Author Summary There is a striking correlation between age and the onset of many diseases, such as cancer, suggesting that the aging process itself can contribute to their development. Cancer is a genetic disease caused by the accumulation of a series of deleterious somatic mutations leading to unchecked proliferation. In humans, it is well established that normal mutation rates are not sufficient to account for the sharp increase in cancer rates in aging populations, suggesting a change in mutation rate is a necessary component of cancer development. Here, we find that the aging process in the budding yeast Saccharomyces cerevisiae leads to an increased rate of homologous recombination within a repetitive DNA sequence element, the ribosomal rDNA array. While these mutational events are initiated primarily at this single locus, they are propagated to the end of the chromosome and thus affect hundreds of genes. These results suggest that the aging process itself could contribute to increasing mutation rates and perhaps to the onset of age-associated disease. of the budding yeast, Saccharomyces cerevisiae. Pedigree analysis revealed that old cells begin to produce offspring that have dramatically higher incidences of genomic instability, which is manifest as an apparent ,100-fold increase in LOH on at least two different chromosomes [11]. Virtually all the LOH occurred via mitotic recombination and gave rise to UPD genotypes. Subsequent analysis revealed that these LOH events were a consequence of loss of mitochondrial DNA in daughter cells, which led to a transient ‘‘crisis’’ state characterized by cell cycle arrest and high mortality [12]. Cells that survived this crisis showed a high frequency of LOH events in their nuclear genome. While yeast cells lacking mitochondrial DNA cannot perform oxidative phosphorylation (respiration), they remain viable by relying on aerobic glycolysis (fermentation). In contrast, most eukaryotic cells retain, and even require, respiration. Thus we were interested in determining whether S. cerevisiae cells that retain respiratory competence throughout their replicative life span (RLS) also exhibit a mutator phenotype. The frequency at which functional mitochon- dria are successfully segregated during cell division varies widely between strains of S. cerevisiae; alleles in over 100 genes can affect mitochondrial DNA transmission frequency [13–15]. Therefore, in this study we sought to use a strain in which respiration competence was faithfully transmitted with increasing replicative age. We recently developed the Mother Enrichment Program (MEP), a genetic program to facilitate replicative aging studies in yeast [16]. The MEP provides an efficient and inducible selection against newborn daughter cells. When the MEP is active, mother cells continue to divide and age normally, while the division of newborn daughter cells is arrested. The MEP provides the opportunity to follow a cohort of mother cells in liquid culture throughout their entire RLS without any requirement for removing progeny cells. Once cells have reached a desired age, the MEP can be switched off and aged mothers will resume production of viable daughters, allowing for colony-based phenotypic analysis. Compared to pedigree analysis, which is done by single-cell micromanipulation, the MEP can dramatically improve the sensitivity of the LOH assay by increasing the sample number of aged cells by over three orders of magnitude. Here we report our finding that replicative age is accompanied by a progressive decline in rDNA array stability, leading to higher incidence of LOH affecting the right arm of chromosome XII. Declining rDNA Stability in Aging Yeast Results MEP strain mother cells produce respiration-competent daughters throughout their replicative life span In order to assess whether MEP strains could be useful for analyzing LOH rates in aging yeast cells, we first examined whether old mother cells produced daughters that retained respiration competence. Specifically, we examined the inheritance of respiration competence in daughter cells of a MEP strain (UCC5185) using pedigree analysis. We found that ,65% of the mother cells produced daughters with respiration competence through their entire RLS (Median of 36 generations, Figure S1A). This is in striking contrast to the strain we originally examined for age-associated LOH (UCC809), where only ,5% of the mother cells produced respiration-competent daughters throughout their life span (Figure S1A and [11–12]). Furthermore, for those UCC5185 mother cells that did produce respiration-incompetent daughters, the median age at which this occurred was significantly later in the mother’s life span (23 generations versus 10 generations in UCC809) (Figure S1B). Together, these results demonstrate that the majority of UCC5185 cells are capable of producing respiration-competent daughters throughout their life span, thus providing the basis for our analysis of LOH in such a population. An age-associated increase in LOH on chromosome XII but not chromosome IV LOH events in diploid cells can be visualized using colony color phenotypes and, when combined with half-sector analysis, the rate of LOH can be determined [17–20]. We used two heterozygous markers that alter colony color when lost: Loss of MET15 function, resulting in a black colony sector, or loss of ADE2 function, resulting in a red colony sector. LOH events that occur within the first cell division after plating will generate sectors that form one half of a colony and can be used as a direct measure of mutation rate. Replicative life span is measured in terms of the number of mitotic divisions an individual cell completes before senescence. To measure LOH rates as mother cells divide and age, logarithmically growing MEP cells were inoculated into rich media containing estradiol and aged in liquid culture. Estradiol activates the MEP, allowing mother cells to divide and age normally while daughter cells are rendered incapable of cell division. Throughout the 95-hour aging period, samples were harvested and washed to remove estradiol, then plated to solid medium for half-sector analysis. Once the MEP is inactivated by removal of estradiol, surviving mother cells can produce viable daughter cells and form colonies. Because aged mother cells represent the only cells in the aging liquid culture capable of forming colonies (Figure 1A and [16]), no fractionation of cell populations was required to isolate aged mothers for the analysis. The vast majority of colonies at each time point grew robustly and were respiration-competent. Respiration-competent colonies were easily distinguished from those incapable of respiration, which show a severe growth defect on glucose media and thus could be excluded from our analysis. We determined the LOH rates in young cells at loci on chromosome IV and chromosome XII simultaneously. By scoring ,20,000 colonies per time point, we measured an LOH rate of 6.6961024 events/cell division at MET15 on chromosome XII and a rate of 1.2561024 events/cell division at an intergenic region on chromosome IV (Figure 1A and 1B). The LOH rate at MET15 was significantly higher than the chromosome IV locus (p = 0.0059, Fisher’s exact test using contingency tables), suggesting that MET15 LOH rate is affected by proximity to a ‘‘hotspot’’. As PLoS Genetics | www.plosgenetics.org 2 March 2011 | Volume 7 | Issue 3 | e1002015