Project description:The yeast Saccharomyces cerevisiae has emerged as a superior model organism. Selection of distinct laboratory strains of S. cerevisiae with unique phenotypic properties, such as superior mating or sporulation efficiencies, has facilitated advancements in research. W303 is one such laboratory strain that is closely related to the first completely sequenced yeast strain, S288C. In this work, we provide a high-quality, annotated genome sequence for W303 for utilization in comparative analyses and genome-wide studies. Approximately 9500 variations exist between S288C and W303, affecting the protein sequences of ?700 genes. A listing of the polymorphisms and divergent genes is provided for researchers interested in identifying the genetic basis for phenotypic differences between W303 and S288C. Several divergent functional gene families were identified, including flocculation and sporulation genes, likely representing selection for desirable laboratory phenotypes. Interestingly, remnants of ancestor wine strains were found on several chromosomes. Finally, as a test of the utility of the high-quality reference genome, variant mapping revealed more accurate identification of accumulated mutations in passaged mismatch repair-defective strains.
Project description:Mutational load is the depression in a population's mean fitness that results from the continual influx of deleterious mutations. Here, we directly estimate the mutational load in a population of haploid Saccharomyces cerevisiae that are deficient for mismatch repair. We partition the load in haploids into two components. To estimate the load due to nonlethal mutations, we measure the competitive fitness of hundreds of randomly selected clones from both mismatch-repair-deficient and -proficient populations. Computation of the mean clone fitness for the mismatch-repair-deficient strain permits an estimation of the nonlethal load, and the histogram of fitness provides an interesting visualization of a loaded population. In a separate experiment, in order to estimate the load due to lethal mutations (i.e. the lethal mutation rate), we manipulate thousands of individual pairs of mother and daughter cells and track their fates. These two approaches yield point estimates for the two contributors to load, and the addition of these estimates is nearly equal to the separately measured short-term competitive fitness deficit for the mismatch-repair-deficient strain. This correspondence suggests that there is no need to invoke direct fitness effects to explain the fitness difference between mismatch-repair-deficient and -proficient strains. Assays in diploids are consistent with deleterious mutations in diploids tending towards recessivity. These results enhance our understanding of mutational load, a central population genetics concept, and we discuss their implications for the evolution of mutation rates.
Project description: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 <i>Saccharomyces cerevisiae</i> 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 (<i>POLE-P286R</i>). Although these cells grew robustly, within 120 generations, all subclones carried truncating or nonsynonymous mutations in the <i>POLE-P286R</i> homologous allele (<i>pol2</i> <i>-P301R</i>) 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 <i>POLE</i> mutator allele and preserve intratumoral genetic diversity for future adaptation.
Project description:Mutation of predicted 3'-->5' exonuclease active site residues of Saccharomyces cerevisiae POL3 DNA polymerase (delta) or deletion of the PMS1 mismatch repair gene lead to relative (to wild type) spontaneous mutation rates of approximately 130 and 41, respectively, measured at a URA3 reporter gene inserted near to a defined replication origin. The POL3 exonuclease-deficient mutant pol3-01 generated most classes of single base mutation in URA3, indicating a broad specificity that generally corresponds to that of the PMS1 system. pol3-01 pms1 haploid cells ceased growth after a few divisions with no unique terminal cell morphology. A pol3-01/pol3-01 pms1/pms1 diploid was viable and displayed an estimated URA3 relative mutation rate of 2 x 10(4), which we calculate to be catastrophically high in a haploid. The relationship between the relative mutation rates of pol3-01 and pms1 was multiplicative, indicating action in series. The PMS1 transcript showed the same cell cycle periodicity as those of a set of DNA replication genes that includes POL3, suggesting PMS1 is co-regulated with these genes. We propose that the POL3 3'-->5' exonuclease and the PMS1 mismatch repair system act on a common pathway analogous to the dnaQ-->mutHLS pathway of DNA replication error correction in Escherichia coli.
Project description:In budding yeast, the MLH1-PMS1 heterodimer is the major MutL homolog complex that acts to repair mismatches arising during DNA replication. Using a highly sensitive mutator assay, we observed that Saccharomyces cerevisiae strains bearing the S288c-strain-derived MLH1 gene and the SK1-strain-derived PMS1 gene displayed elevated mutation rates that conferred a long-term fitness cost. Dissection of this negative epistatic interaction using S288c-SK1 chimeras revealed that a single amino acid polymorphism in each gene accounts for this mismatch repair defect. Were these strains to cross in natural populations, segregation of alleles would generate a mutator phenotype that, although potentially transiently adaptive, would ultimately be selected against because of the accumulation of deleterious mutations. Such fitness "incompatibilities" could potentially contribute to reproductive isolation among geographically dispersed yeast. This same segregational mutator phenotype suggests a mechanism to explain some cases of a human cancer susceptibility syndrome known as hereditary nonpolyposis colorectal cancer, as well as some sporadic cancers.
Project description:Transcriptional profiling of Saccharomyces cerevisiae cells comparing the W303-1A wildtype with the W303-1A double mutant for MSN2 and MSN4 during zinc deficient conditions Keywords: Genetic modification with zinc limitation Two condition experiment, W303-1A vs W303-1A delta MSN2, MSN4. Biological replicates: 2 wildtype, 2 knock-out, independently grown and harvested.
Project description:DNA replication fidelity relies on base selectivity of the replicative DNA polymerases, exonucleolytic proofreading, and postreplicative DNA mismatch repair (MMR). Ultramutated human cancers without MMR defects carry alterations in the exonuclease domain of DNA polymerase ? (Pol?). They have been hypothesized to result from defective proofreading. However, modeling of the most common variant, Pol?-P286R, in yeast produced an unexpectedly strong mutator effect that exceeded the effect of proofreading deficiency by two orders of magnitude and indicated the involvement of other infidelity factors. The in vivo consequences of many additional Pol? mutations reported in cancers remain poorly understood. Here, we genetically characterized 13 cancer-associated Pol? variants in the yeast system. Only variants directly altering the DNA binding cleft in the exonuclease domain elevated the mutation rate. Among these, frequently recurring variants were stronger mutators than rare variants, in agreement with the idea that mutator phenotype has a causative role in tumorigenesis. In nearly all cases, the mutator effects exceeded those of an exonuclease-null allele, suggesting that mechanisms distinct from loss of proofreading may drive the genome instability in most ultramutated tumors. All mutator alleles were semidominant, supporting the view that heterozygosity for the polymerase mutations is sufficient for tumor development. In contrast to the DNA binding cleft alterations, peripherally located variants, including a highly recurrent V411L, did not significantly elevate mutagenesis. Finally, the analysis of Pol? variants found in MMR-deficient tumors suggested that the majority cause no mutator phenotype alone but some can synergize with MMR deficiency to increase the mutation rate.
Project description:Hereditary nonpolyposis colorectal cancer (HNPCC) is associated with defects in DNA mismatch repair. Mutations in either hMSH2 or hMLH1 underlie the majority of HNPCC cases. Approximately 25% of annotated hMSH2 disease alleles are missense mutations, resulting in a single change out of 934 amino acids. We engineered 54 missense mutations in the cognate positions in yeast MSH2 and tested for function. Of the human alleles, 55% conferred strong defects, 8% displayed intermediate defects, and 38% showed no defects in mismatch repair assays. Fifty percent of the defective alleles resulted in decreased steady-state levels of the variant Msh2 protein, and 49% of the Msh2 variants lost crucial protein-protein interactions. Finally, nine positions are predicted to influence the mismatch recognition complex ATPase activity. In summary, the missense mutations leading to loss of mismatch repair defined important structure-function relationships and the molecular analysis revealed the nature of the deficiency for Msh2 variants expressed in the tumors. Of medical relevance are 15 human alleles annotated as pathogenic in public databases that conferred no obvious defects in mismatch repair assays. This analysis underscores the importance of functional characterization of missense alleles to ensure that they are the causative factor for disease.
Project description:Although it is clear that postreplicative DNA mismatch repair (MMR) plays a critical role in maintaining genomic stability in nearly all forms of life surveyed, much remains to be understood about the genome-wide impact of MMR on spontaneous mutation processes and the extent to which MMR-deficient mutation patterns vary among species. We analyzed spontaneous mutation processes across multiple genomic regions using two sets of mismatch repair-deficient (msh-2 and msh-6) Caenorhabditis elegans mutation-accumulation (MA) lines and compared our observations to mutation spectra in a set of wild-type (WT), repair-proficient C. elegans MA lines. Across most sequences surveyed in the MMR-deficient MA lines, mutation rates were approximately 100-fold higher than rates in the WT MA lines, although homopolymeric nucleotide-run (HP) loci composed of A:T base pairs mutated at an approximately 500-fold greater rate. In contrast to yeast and humans where mutation spectra vary substantially with respect to different specific MMR-deficient genotypes, mutation rates and patterns were overall highly similar between the msh-2 and msh-6 C. elegans MA lines. This, along with the apparent absence of a Saccharomyces cerevisiae MSH3 ortholog in the C. elegans genome, suggests that C. elegans MMR surveillance is carried out by a single Msh-2/Msh-6 heterodimer.