A modified fluctuation assay reveals a natural mutator phenotype that drives mutation spectrum variation within Saccharomyces cerevisiae

Although studies of Saccharomyces cerevisiae have provided many insights into mutagenesis and DNA repair, most of this work has focused on a few laboratory strains. Much less is known about the phenotypic effects of natural variation within S. cerevisiae's DNA repair pathways. Here, we use natural polymorphisms to detect historical mutation spectrum differences among several wild and domesticated S. cerevisiae strains. To determine whether these differences are likely caused by genetic mutation rate modifiers, we use a modified fluctuation assay with a CAN1 reporter to measure de novo mutation rates and spectra in 16 of the analyzed strains. We measure a 10-fold range of mutation rates and identify two strains with distinctive mutation spectra. These strains, known as AEQ and AAR, come from the panel's 'Mosaic beer' clade and share an enrichment for C>A mutations that is also observed in rare variation segregating throughout the genomes of several Mosaic beer and Mixed origin strains. Both AEQ and AAR are haploid derivatives of the diploid natural isolate CBS 1782, whose rare polymorphisms are enriched for C>A as well, suggesting that the underlying mutator allele is likely active in nature. We use a plasmid complementation test to show that AAR and AEQ share a mutator allele in the DNA repair gene OGG1, which excises 8-oxoguanine lesions that can cause C>A mutations if left unrepaired.

Spontaneous Polyploids and Antimutators Compete During the Evolution of Saccharomyces cerevisiae Mutator Cells

Mutations affecting DNA polymerase exonuclease domains or mismatch repair (MMR) generate “mutator” phenotypes capable of driving tumorigenesis. Cancers with both defects exhibit an explosive increase in mutation burden that appears to reach a threshold, consistent with selection acting against further mutation accumulation. In Saccharomyces cerevisiae haploid yeast, simultaneous defects in polymerase proofreading and MMR select for “antimutator” mutants that suppress the mutator phenotype. We report here that spontaneous polyploids also escape this “error-induced extinction” and routinely outcompete antimutators in evolved haploid cultures. We performed similar experiments to explore how diploid yeast adapt to the mutator phenotype. We first evolved cells with homozygous mutations affecting polymerase δ proofreading and MMR, which we anticipated would favor tetraploid emergence. While tetraploids arose with a low frequency, in most cultures, a single antimutator clone rose to prominence carrying biallelic mutations affecting the polymerase mutator alleles. Variation in mutation rate between subclones from the same culture suggests that there exists continued selection pressure for additional antimutator alleles. We then evolved diploid yeast modeling MMR-deficient cancers with the most common heterozygous exonuclease domain mutation (POLE-P286R). Although these cells grew robustly, within 120 generations, all subclones carried truncating or nonsynonymous mutations in the POLE-P286R homologous allele (pol2-P301R) that suppressed the mutator phenotype as much as 100-fold. Independent adaptive events in the same culture were common. Our findings suggest that analogous tumor cell populations may adapt to the threat of extinction by polyclonal mutations that neutralize the POLE mutator allele and preserve intratumoral genetic diversity for future adaptation.

Spontaneous polyploids and antimutators compete during the evolution of mutator cells

Heterozygous mutations affecting DNA polymerase (Pol) exonuclease domains and homozygous inactivation of mismatch repair (MMR) each generate “mutator” phenotypes capable of driving tumorigenesis. Cancers with both defects exhibit an explosive increase in mutation burden that appears to reach a threshold, consistent with selection acting against further mutation accumulation. In haploid yeast, simultaneous defects in polymerase proofreading and MMR select for “antimutator” mutants that suppress the mutator phenotype. We report here that spontaneous polyploids also escape this “error-induced extinction” and routinely out-compete antimutators in evolved haploid cultures. We performed similar experiments to explore how diploid yeast adapt to the mutator phenotype. We first evolved cells with homozygous mutations affecting proofreading and MMR, which we anticipated would favor tetraploid emergence. While tetraploids arose with a low frequency, in most cultures, a single antimutator clone rose to prominence carrying biallelic mutations affecting the polymerase mutator alleles. Variation in mutation rate between subclones from the same culture suggests there exists continued selection pressure for additional antimutator alleles. We then evolved diploid yeast modeling MMR-deficient cancers with the most common heterozygous exonuclease domain mutation (POLE-P286R). Although these cells grew robustly, within 120 generations, all subclones carried truncating or nonsynonymous mutations in the POLE-P286R homologous allele (pol2-P301R) that suppressed the mutator phenotype as much as 100-fold. Independent adaptive events in the same culture were common. Our findings suggest that analogous tumor cell populations may adapt to the threat of extinction by polyclonal mutations that neutralize the POLE mutator allele and preserve intra-tumoral genetic diversity for future adaptation.

Known mutator alleles do not markedly increase mutation rate in clinical Saccharomyces cerevisiae strains

Natural selection has the potential to act on all phenotypes, including genomic mutation rate. Classic evolutionary theory predicts that in asexual populations, mutator alleles, which cause high mutation rates, can fix due to linkage with beneficial mutations. This phenomenon has been demonstrated experimentally and may explain the frequency of mutators found in bacterial pathogens. By contrast, in sexual populations, recombination decouples mutator alleles from beneficial mutations, preventing mutator fixation. In the facultatively sexual yeast Saccharomyces cerevisiae , segregating alleles of MLH1 and PMS1 have been shown to be incompatible, causing a high mutation rate when combined. These alleles had never been found together naturally, but were recently discovered in a cluster of clinical isolates. Here we report that the incompatible mutator allele combination only marginally elevates mutation rate in these clinical strains. Genomic and phylogenetic analyses provide no evidence of a historically elevated mutation rate. We conclude that the effect of the mutator alleles is dampened by background genetic modifiers. Thus, the relationship between mutation rate and microbial pathogenicity may be more complex than once thought. Our findings provide rare observational evidence that supports evolutionary theory suggesting that sexual organisms are unlikely to harbour alleles that increase their genomic mutation rate.

The effect of known mutator alleles in a cluster of clinicalSaccharomyces cerevisiaestrains

Natural selection has the potential to act on all phenotypes, including genomic mutation rate. Classic evolutionary theory predicts that in asexual populations, mutator alleles, which cause high mutation rates, can fix due to linkage with beneficial mutations. This phenomenon has been demonstrated experimentally and may explain the frequency of mutators found in bacterial pathogens. In contrast, in sexual populations, recombination decouples mutator alleles from beneficial mutations, preventing mutator fixation. In the facultatively sexual yeastSaccharomyces cerevisiae, segregating alleles ofMLH1andPMS1have been shown to be incompatible, causing a high mutation rate when combined. These alleles had never been found together naturally, but were recently discovered in a cluster of clinical isolates. Here we report that the incompatible mutator allele combination only marginally elevates mutation rate in these clinical strains. Genomic and phylogenetic analyses provide no evidence of a historically elevated mutation rate. We conclude that the effect of the mutator alleles is dampened by background genetic modifiers. Thus, the relationship between mutation rate and microbial pathogenicity may be more complex than once thought. Our findings provide rare observational evidence that supports evolutionary theory suggesting that sexual organisms are unlikely to harbor alleles that increase their genomic mutation rate.

Effects of antibiotic resistance alleles on bacterial evolutionary responses to viral parasites

Antibiotic resistance has wide-ranging effects on bacterial phenotypes and evolution. However, the influence of antibiotic resistance on bacterial responses to parasitic viruses remains unclear, despite the ubiquity of such viruses in nature and current interest in therapeutic applications. We experimentally investigated this by exposing various Escherichia coli genotypes, including eight antibiotic-resistant genotypes and a mutator, to different viruses (lytic bacteriophages). Across 960 populations, we measured changes in population density and sensitivity to viruses, and tested whether variation among bacterial genotypes was explained by their relative growth in the absence of parasites, or mutation rate towards phage resistance measured by fluctuation tests for each phage. We found that antibiotic resistance had relatively weak effects on adaptation to phages, although some antibiotic-resistance alleles impeded the evolution of resistance to phages via growth costs. By contrast, a mutator allele, often found in antibiotic-resistant lineages in pathogenic populations, had a relatively large positive effect on phage-resistance evolution and population density under parasitism. This suggests costs of antibiotic resistance may modify the outcome of phage therapy against pathogenic populations previously exposed to antibiotics, but the effects of any co-occurring mutator alleles are likely to be stronger.

Competition between high- and higher-mutating strains of Escherichia coli

Experimental studies have shown that a mutator allele can readily hitchhike to fixation with beneficial mutations in an asexual population having a low, wild-type mutation rate. Here, we show that a genotype bearing two mutator alleles can supplant a population already fixed for one mutator allele. Our results provide experimental support for recent theory predicting that mutator alleles will tend to accumulate in asexual populations by hitchhiking with beneficial mutations, causing an ever-higher genomic mutation rate.

Fitness Evolution and the Rise of Mutator Alleles in Experimental Escherichia coli Populations

We studied the evolution of high mutation rates and the evolution of fitness in three experimental populations of Escherichia coli adapting to a glucose-limited environment. We identified the mutations responsible for the high mutation rates and show that their rate of substitution in all three populations was too rapid to be accounted for simply by genetic drift. In two of the populations, large gains in fitness relative to the ancestor occurred as the mutator alleles rose to fixation, strongly supporting the conclusion that mutator alleles fixed by hitchhiking with beneficial mutations at other loci. In one population, no significant gain in fitness relative to the ancestor occurred in the population as a whole while the mutator allele rose to fixation, but a substantial and significant gain in fitness occurred in the mutator subpopulation as the mutator neared fixation. The spread of the mutator allele from rarity to fixation took >1000 generations in each population. We show that simultaneous adaptive gains in both the mutator and wild-type subpopulations (clonal interference) retarded the mutator fixation in at least one of the populations. We found little evidence that the evolution of high mutation rates accelerated adaptation in these populations.