A new dominant peroxiredoxin allele identified by whole-genome re-sequencing of random mutagenized yeast causes oxidant-resistance and premature aging

Abstract

The combination of functional genomics with next generation sequencing facilitates new experimental strategies for addressing complex biological phenomena. Here, we report the identification of a gain-of-function allele of peroxiredoxin (thioredoxin peroxidase, Tsa1p) via whole-genome re-sequencing of a dominantSaccharomyces cerevisiae mutant obtained by chemical mutagenesis. Yeast strain K6001, a screening system for lifespan phenotypes, was treated with ethyl methanesulfonate (EMS). We isolated an oxidative stress-resistant mutant (B7) which transmitted this phenotype in a background-independent, monogenic and dominant way. By massive parallel pyrosequencing, we generated an 38.8 fold whole-genome coverage of the strains, which differed in 12,482 positions from the reference (S288c) genome. Via a subtraction strategy, we could narrow this number to 13 total and 4 missense nucleotide variations that were specific for the mutant. Via expression in wild type backgrounds, we show that one of these mutations, exchanging a residue in the peroxiredoxin Tsa1p, was responsible for the mutant phenotype causing background-independent dominant oxidative stress-resistance. These effects were not provoked by altered Tsa1p levels, nor could they be simulated by deletion, haploinsufficiency or over-expression of the wild-type allele. Furthermore, via both a mother-enrichment technique and a micromanipulation assay, we found a robust premature aging phenotype of this oxidant-resistant strain. Thus, TSA1-B7 encodes for a novel dominant form of peroxiredoxin, and establishes a new connection between oxidative stress and aging. In addition, this study shows that the re-sequencing of entire genomes is becoming a promising alternative for the identification of functional alleles in approaches of classic molecular genetics.

Confirmation of the cellular targets of benomyl and rapamycin using next-generation sequencing of resistant mutants in S. cerevisiae

“…When we compared the pdr1Δ sequence with that of the reference genome, ~20% of the 128 SNPs in the putatively haploid pdr1Δ appeared heterozygous. The detection of non-uniform SNPs in a haploid organism is consistent with reports from other genome-wide studies in yeast[24], in which spurious diploidization and transient polysomy has been known to occur during or prior to selection[34, 35]. Indeed, all of the SNPs that appeared heterozygous in the parental strain were also found at a similar allele frequency in the drug-selected pools.…”

“…For example, the target of a new anti-tuberculosis drug was identified by whole-genome sequencing of resistant clones[22], and NGS approaches have been used to identify mutations responsible for echinocandin resistance in Candida galbrata [23]. Deep sequencing was also used to identify mutations that confer resistance to oxidative stress in S. cerevisiae [24]. …”

Peroxiredoxins, gerontogenes linking aging to genome instability and cancer

“…Prx deficiency results in accelerated aging in yeast, worms, flies, and rodents (Neumann et al 2003;Olahova et al 2008;Lee et al 2009;Timmermann et al 2010). Moreover, the yeast and rat Prxs, Tsa1 and PrxII, are becoming increasingly hyperoxidized during aging in the absence of external peroxide stress (Musicco et al 2009;Molin et al 2011), indicating that Prx inactivation may be a common phenomenon in different aging organisms.…”

Thioredoxins, Glutaredoxins, and Peroxiredoxins—Molecular Mechanisms and Health Significance: from Cofactors to Antioxidants to Redox Signaling

“…Mutations in various genes that interfere with the aging process have been characterized. These encode for proteins functioning either in the regulation of energy use or in redox regulation and signaling, including, for instance, catalase (770) and Prx (194,775). Various model organisms, including Sacharomyces cerevisiae, Cenorhabtitis elegans, Drosophily melanogaster, or Mus musculus, depleted from SOD (446), Trx (748), TrxR Prx, and PDIs (250), were characterized by a reduced lifespan, reviewed in (334).…”