA lesion, the seDSB is repaired by SCR requiring key HR proteins such as MRN-Ctp1 and Rad51, followed by resolution of the Holliday junction or D-loop by Mus81-Eme1 endonuclease.