Additional file 1 of Global analysis of suppressor mutations that rescue human genetic defects

Additional file 1: Fig. S1. Literature curation process. Fig. S2. Suppressor genes are important for maintaining health and cellular fitness. Fig. S3. Overlap with other interaction networks. Fig. S4. Functional connections between query and suppressor genes. Fig. S5. General mechanistic classes of suppression. Fig. S6. Query gene knockout is associated with large variation in fitness across cell lines. Fig. S7. Suppressor gene prediction

Validated Conformational Ensembles Are Key for the Successful Prediction of Protein Complexes

Conformational fluctuations in proteins play key roles in their functions and interactions. In this work, validated conformational ensembles for ubiquitin have been used in docking trials. The ensembles were used in a systematic predictive study of known ubiquitin complexes by applying a cross-docking strategy against the bound structure of each partner. The global docking predictions obtained with the complete ubiquitin ensembles were significantly better than those obtained with the crystallographic structure of free ubiquitin. Importantly, in all cases we identified an individual ensemble member that performed equally well, or even better, than the bound structure of ubiquitin. These results unequivocally demonstrate that, for proteins that recognize binding partners by conformational selection, the availability of conformational ensembles can greatly improve the performance of automatic docking predictions. Our results highlight the need for docking methodologies to capitalize on validated ensemble representations of biomacromolecules

Genetic Interactions Implicating Postreplicative Repair in Okazaki Fragment Processing

<div><p>Ubiquitination of the replication clamp proliferating cell nuclear antigen (PCNA) at the conserved residue lysine (K)164 triggers postreplicative repair (PRR) to fill single-stranded gaps that result from stalled DNA polymerases. However, it has remained elusive as to whether cells engage PRR in response to replication defects that do not directly impair DNA synthesis. To experimentally address this question, we performed synthetic genetic array (SGA) analysis with a ubiquitination-deficient K164 to arginine (K164R) mutant of PCNA against a library of <i>S</i>. <i>cerevisiae</i> temperature-sensitive alleles. The SGA signature of the K164R allele showed a striking correlation with profiles of mutants deficient in various aspects of lagging strand replication, including <i>rad27Δ</i> and <i>elg1Δ</i>. Rad27 is the primary flap endonuclease that processes 5’ flaps generated during lagging strand replication, whereas Elg1 has been implicated in unloading PCNA from chromatin. We observed chronic ubiquitination of PCNA at K164 in both <i>rad27Δ</i> and <i>elg1Δ</i> mutants. Notably, only <i>rad27Δ</i> cells exhibited a decline in cell viability upon elimination of PRR pathways, whereas <i>elg1Δ</i> mutants were not affected. We further provide evidence that K164 ubiquitination suppresses replication stress resulting from defective flap processing during Okazaki fragment maturation. Accordingly, ablation of PCNA ubiquitination increased S phase checkpoint activation, indicated by hyperphosphorylation of the Rad53 kinase. Furthermore, we demonstrate that alternative flap processing by overexpression of catalytically active exonuclease 1 eliminates PCNA ubiquitination. This suggests a model in which unprocessed flaps may directly participate in PRR signaling. Our findings demonstrate that PCNA ubiquitination at K164 in response to replication stress is not limited to DNA synthesis defects but extends to DNA processing during lagging strand replication.</p></div

Overexpression of <i>EXO1</i> does not alter PCNA ubiquitination under conditions that cause ssDNA gap formation.

<p>(A) UV treatment leads to the formation of ssDNA gaps in replicating cells [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005659#pgen.1005659.ref012" target="_blank">12</a>]. Overexpression of <i>EXO1</i> has no impact on ssDNA gap formation. The red triangles indicate UV-induced lesions. RPA-coated ssDNA is marked as RPA-ssDNA. (B) Cells carrying gal-EV or gal-<i>EXO1</i> plasmids were grown to OD₆₀₀ = 0.600 at 25°C in raffinose containing medium lacking uracil. Galactose was then added to a final concentration of 2% and the cells were grown an additional 2 h at 25°C. Each culture was then split into 3 parts and treated with either 0, 50, or 100 J/m² UV and left to recover for 40 min at 25°C before harvesting. His₆-PCNA was purified under denaturing conditions and analyzed by western blot with antibodies specific to PCNA, ubiquitin, and SUMO as indicated. (C) Inefficient priming along the lagging strand template leads to the formation of RPA-coated ssDNA gaps (RPA-ssDNA) in <i>pol1</i> mutants [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005659#pgen.1005659.ref023" target="_blank">23</a>,<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005659#pgen.1005659.ref064" target="_blank">64</a>,<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005659#pgen.1005659.ref066" target="_blank">66</a>]. Overexpression of <i>EXO1</i> has no impact on ssDNA gap formation. (D) Wild-type and <i>pol1-1</i> cells carrying gal-EV or gal-<i>EXO1</i> plasmids were grown to OD₆₀₀ = 0.600 at 25°C in raffinose containing medium lacking uracil. Galactose was then added to a final concentration of 2% and the cultures we shifted to 35°C for 3 h before harvesting. His₆-PCNA was purified under denaturing conditions and analyzed by western blot with antibodies specific to PCNA, ubiquitin, and SUMO as indicated.</p

The PCNA-K164R SGA profile exhibits a limited set of direct genetic interactions but correlates strongly with other replication mutants.

<p>(A) Heat map of selected significant negative genetic interactions identified by SGA against the TS array for 2 independently isolated PCNA-K164R mutants. PCNA-WT and PCNA-DAmP are shown for comparison. The scale indicates epsilon- (ε-) values for the reported genetic interactions with negative interactions in red and positive interactions in green. (B) Heat map denoting the strength of correlation (measured by PCC) between PCNA-WT, PCNA-DAmP, PCNA-K164R clone 1 (Cl. 1) and PCNA-K164R clone 2 (Cl. 2) signatures against the TS array with the SGA signatures of the indicated strains. The scale denotes the strength of correlation between the indicated profiles with green being positive correlation and red being negative (C) Top 15 GO terms (<a href="http://www.ebi.ac.uk/QuickGO/" target="_blank">http://www.ebi.ac.uk/QuickGO/</a>) for alleles with PCC > 0.15 with the PCNA-K164R Cl. 1 profile against the FG array. This list is derived from a representative allele randomization (randomization 1 in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005659#pgen.1005659.s009" target="_blank">S5 Table</a>). Nine other randomizations were performed with similar results (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005659#pgen.1005659.s009" target="_blank">S5 Table</a>).</p

Lagging strand replication mutants that correlate strongly with PCNA-DAmP and PCNA-K164R.

<p>PCC values for mutant alleles of lagging strand replication genes with TS array signatures that correlated strongly with PCNA-DAmP, PCNA-K164R Cl. 1, and PCNA-K164R Cl.2.</p><p>Lagging strand replication mutants that correlate strongly with PCNA-DAmP and PCNA-K164R.</p

<i>rad27Δ pol30-K164R</i> double mutants exhibit increased checkpoint activation and cell cycle arrest.

<p>(A) The indicated strains were grown to OD₆₀₀ = 0.600 at 25°C and then shifted to 37°C for 4 h. Samples were harvested immediately before the temperature shift and every hour for 4 h afterward. Protein was subsequently extracted by TCA precipitation. Extracts were fractionated by SDS-PAGE and analyzed by western blot with anti-Rad53 and anti-tubulin antibodies. Tubulin served as a loading control. (B) Aliquots of the same cultures from (A) were analyzed for DNA content by flow cytometry. Red arrows indicate the G1 peaks of <i>rad27Δ</i> and <i>rad27Δ pol30-K164R</i> after 3 and 4 h at 37°C for comparison. Percentages indicate the percent of all cells analyzed that fall within the highlighted area. Green regions indicate G1 phase peaks while red regions mark late S and G2/M phases.</p

TLS and template switching are redundant in promoting <i>rad27Δ</i> viability.

<p>(A and B) 10-fold serial dilutions of the indicated strains were incubated 1.5 or 2.5 days on YPD plates at varying temperatures. (C) Bars indicate the rate of mutation at the <i>CAN1</i> locus for the indicated strains. Each measurement represents the median of at least 14 independent determinations. Significance was determined using the Mann-Whitney U test.</p

Legislative Documents

Also, variously referred to as: Senate bills; Senate documents; Senate legislative documents; legislative documents; and General Court documents

AdditionalDataS5

This file contains the complete list of yeast strains present on the diagnostic array, which was used for genetic interaction screens in this study