Chemical Suppression of Defects in Mitotic Spindle Assembly, Redox Control, and Sterol Biosynthesis by Hydroxyurea

We describe the results of a systematic search for a class of hitherto-overlooked chemical-genetic interactions in the Saccharomyces cerevisiae genome, which exists between a detrimental genetic mutation and a chemical/drug that can ameliorate, rather than exacerbate, that detriment. We refer to this type of interaction as “chemical suppression.” Our work was driven by the hypothesis that genome instability in a certain class of mutants could be alleviated by mild replication inhibition using chemicals/drugs. We queried a collection of conditionally lethal, i.e., temperature-sensitive, alleles representing 40% of the yeast essential genes for those mutants whose growth defect can be suppressed by hydroxyurea (HU), known as a potent DNA replication inhibitor, at the restrictive temperature. Unexpectedly, we identified a number of mutants defective in diverse cellular pathways other than DNA replication. Here we report that HU suppresses selected mutants defective in the kinetochore-microtubule attachment pathway during mitotic chromosome segregation. HU also suppresses an ero1-1 mutant defective for a thiol oxidase of the endoplasmic reticulum by providing oxidation equivalents. Finally, we report that HU suppresses an erg26-1 mutant defective for a C-3 sterol dehydrogenase through regulating iron homeostasis and in turn impacting ergosterol biosynthesis. We further demonstrate that cells carrying the erg26-1 mutation show an increased rate of mitochondrial DNA loss and delayed G1 to S phase transition. We conclude that systematic gathering of a compendium of “chemical suppression” of yeast mutants by genotoxic drugs will not only enable the identification of novel functions of both chemicals and genes, but also have profound implications in cautionary measures of anticancer intervention in humans.

(A) The majority of the mutants whose temperature-sensitivity was rescued by HU cannot be rescued by nutrient limitation-induced cell cycle delay. Growth media with different carbon sources, identical in each of the three panels under different temperatures, are as shown. Plates were photographed after three days incubation at the respective temperatures. The order of strains on each plate is indicated by the key on the left. (B) Overexpression of the Rad53 kinase does not rescue the temperature sensitivity of the mutants suppressed by HU. WT, ipl1-2, spc105-4, and tub4-Y445D strains were transformed with the pRS316, YEp352, or pJA-98 (GAL-RAD53) plasmids bearing URA3 prototrophic marker. pRS316 and YEp352 are empty plasmid controls for the pJA-98(GAL-RAD53) plasmid. The resulting transformants were streaked on SC medium lacking uracil with either 2% glucose (repressed for RAD53 expression) or 3% galactose (induced for RAD53 expression). The plates were incubated at either 25°C or 37°C for two days before photographing. The keys to the strains on each plate are shown at the bottom of the panel. A 30°C 34.5°C 37°C

Figure S3
Thymidine limitation does not suppress most of the temperature-sensitive alleles that are suppressed by HU. Serial diluted cells were spotted on YPD media containing the indicated concentrations of thymidine and incubated at the indicated temperatures for 2-3 days before photographing. YPD medium containing 4 mM thymidine and 10 mM HU served as a control.
A HU specifically suppresses erg26-1, not other ergosterol mutants in the temperature-sensitive strain collection. The indicated strains were streaked on YPD medium with or without 10 mM HU and incubated at the indicated temperatures for two to three days before photographing.
A. McCulley et al.

SI
Table S1 Sequence of PCR and sequencing primers for ERO1 and ERG26.

mM HU Mutation Gene function prp9-ts
Subunit of the SF3a splicing factor complex, required for spliceosome assembly; acts after the formation of the U1 snRNP-pre-mRNA complex sup35-td Translation termination factor eRF3, has a role in mRNA deadenylation and decay; altered protein conformation creates the [PSI(+)] prion that alters translational fidelity and results in a nonsense suppressor phenotype tid3-1 (ndc80) Component of the evolutionarily conserved kinetochore-associated Ndc80 complex; conserved coiled-coil protein involved in chromosome segregation, spindle checkpoint activity, kinetochore assembly and clustering 50 mM HU Mutation Gene function lst8-6, -15 Component of the TOR signaling pathway mvd1-1296 ERG19, Mevalonate pyrophosphate decarboxylase, involved in the biosynthesis of isoprenoids and sterols, including ergosterol nop2-3 Probable RNA m(5)C methyltransferase, essential for processing and maturation of 27S pre-rRNA and large ribosomal subunit biogenesis; localized to the nucleolus; constituent of 66S preribosomal particles tsc3-2 Protein involved in sphingolipid biosynthesis 100 mM HU Mutation Gene function act1-111 Actin, structural protein involved in multiple cytoskeletal functions arp3-G302Y, -H161A, -G15C Component of the Arp2/3 complex, a highly conserved actin nucleation center required for the motility and integrity of actin patches cdc2-1 DNA polymerase delta cdc8-1 Thymidylate and uridylate kinase, de novo biosynthesis of pyrimidine deoxyribonucleotides; converts dTMP to dTDP and dUMP to dUTP cks1-35 Cyclin-dependent protein kinase regulatory subunit and adaptor esa1-1851 Catalytic subunit of the histone acetyltransferase complex (NuA4) gpi8-ts Subunit of the glycosylphosphatidylinositol transamidase complex las17-13 Actin assembly factor, activates the Arp2/3 protein complex mps3-7 Nuclear envelope protein required for SPB duplication and nuclear fusion nse1-16 Component of the SMC5-SMC6 complex nse4-ts2 Component of the SMC5-SMC6 complex pob3-Q308K Subunit of the heterodimeric FACT complex (Spt16p-Pob3p) pre2-2 Beta 5 subunit of the 20S proteasome rfa1-M2 Subunit of heterotrimeric Replication Protein A that binds ssDNA