A sub-lethal canavanine concentration induced mutagenesis in culture that involves Msn2-Msn4, Rev1, Rev3 and Ku.

<p>(A) 2.5 µg/ml canavanine is not lethal to cells of the various mutants examined in these experiments. Average plating efficiencies and standard deviations of at least three cultures for each strain are shown. (B) Canavanine induces mutagenesis in culture, generating FOA^R mutants. Canavanine-induced mutagenesis is partially reduced in strains deleted for <i>MSN2</i> and <i>MSN4</i> (<i>msnΔ</i>) or <i>REV1</i> and fully abolished in strains deleted for <i>REV3</i> or <i>YKU80</i>. Also, <i>MSN2</i> and <i>MSN4</i> have different genetic interactions with <i>REV1</i> and <i>REV3</i>/<i>YKU80</i> in this process: <i>rev1Δ</i> is further compromised for canavanine-induced mutagenesis by <i>msnΔ</i>, while <i>rev3Δ</i> and <i>yku80Δ</i> are rescued by the <i>msnΔ</i> mutant. Mutation rates were calculated from 80 to 300 cultures for each genotype; error bars show 95% confidence intervals. (C) A simple model, consistent with data in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003680#pgen-1003680-g005" target="_blank">Figure 5B</a>, proposes that the ESR functions upstream of Rev3 and Yku80 in canavanine-induced mutagenesis.</p

Two types of environmental stress known to cause mutagenesis do not require <i>MSN2</i> and <i>MSN4</i>.

<p>(A) Viability of the <i>msnΔ</i> strain is not significantly affected relative to that of the WT strain by growth in the presence of 1 M NaCl or 100 mM HU. Average plating efficiencies and standard deviations of at least three cultures for each strain are shown. (B) Osmotic stress in culture induces mutation or <i>can1</i> and generation of FOA^R mutants both in WT and <i>msnΔ</i> strains. (C) DNA replication stress induces mutation of <i>can1</i> more strongly than FOA^R mutagenesis, and neither process depends on <i>MSN2</i> and <i>MSN4</i>. Plotted are mutation rates and 95% confidence intervals calculated from fluctuation experiments using at least 40 cultures for canavanine resistance and at least 80 cultures for FOA resistance.</p

The Yeast Environmental Stress Response Regulates Mutagenesis Induced by Proteotoxic Stress

<div><p>Conditions of chronic stress are associated with genetic instability in many organisms, but the roles of stress responses in mutagenesis have so far been elucidated only in bacteria. Here, we present data demonstrating that the environmental stress response (ESR) in yeast functions in mutagenesis induced by proteotoxic stress. We show that the drug canavanine causes proteotoxic stress, activates the ESR, and induces mutagenesis at several loci in an ESR-dependent manner. Canavanine-induced mutagenesis also involves translesion DNA polymerases Rev1 and Polζ and non-homologous end joining factor Ku. Furthermore, under conditions of chronic sub-lethal canavanine stress, deletions of Rev1, Polζ, and Ku-encoding genes exhibit genetic interactions with ESR mutants indicative of ESR regulating these mutagenic DNA repair processes. Analyses of mutagenesis induced by several different stresses showed that the ESR specifically modulates mutagenesis induced by proteotoxic stress. Together, these results document the first known example of an involvement of a eukaryotic stress response pathway in mutagenesis and have important implications for mechanisms of evolution, carcinogenesis, and emergence of drug-resistant pathogens and chemotherapy-resistant tumors.</p></div

Western blot showing that the ER chaperone Kar2 is induced in response to treatment with canavanine in culture (‘Can’: cells were cultured in the presence of 100 µg/ml canavanine for 6 hours) but not to osmotic stress (‘Osm’: cells were cultured in the presence of 0.5M NaCl for 6 hours) or glucose starvation (‘NG’ = no glucose: cells were cultured in medium lacking glucose for 6 hours).

<p>Both WT and <i>msn2Δ msn4Δ</i> (<i>msnΔ</i>) strains exhibited a canavanine-dependent increase in Kar2 levels. Msh2-13xMyc served as loading control.</p

Different types of proteotoxic stress have different consequences on promoting formation canavanine-resistant versus FOA-resistant mutants and differentially involve Msn2-Msn4.

<p>(A) Graph showing cell viability of the WT and <i>msnΔ</i> strains in the presence of indicated concentrations of radicicol, PFPA, and tunicamycin (TN). (B) Mutation rates in culture to generate canavanine-resistant (left) or FOA-resistant (right) mutants in the absence and presence of 25 µg/ml radicicol. Radicicol did not induce <i>can1</i> mutagenesis but did induce FOA^R mutant formation in a manner that was independent of <i>MSN2</i> and <i>MSN4</i>. (C) Mutation rates in culture to generate canavanine-resistant (left) or FOA-resistant (right) mutants in the absence and presence of 50 µg/ml PFPA. PFPA induced <i>can1</i> mutagenesis by two-fold (consistent with <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003680#pgen.1003680-Davies1" target="_blank">[41]</a>) in the WT and <i>msnΔ</i> strains. However, PFPA induced FOA^R mutations only in the <i>msnΔ</i> mutant, indicating that Msn2-Msn4 function to suppress PFPA-induced mutagenesis at some loci. (D) Mutation rates in culture to generate canavanine-resistant (left) or FOA-resistant (right) mutants in the absence and presence of 1 µg/ml tunicamycin (TN). Tunicamycin did not significantly affect <i>can1</i> mutagenesis, but did induce FOA^R mutations only in the WT strain, showing that Msn2-Msn4 can promote tunicamycin-induced mutagenesis.</p

<i>CAN1</i> mutations can occur after cells are plated on canavanine medium.

<p>(A) Cultures incubated on canavanine plates produced <i>can1</i> mutant colonies, some of which were very small (black arrowheads). (B) “Large” colony data from >200 independent cultures fit Luria-Delbrück distribution (L-D) better than Poisson distribution, while “small” colony data fit Poisson better than L-D. Correlation coefficients between data and theoretical distributions that best fit the data are shown. (C) Reconstruction experiment demonstrating that small canavanine-resistant colonies are not inherently slow growing. All of the <i>can1</i> colonies originated from seeded <i>can1</i> cells and all gave rise to large colonies, irrespective of whether they originally came from large or small <i>can1</i> colonies. (D) NHEJ and DNA polymerase ζ affect canavanine-induced mutagenesis. <i>yku80Δ</i> reduced post-plating mutation (right panel) while leaving mutation in culture unaffected (left panel), while <i>rev3Δ</i> reduced both <i>CAN1</i> mutation in culture and post-plating. Error bars represent 95% confidence intervals for mutation rates in culture and standard deviations for post-plating mutation frequencies (*, P<0.05, Student's t-test).</p

The ESR plays a role in canavanine-induced <i>can1</i> mutation.

<p>(A) and (B) Nuclear Msn2-GFP localization increased in canavanine-sensitive cells after plating them on canavanine, reflecting ESR activation. For the graph in (B) 20 to 55 cells were analyzed for each condition and the differences between <i>CAN1</i> and <i>can1-100</i> strains at 6 and 12 hours were highly statistically significant (Fisher exact test P<10⁻⁵). (C) <i>msnΔ</i> greatly reduced post-plating <i>CAN1</i> mutation (**, P = 0.005, Student's t-test; averages and standard deviations of three experiments are shown) but not mutation in culture (calculated from L-D distributions of large <i>can1</i> mutants; error bars show 95% confidence intervals). (D) Cell survival and proliferation on canavanine medium were not significantly affected by <i>msnΔ</i>. Averages and standard deviations of three to nine experiments are shown.</p

Mutation spectra of <i>can1</i> mutants generated on canavanine plates exhibit several significant differences with those generated in culture.

<p>(A) The overall distribution of different <i>can1</i> mutation categories is not altered between pre-plating(large) and post-plating (small) <i>can1</i> mutants. The “other” category contains multiple mutations per <i>can1</i> ORF, usually clustered together. (B) Pre-plating (large) but not post-plating (small) <i>can1</i> mutants contained transcription-associated mutation (TAM) hotspots. Lippert et al identified the five main TAM hotspots in <i>CAN1</i>, with ATΔ at CATAT at position 1127 being the most frequent in both their study and ours <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003680#pgen.1003680-Lippert1" target="_blank">[39]</a>. (C) Post-plating mutants were characterized by a significant increase in single nucleotide deletions (predominantly occurring in mononucleotide runs; <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003680#pgen.1003680.s005" target="_blank">Table S1</a>). (D) Base pair substitution spectra differed significantly between pre- and post-plating mutants.</p

Mutation spectra of pre- and post-plating <i>can1</i> mutants generated in the <i>msnΔ</i> strain.

<p>(A) The overall distribution of different <i>can1</i> mutation categories is not altered between pre-plating (large) and post-plating (small) <i>can1</i> mutants derived from the <i>msnΔ</i> strain. (B) As in the WT strain, pre-plating (large) but not post-plating (small) <i>can1</i> mutants derived from the <i>msnΔ</i> strain contained transcription-associated mutation (TAM) hotspots <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003680#pgen.1003680-Lippert1" target="_blank">[39]</a>. (C) In contrast to the WT strain where there was a significant shift in the types of deletions observed in the large vs small <i>can1</i> colonies (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003680#pgen-1003680-g002" target="_blank">Figure 2C</a>), there was no such shift in the <i>msnΔ</i> mutant. (D) Also unlike the WT strain, in the <i>msnΔ</i> mutant there was no significant change in the types of base pair substitutions recovered from pre- and post-plating <i>can1</i> mutants.</p

MaPS identifies <i>RAP1</i> as the gene whose product interacts with the Rap1 binding site.

<p>The yeast genomic DNA phage display library was selected for three rounds against a double-stranded oligonucleotide and PCR products of an upstream region containing Rap1 binding sites. The selected population of phage were profiled through microarray hybridization. Displayed is the distribution of the mean percentile rank for five independent such selections performed. The ORF corresponding to Rap1 had the highest mean percentile rank out of a total of 6242 ORFs queried on the array.</p