The majority of the selected proteins modulate the recovery after damage from the three compounds MMS, 4-NQO and t-BuOOH.

<p>A) RNA levels of shRNA targeted genes in 293T cells were measured by qRT-PCR and compared to cells infected with non-silencing control shRNA. B) Survival of cells depleted of target proteins exposed to three DNA damaging agents as revealed by heatmap. The color represents sensitivity to the damaging agent compared to the cell lines with non-silenced targets. ++ indicate high resistance. + low resistance, − high sensitivity, − low sensitivity. C) Knock-down of human homologs of non-toxicity modulating proteins in yeast, as measured by qRT-PCR. D) Survival of cells depleted of human homologs of non-toxicity modulating proteins in yeast. Colors and symbols are the same as in B.</p

Response to MMS relies on autophagy.

<p>A) Model for induction and inhibition of autophagy. 3-methyladenine (3MeA) inhibits the formation of autophagosomes and bafiloycin A1 inhibits the acidification of the lysosomes leading to an accumulation of autophagosomes. LC3 is a marker of autophagosomes, here was tagged with GFP. B) Inhibition of autophagy decreases survival, both with 3MeA and BA1. C) Autophagy as seen by the formation of LC3-GFP-puncta showing autophagosomes in treated cells (top panel). A subset of the LC3-GFP-puncta co-stain (white arrows) with the acidic vesicles labeled by Lysotracker Red (white and red arrows) (bottom panel). D) Significant induction of autophagy after MMS treatment (1.2 µM). E–F) MMS induces autophagy in a dose and time dependent manner, whereas accumulation of autophagosomes by BA1 is not affected by MMS.</p

Autophagic response to MMS is modulated by ATP6V1D and ZFYVE20.

<p>A) Inhibition of autophagy further sensitizes the cells that have been depleted of ZFYVE20 and APT6V1D to MMS. B) The formation of autophagosomes after MMS treatment is visible in a background of cytoplasmic GFP. C) MMS induces autophagy in cells that is dependent on ZFYVE20. The statistical significance of the difference between each condition and its untreated control is indicated by asterisks (* p<0.05, ** p<0.01).</p

Human interaction network shows high connectivity among putative human toxicity-modulating proteins homologous to toxicity-modulating proteins in yeast.

<p>The largest connected component of the human interactome selected from yeast orthologs being required for damage recovery after treatment with MMS, 4NQO, t-BuOOH and UV <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037368#pone.0037368-Begley2" target="_blank">[13]</a>. The circles represent: red – proteins with toxicity-modulating yeast homologs targeted for silencing in this study; grey – proteins with toxicity-modulating yeast homologs not targeted in this study; blue – proteins with non-toxicity-modulating yeast homologs targeted in this study; green –proteins specific for mammalian telomere maintenance targeted in this study. An interactive version of this figure is available at <a href="http://www.bionut.ki.se/users/pesv/MIT/fig1.html" target="_blank">http://www.bionut.ki.se/users/pesv/MIT/fig1.html</a>.</p

Tryptophan biosynthesis rescues cells from ROS.

<p>A) A schematic of the tryptophan biosynthesis pathway <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073736#pone.0073736-Braus1" target="_blank">[29]</a>. PRA: N-(5′-phospohribosyl)-anthsranilate, CDRP: 1-(o-carboxyphenylamino)-1-desoxyribuose-5-phosphate. B) Compound sensitivity of selected mutant strains were analyzed by spot assay. Strains were grown in liquid YPD+G418 overnight at 30°C and then diluted in YPD. Ten-fold serial dilutions of each yeast culture was spotted onto YPD plates in the absence (control) and presence of the different compounds: MMS (0.006%), MEN (40 µM), HYQ (3 mg ml⁻¹), and tBuOOH (0.75 mM). Plates were incubated at 30°C and growth was recorded after 48 h exposure.</p

The difference between alkylating and oxidizing agents can be explained by fitness profiles of the strains.

<p>A) Two-dimensional hierarchical clustering of fitness ratio (median log ratio of exposed/control) results using the strains sensitive after 10 and 20 generation times upon exposure to different chemicals. Compounds and doses are plotted across the horizontal axis. On the vertical axis, a subset of 508 strains with reduced fitness is shown. B) Protein-protein interaction networks with >5 toxicity-modulating proteins. The colors (explained in legend, same as labels in A) within the pie charts indicate the contribution of each of the eight compounds. Alkylating agents represented in shades of yellow-red, oxidizing agents in shades of blue and non-genotoxic compounds in green.</p

Functional enrichment reveals an alkylating agent-specific DNA repair and cell cycle dependency.

<p>Gene-annotation enrichment analysis heat map and clustering for sensitive strains to different compounds at early (10 generation times) or late (20 generation times) timepoints. Heat map colors correspond to the –log10 of the p-values.</p

Experimental workflow for barcoded genomic phenotyping.

<p>Schematic representation of A) experimental design (t is the time of cell harvest, which was at 10 or 20 generation times) and B) analysis and filtering of high-throughput sequencing data.</p

List of selected compounds and doses used in this study.

<p>List of selected compounds and doses used in this study.</p

Overexpression of CENP-A^Cnp1 causes toxicity in FACT mutants.

<p>(A) Viability of wild-type (wt) and <i>spt16-18</i> cells expressing additional CENP-A^Cnp1 or H3 at low (nmt81-CENP-A^Cnp1, nmt81-H3) or medium (nmt41-CENP-A^Cnp1, nmt41-H3) levels compared to empty vector. Cells were grown at 27°C which is semi-permissive for <i>spt16-18</i>. Phloxine B plates stain dead cells red. (B) Viability of wt, <i>pob3</i>Δ and <i>spt16-17</i> cells expressing additional CENP-A^Cnp1 at low (nmt81-CENP-A^Cnp1) or medium (nmt41-CENP-A^Cnp1) levels compared to empty vector at indicated temperatures. (C) Viability of wt and <i>spt16-18</i> cells expressing GFP-CENP-A^Cnp1 from integrated pREP41-GFP-<i>cnp1</i>⁺ (nmt41-GFP-CENP-A^cnp1) compared to no GFP-CENP-A^Cnp1 control. (D) Western analysis of GFP-CENP-A^Cnp1 levels in wt and <i>spt16-18</i> cells expressing GFP-CENP-A^Cnp1 under endogenous, nmt81 or nmt41 promoter (upper panel). The intensities of GFP-CENP-A^Cnp1 and TAT-1 (alpha-tubulin) signals were measured using LICOR Odyssey Infrared Imaging System software (Li-COR Bioscience) and the relative intensities of GFP-CENP-A^Cnp1/TAT-1 were quantified (bottom panel). GFP-CENP-A^Cnp1 was expressed for 24 h at 25°C before harvest.</p