Defining the impact of mutation accumulation on replicative lifespan in yeast using cancer-associated mutator phenotypes

Abstract

Mutations accumulate within somatic cells and have been proposed to contribute to aging. It is unclear what level of mutation burden may be required to consistently reduce cellular lifespan. Human cancers driven by a mutator phenotype represent an intriguing model to test this hypothesis, since they carry the highest mutation burdens of any human cell. However, it remains technically challenging to measure the replicative lifespan of individual mammalian cells. Here, we modeled the consequences of cancer-related mutator phenotypes on lifespan using yeast defective for mismatch repair (MMR) and/or leading strand (Polε) or lagging strand (Polδ) DNA polymerase proofreading. Only haploid mutator cells with significant lifetime mutation accumulation (MA) exhibited shorter lifespans. Diploid strains, derived by mating haploids of various genotypes, carried variable numbers of fixed mutations and a range of mutator phenotypes. Some diploid strains with fewer than two mutations per megabase displayed a 25% decrease in lifespan, suggesting that moderate numbers of random heterozygous mutations can increase mortality rate. As mutation rates and burdens climbed, lifespan steadily eroded. Strong diploid mutator phenotypes produced a form of genetic anticipation with regard to aging, where the longer a lineage persisted, the shorter lived cells became. Using MA lines, we established a relationship between mutation burden and lifespan, as well as population doubling time. Our observations define a threshold of random mutation burden that consistently decreases cellular longevity in diploid yeast cells. Many human cancers carry comparable mutation burdens, suggesting that while cancers appear immortal, individual cancer cells may suffer diminished lifespan due to accrued mutation burden.

DNA polymerase δ proofreads errors made by DNA polymerase ε

“…The exonuclease activity of both polymerases can be abolished by alanine substitutions at the conserved carboxylate residues in the ExoI motif FDIET/C (17,18). The resulting mutator phenotype of the Polδ-exo − variant is an order of magnitude stronger than the phenotype of the analogous Pole-exo − variant (2,(17)(18)(19)(20)(21)(22)(23)(24)(25). Furthermore, haploid yeast deficient in Polδ proofreading do not survive when DNA mismatch repair (MMR) is also inactivated with the death attributed to an excessive level of mutagenesis (26).…”

Spontaneous polyploids and antimutators compete during the evolution of mutator cells

“…AH5610 was constructed for this study by transforming AH0401 with a LEU2 PCR product amplified from pRS416 with Msh2U and Msh2D [19]. AH2601 is a previously described POL3/URA3::pol3-01 MSH6/msh6Δ::LEU2 strain [18]. All strains derived from mating between pol3-01 msh6Δ spores used in the evolution experiment were designated AH164_NN, where “NN” refers to their coordinate in the 96-well plate.…”

“…We performed whole genome sequencing of subclones from the AH164 evolved cultures as described [18] except that we used a variant frequency cutoff of 0.1 for SNPs to demonstrate that the sequenced genomes were indeed from 2n and not 4n cells (S2 Dataset). We used a variant frequency cutoff of 0.3 for indels to minimize false positives at repetitive sequences due to frameshift errors during library preparation.…”

“…High mutation loads may inherently restrict cellular longevity. Recent lifespan experiments in yeast demonstrate that strong diploid mutators exhibit a form of genetic anticipation: the longer a lineage exists, the higher the mutation burden, and the shorter the cellular lifespan becomes [18].…”

Loss of vacuolar acidity results in iron sulfur cluster defects and divergent homeostatic responses during aging inSaccharomyces cerevisiae

“…We purified genomic DNA from the pooled cells using a Quick-DNA Fungal/Bacterial Miniprep kit (Zymo Research). We performed whole genome sequencing of the two pools essentially as described (Lee et al, 2019), except that we used a variant frequency cutoff of 0.8 to identify SNPs. There was a single SNP in the suppressor pool, which was present in 20 out of 20 reads and completely absent from the non-suppressed pool.…”