Evidence that mutation accumulation does not cause aging in Saccharomyces cerevisiae

Abstract

The concept that mutations cause aging phenotypes could not be directly tested previously due to inability to identify age-related mutations in somatic cells and determine their impact on organismal aging. Here, we subjected Saccharomyces cerevisiae to multiple rounds of replicative aging and assessed de novo mutations in daughters of mothers of different age. Mutations did increase with age, but their low numbers, < 1 per lifespan, excluded their causal role in aging. Structural genome changes also had no role. A mutant lacking thiol peroxidases had the mutation rate well above that of wild-type cells, but this did not correspond to the aging pattern, as old wild-type cells with few or no mutations were dying, whereas young mutant cells with many more mutations continued dividing. In addition, wild-type cells lost mitochondrial DNA during aging, whereas shorter-lived mutant cells preserved it, excluding a causal role of mitochondrial mutations in aging. Thus, DNA mutations do not cause aging in yeast. These findings may apply to other damage types, suggesting a causal role of cumulative damage, as opposed to individual damage types, in organismal aging.

Keywords: DNA damage; aging; lifespan; mitochondria; mutations; thiol peroxidase; yeast.

Fig 1

Experimental design of the pedigree assay. Virgin mothers (shown as initial mother in the figure) were subjected individually to replicative lifespan, and their 5th and last daughters isolated to give rise to individual clones. Virgin mothers from these clones were again subjected to the replicative lifespan procedure, isolating either 5th (from the ‘young’ lineage) or last (from the ‘old’ lineage) daughters. This procedure was repeated six times for four independent WT and four independent Δ8 lines, followed by genome sequencing of the respective colonies following the 2nd, 4th, and 6th cycles (shown by red squares). In total, 48 genomes of individual cell-derived lineages were sequenced and analyzed.

Fig 2

Mutation accumulation does not cause aging in yeast. (A) Overall experimental design of the study. Individual yeast cells (mothers) were subjected to replicative aging, and their daughters were collected at different ages of mother cells. These cells gave rise to colonies, from which new mother cells were taken and subjected to a second round of the aging procedure. The genomes of young and old lines after 2, 4, and 6 cycles were sequenced. See Fig.1 for details. (B) Average number of mutations per aging cycle of young and old WT cells. (C) Average number of mutations per aging cycle of young and old Δ8 cells. Error bars represent the standard error of mean. (D) Mean mutation rate (number of mutations per line divided by the number of cell divisions and the length of the genome). The graph shows the mean of four lines in each experiment, and error bars are standard errors. P-values for each comparison were calculated using the Wilcoxon rank-sum test.

Fig 3

Mitochondrial content is higher in Δ8 cells and does not decrease during aging. Genomes of individual clones (four young and four old at each cycle) were sequenced following 2, 4, and 6 cycles of aging. X_mt coverage (mitochondrial genome) divided by X_nuc coverage (nuclear genome) is shown. (A) Mitochondrial genome abundance of young WT and Δ8 cells following 2 and 6 cycles. (B) Mitochondrial genome abundance of old WT and Δ8 cells following 2 and 6 cycles.