Sodium Selenide Toxicity Is Mediated by O2-Dependent DNA Breaks

Hydrogen selenide is a recurrent metabolite of selenium compounds. However, few experiments studied the direct link between this toxic agent and cell death. To address this question, we first screened a systematic collection of Saccharomyces cerevisiae haploid knockout strains for sensitivity to sodium selenide, a donor for hydrogen selenide (H2Se/HSe−/Se2−). Among the genes whose deletion caused hypresensitivity, homologous recombination and DNA damage checkpoint genes were over-represented, suggesting that DNA double-strand breaks are a dominant cause of hydrogen selenide toxicity. Consistent with this hypothesis, treatment of S. cerevisiae cells with sodium selenide triggered G2/M checkpoint activation and induced in vivo chromosome fragmentation. In vitro, sodium selenide directly induced DNA phosphodiester-bond breaks via an O2-dependent reaction. The reaction was inhibited by mannitol, a hydroxyl radical quencher, but not by superoxide dismutase or catalase, strongly suggesting the involvement of hydroxyl radicals and ruling out participations of superoxide anions or hydrogen peroxide. The •OH signature could indeed be detected by electron spin resonance upon exposure of a solution of sodium selenide to O2. Finally we showed that, in vivo, toxicity strictly depended on the presence of O2. Therefore, by combining genome-wide and biochemical approaches, we demonstrated that, in yeast cells, hydrogen selenide induces toxic DNA breaks through an O2-dependent radical-based mechanism

A Ca2+-regulated deAMPylation switch in human and bacterial FIC proteins

In many AMPylating FIC proteins a structurally conserved glutamate represses AMPylation. Here, the authors show that this glutamate supports deAMPylation in Enterococcus faecalis FIC (EfFIC), and that EfFIC switches from AMPylation to deAMPylation by binding Ca2+ at distinct sites

The recruitment of RNA polymerase I on rDNA is mediated by the interaction of the A43 subunit with Rrn3

RNA polymerase I (Pol I) is dedicated to transcription of the large ribosomal DNA (rDNA). The mechanism of Pol I recruitment onto rDNA promoters is poorly understood. Here we present evidence that subunit A43 of Pol I interacts with transcription factor Rrn3: conditional mutations in A43 were found to disrupt the transcriptionally competent Pol I–Rrn3 complex, the two proteins formed a stable complex when co-expressed in Escherichia coli, overexpression of Rrn3 suppressed the mutant phenotype, and A43 and Rrn3 mutants showed synthetic lethality. Consistently, immunoelectron microscopy data showed that A43 and Rrn3 co-localize within the Pol I–Rrn3 complex. Rrn3 has several protein partners: a two-hybrid screen identified the C-terminus of subunit Rrn6 of the core factor as a Rrn3 contact, an interaction supported in vitro by affinity chromatography. Our results suggest that Rrn3 plays a central role in Pol I recruitment to rDNA promoters by bridging the enzyme to the core factor. The existence of mammalian orthologues of A43 and Rrn3 suggests evolutionary conservation of the molecular mechanisms underlying rDNA transcription in eukaryotes

Analysis of cell-cycle phases distribution in asynchronous cultures of wild-type (wt) and <i>rad52</i>

<p>Δ <b>strains.</b> Before analysis by flow cytometry, cells were grown for 2 h in YTD in the presence of the indicated concentrations of Cpt or sodium selenide. The histogram shows the number of cells (counts) corresponding to each fluorescence intensity (in arbitrary units). The percentages of G1 and G2/M subpopulations are indicated above the corresponding peaks. The boxes at the top of the panels indicate the fluorescence intensity intervals chosen to calculate G1 and G2/M cell populations.</p

Ranking of genes (1 to 30) based on relative fitness defects calculated for deletion strains after treatment with sodium selenide.

a<p>rf: relative fitness defect.</p>b<p>DSB : genes of the list encoding proteins implicated in cellular response to DSBs (HR and DNA damage checkpoint) are labeled with +.</p>c<p>Cpt: genes of the list having a low rank in a screen with Cpt, as reported by Hillenmeyer <i>et al</i>. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036343#pone.0036343-Hillenmeyer1" target="_blank">[45]</a>. Genes ranking under 30 are labeled with +, genes ranking between 31 and 150 are labeled with #.</p>d<p>γ-rays: genes ranking under 30 in a γ-rays screen, as reported by Game <i>et al</i>. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036343#pone.0036343-Game1" target="_blank">[30]</a> (data regarding higher ranks are no more available on the referenced web site).</p>e<p>GSH: the designated proteins are involved in glutathione homeostasis.</p

PFGE analysis of the effect of sodium selenide on DNA integrity <i>in vivo</i>.

<p>Exponentially growing <i>S. cerevisiae</i> cells were incubated for 1 h in the presence of Na₂Se at the indicated concentrations. (A) Chromosomal DNA was extracted, separated by PFGE and stained with ethidium bromide. (B) Cell survival was measured in parallel by counting colony-forming units. Aliquots of the cultures were diluted 10,000-fold in water and 200 µl of these dilutions were plated in duplicate onto YTD agar plates. Results are expressed as cell death percentages (mean value and range of the duplicate measurements) relative to the number of survivors in the culture without sodium selenide (circles). To quantify the levels of DNA breaks in cells grown in the presence of sodium selenide in solution, the intensities of the four bands corresponding to the four largest chromosomes (indicated by stars in panel A) were determined by gel optical scanning. The intensities of the bands in the presence of Na₂Se were expressed as percentages of the corresponding intensities in the control without Na₂Se. Next, in each Na₂Se condition, the four obtained percentage values were averaged and a standard deviation was calculated (squares).</p

ESR analysis of the oxidation of sodium selenide in the presence of dioxygen.

<p>Before measurements, DEPMPO spin trapper (at a final concentration of 120 mM in 400 µl-samples) was incubated for 5 min at 37°C with the following compounds. (<b>A</b>) 10 mM H₂O₂+40 µM Fe(II)-EDTA+SOD (40 units). (<b>B</b>) 10 mM H₂O₂+40 µM Fe(II)-EDTA+150 mM mannitol+SOD (40 units). (<b>C</b>) 100 µM Na₂Se. (<b>D</b>) 100 µM Na₂Se+SOD (40 units)+catalase (200 units). (<b>E</b>) 100 µM Na₂Se+150 mM mannitol+SOD (40 units)+catalase (200 units). The ordinate scale is the same for spectra (<b>A</b>) and (<b>B</b>) and for spectra (<b>C</b>), (<b>D</b>) and (<b>E</b>).</p