A Genetic Screen Highlights a Key Role of Recombinational Repair and Checkpoint Genes in Resistance to Sodium Selenide
To identify cellular processes involved in S. cerevisiae
resistance to hydrogen selenide, we analyzed the sensitivity of a collection of approximately 5000 isogenic haploid knockout mutants to sodium selenide, a donor of hydrogen selenide 
. Because oxidation of hydrogen selenide is rapid in an oxygenated solution (half-life <2 min in rich YTD medium at 30°C and pH 6.0), the selenide stress challenge was performed by renewing sodium selenide addition every 4 h. Two experiments were performed with 1 or 2 µM sodium selenide final concentrations, respectively. At such concentrations, the doubling time of the wild-type strain was increased by 30–40%. Cells were collected after 16 and 27 h. DNA barcode regions of the various strains were amplified by PCR and labeled with Cy3 or Cy5 fluorophores (Figure S1
). Hybridization of these PCR products on Agilent barcode microarrays was used to derive relative fitness defect estimates for individual mutants (see Materials and Methods
As shown in Table S1
, the results were similar for the two concentrations of Na2
Se used (1 and 2 µM). Thus, the two sets of data were fused. The resulting distribution of the fitness values was asymmetric with a long tail on the negative side, as expected from a selective effect of sodium selenide treatment on a subset of the mutants from the collection (Figure S2A
). Because of this asymmetry and of the broader-than-Gaussian shape of the density distribution, a z-type statistics could not be used to analyze the results. Instead, we examined the ranks of the values (see for the annotated list of the 30 most selenide-sensitive deletion strains, and Table S1
for the full list).
Ranking of genes (1 to 30) based on relative fitness defects calculated for deletion strains after treatment with sodium selenide.
Two criteria validated the screen a posteriori
. First, we found similar sensitivities to sodium selenide when two independent disruptions of a same gene were available and compared. This was the case with overlapping ORFs (Figure S2B
, see also, for example, MMS4
, or FIG4
in ). Secondly, deletion mutants for genes coding for distinct subunits of a same protein complex often ranked similarly. This was the case of the Rad55p-Rad57p complex (rank 2 for RAD57
and 45 for RAD55
), the Mus81p-Mms4p complex (rank 26 for MUS81
and 52 for MMS4
) and the Vac14p-Fig4p complex (rank 29 for VAC14
and 32 for FIG4
). Notably, out of the 9 nonessential genes encoding subunits of the Swr1 complex, 7 (SWR1, YAF9
) belonged to the 100 first ranked genes.
Strikingly, our screen revealed that 29% of the 28 genes belonging to the HR pathway ranked in the first 30 (with a p-value of 4.2·10−14
), and 75% under 250 (p
) (). In particular, all genes encoding proteins involved in synapsis (Rad51p, Rad52p, Rad54p, Rad55p and Rad57p) ranked below 53 (p
) with a median rank of 27. Similarly, 73% of the 11 genes associated with checkpoint activation were found in the first 250 ranks (p
), while 45% ranked in the first 30 (p
), with particularly low ranks for DDC1
(8, 9, 10, 13 and 23, respectively). Among the genes with a ranking lower than 100, were also found the 2 genes encoding the Mus81p-Mms4p complex involved in the resolution of Holliday junctions 
and, as already mentioned, 7 genes encoding subunits of the Swr1 complex responsible for chromatin remodeling during DSB repair 
. These results establish that HR and checkpoint genes are important for cell resistance to selenide and suggest that genotoxicity resulting from DSB formation is a key component of selenide toxicity. In favor of DSB occurrence in vivo
upon sodium selenide treatment, the results of our genetic screen resemble other results obtained using ionizing radiation or Cpt, two agents known to induce DSBs 
Sodium selenide sensitivity distribution of mutants for genes involved in DNA damage and oxidative stress responses.
None of the genes encoding the Mre11p-Rad50p-Xrs2p (MRX) complex and the nuclease Sae2p, which cooperate in resection initiation, emerged in the first 200 ranks. Possibly, as already observed for the repair of multiple DSBs induced by HO endonuclease 
, MRX-dependent resection initiation plays a facultative role in the DNA repair after sodium selenide treatment. Moreover, none of the base excision repair (BER, 7 genes) and non-homologous end joining (NHEJ) genes (6 genes) were in the first 250 ranks (median ranks of 1774 and 2505, respectively) (). In between, nucleotide excision repair (NER, 10 genes) and post-replicative repair (PRR, 9 genes) showed an intermediate distribution, with (i) no gene ranking under 30, (ii) 20% of NER genes and 22% of PRR genes between positions 31 and 250, and (iii) overall median ranks of 1393 (NER) and 1435 (PRR). Thus, after a sodium selenide challenge, HR dominates other repair pathways in cell survival. This advocates once more for the importance of DSBs in the toxicity of selenide.
The genes of γ-glutamylcysteine synthethase, glutathione reductase and glutaredoxin 1 belong to the first 10 ranked genes. That of glutaredoxin 3 and TSA1
, the gene of a thioredoxin peroxidase, have ranks 31 and 65, respectively (). The low ranks of these genes, which are involved in the oxidative stress pathway, indicates that redox potential is also an important factor in the cell resistance to sodium selenide. Indeed, reduced gluthatione, glutaredoxin and thioredoxin are antioxidants preventing damages caused by reactive oxygen species such as free radicals and peroxides. These thiol-containing agents can also be involved in the recycling of protein disulfides or protein-selenotrisulfides produced in the course of H2
Se redox cycling 
G2/M Cell Cyle Checkpoint is Activated by Sodium Selenide Treatment
The genome-wide screen suggested the induction of DSBs by sodium selenide. Consequently, a cell-cycle arrest should be observed. Indeed, the cell-cycle progression of cells exposed to genotoxic agents can be perturbed in two ways, depending on the effect on the replication process. On the one hand, DNA damages which block the replication forks (such as some base oxidations, base methylations, intra- or inter-strand cross-links…) trigger the activation of an intra-S checkpoint and the S-phase is prolonged until DNA repair occurs 
. On the other hand, in the presence of SSBs or DSBs, replication forks occasionally collapse. However, replication fork block does not occur, and checkpoint activation does not interfere with S-phase progression. As a result, cell-cycle blockage and DNA repair are delayed to G2 
To investigate whether selenide induced a cell-cycle arrest, we followed cell-cycle progression of the wild-type strain and of its isogenic rad52
Δ mutant. RAD52
inactivation is known to impair the HR pathway 
. Cpt, which induces DNA breaks, was used as control. Cell-cycle progression of cells incubated during 2 h with 40 µM Cpt or with 0.5 or 2 µM Na2
Se was monitored using flow cytometry (). In the absence of toxic agent, both wild-type and rad52
Δ strains were predominantly in the G2/M phase (at least 62% of the cells possessed a 2 n DNA content). As expected, Cpt treatment of the wild-type strain caused an increase in G2/M percentage (+8%) paralleled by a comparable decrease in G1 percentage. The block in G2/M was amplified upon RAD52
inactivation (+26% of G2/M cells) and appeared to be more time-stable, as indicated by near complete disappearance of G1 cells.
Analysis of cell-cycle phases distribution in asynchronous cultures of wild-type (wt) and rad52
When the wild-type strain was exposed to sodium selenide, the G2/M population increased. At the highest concentration of Na2Se (2 µM), the effect was dramatic. The G2/M population increased by 19% whereas the G1 population dropped to 6%, and quasi-complete synchronization of the cell population occurred. Importantly, at the lowest used concentration of Na2Se (0.5 µM), the increase in the G2/M percentage was nearly three times higher with the rad52Δ strain than with the wild-type strain (22% and 8%, respectively), indicating that selenide-induced cell-cycle blockage is amplified by RAD52 inactivation. The similarity of the effects of Cpt and sodium selenide on G2/M checkpoint activation, both with the wild-type and rad52Δ strains, is consistent with DSB being the major DNA damage caused by selenide.
Sodium Selenide Induces Double-strand Breaks in vivo
These results prompted us to determine if DSBs were indeed produced in cells treated with sodium selenide. S. cerevisiae cells grown in minimal medium were incubated in the absence or in the presence of increasing concentrations of Na2Se. After 1 h of incubation, cells were included into agarose plugs and their chromosomes were analyzed by PFGE (). Cell survival after the treatment was also measured ().
PFGE analysis of the effect of sodium selenide on DNA integrity in vivo.
In the absence of sodium selenide, yeast chromosomes migrated as discrete bands. In the presence of sodium selenide, the intensities of the chromosome bands decreased, whereas smears corresponding to shorter DNA fragments accumulated. This DNA fragmentation was accompanied by a decrease in cell viability. To estimate the level of damaged chromosomes in each growth condition, we quantified the bands corresponding to the largest chromosomes (). For smaller chromosomes, bands could not be distinguished from the breakage products of the longer chromosomes. Comparison of the two curves shown in indicates that the increase in chromosome damage was very similar to the variation of cell death. We conclude that, in the presence of Na2Se in the culture medium, DSBs are produced and that the rate of DSB formation tightly correlates with the rate of cell death.
Selenide, but not Selenite, Breaks Phosphodiester Bonds in vitro
The DNA breaks we observed in vivo can be caused by hydrogen selenide either directly or indirectly. To help distinguish between these two possibilities, we asked whether addition of Na2Se broke DNA in a minimal system consisting of supercoiled DNA in an oxygenated phosphate buffer. In this system, conversion of the supercoiled plasmid into its nicked form which has a lower electrophoretic mobility allows to detect SSBs. Electrophoresis conditions were set up to separate supercoiled, nicked and linear pNOY102 plasmid DNAs (compare lanes 1, 8 and 9 in ).
Effect of sodium selenide on DNA integrity in vitro.
When incubated for 1 h at 37°C in the presence of fresh Na2
Se (15 µM), the plasmid was almost entirely converted into its nicked form (compare lanes 10 and 8 in ). In contrast, sodium selenite did not break DNA (lane 2) unless it was mixed with glutathione (lane 4), a condition known to convert selenite into hydrogen selenide 
. Thus, we conclude that selenide is sufficient to break phosphodiester bonds, whereas selenite alone has no detectable effect.
Free Radical Production is Required for Selenide to Nick DNA
To determine whether ROS were involved in selenide-induced DNA breakage, the above experiment was repeated in the presence of a radical quencher or of detoxication enzymes. Before addition of sodium selenide or sodium selenite, the plasmid was mixed with either SOD (which converts superoxide anions O2•− into H2O2 and O2), catalase (which converts H2O2 into H2O plus O2) or mannitol (a quencher of various radicals including •OH, but, importantly, not O2•-). The electrophoretic profiles of plasmid DNA samples recorded after treatment with either sodium selenide or sodium selenite plus glutathione were not modified by the presence of catalase or SOD (compare lanes 6 and 7 to lane 4, and lanes 12 and 13 to lane 10 in ). On the other hand, in the presence of mannitol, the profiles remained similar to that observed when sodium selenide was omitted (compare lanes 4 and 11 to lane 8 in ). These data indicate that one or several free radicals, with the exception of O2•−, are involved in the selenide-induced DNA damaging reaction.
ESR spectroscopy was used to monitor the production of radicals from sodium selenide. Reaction of the spin trapper DEPMPO with •
OH radicals leads to an oxidized stable form (DEPMPO-OH) which has a characteristic 8-peak spectrum 
. Superoxide anions also react with DEPMPO, but the resulting modified spin trapper (DEPMPO-OOH) displays a clearly distinct ESR spectrum 
We first carried out a control ESR analysis after incubation of DEPMPO with H2O2 and Fe(II)-EDTA. This mixture is expected to lead to •OH production through the Fenton reaction. Consistent with •OH formation in this control experiment, the spectrum of DEPMPO-OH was observed (). In the presence of mannitol (150 mM) in the mixture, the magnitude of the spectrum was strongly reduced (). This confirmed that the spectrum in indeed reflected •OH production.
ESR analysis of the oxidation of sodium selenide in the presence of dioxygen.
To analyze radical production in the presence of selenide, DEPMPO was incubated in the presence of Na2
Se (100 µM). The eight-peak spectrum characteristic of DEPMPO-OH was obtained (). Because DEPMPO-OOH can slowly convert to DEPMPO-OH 
, we performed the same experiment in the presence of SOD to make sure that the DEPMPO-OH spectrum did not arise from a conversion of DEPMPO-OOH to DEPMPO-OH. We also added catalase to the reaction mixture to preclude formation of hydroxyl radicals from SOD-produced H2
. Indeed, parasitic generation of •
OH might have occurred in the case of contamination of the solution by trace amounts of transition metals. Under these conditions (SOD and catalase), the eight-peak spectrum of DEPMPO-OH was again obtained (). Similarly to what we observed in the Fenton reaction control experiment, the amplitude of the spectrum strongly decreased when the mixture of DEPMPO and Na2
Se was supplemented with mannitol (). Thus, we conclude that the reaction of selenide with dioxygen produces free radicals, likely •
OH radicals, and that if superoxide ions were produced, their concentration remained below the detection threshold.
DNA Damage by Sodium Selenide Requires the Presence of Dioxygen
To establish whether the DNA-damaging reaction required the presence of dioxygen, we compared the rates of selenide-induced SSB formation under aerobic and anaerobic conditions, in vitro. In the experiment shown in , increasing amounts of Na2Se were added to the pNOY102 plasmid DNA solution. Incubations were performed inside a glove box with dioxygen partial pressure continuously lower than 5 ppm. DNA solutions were prepared in the glove box in either a deoxygenated buffer (anaerobic conditions) or an oxygenated one (aerobic conditions). The reaction was initiated by mixing the DNA solutions with Na2Se, and quenched after 1 min by addition of 150 mM mannitol.
In the oxygenated buffer, in agreement with the results presented in , addition of increasing amounts of sodium selenide caused progressive nicking of supercoiled DNA, as shown by the decreasing intensity of the corresponding gel band (, right side). The nicking of plasmid DNA was nearly complete in the presence of 0.1 mM Na2Se. In contrast, supercoiled DNA remained unmodified by sodium selenide in deoxygenated buffer (, left side). We conclude that selenide-induced DNA breakage strictly requires the presence of dioxygen.
Effect of dioxygen on the in vitro Na2Se-induced DNA nicking.
Dioxygen in the Culture Medium Potentiates the Toxicity of Sodium Selenide in vivo
Because selenide requires dioxygen to nick DNA in vitro, we asked whether dioxygen was necessary for selenide to cause death of yeast cells. The wild-type S. cerevisiae strain was exposed to various concentrations of Na2Se (0–50 µM) for 5 min, under aerobic or anaerobic conditions. To estimate short-term viability, cells were plated on rich medium just after the treatment and their ability to form colonies was determined. As expected, in the presence of dioxygen, Na2Se concentrations higher than 5 µM induced a significant loss of viability (). When cells were maintained in strict anaerobic conditions, cell death was no longer observed in the presence of sodium selenide. This experiment establishes that selenide toxicity indeed implies an oxygen-dependent mechanism.
Effect of dioxygen on S. cerevisiae Na2Se-induced lethality.