Figure 1. Age-associated LOH events are a result of recombi- nation within the rDNA array. A) Right y-axis: LOH rates at MET15 (open boxes) reported as total LOH events per cell division. Error bars indicate Standard Error of the Mean (SEM). Left y-axis: Percent viability of mother cells in the aging culture (grey line). B) Right y-axis: LOH rates at Chromosome IV (black line) reported as total LOH events per cell division. Error bars indicate SEM. Left y-axis: Percent viability of mother cells in the aging culture (closed circles). C) A schematic of marker placement on Chromosome XII used to determine homologous recombination break points. D) Table indicating the relative sizes of intervals between markers and the proportion of LOH events that originate within each interval. doi:10.1371/journal.pgen.1002015.g001 mother cells were aged in liquid culture, a robust and significant increase in LOH rates associated with increasing replicative age was observed at the MET15 locus (Figure 1A). This increase was significant at 45 hours (p,0.0001, Fisher’s exact test using contingency tables), a time point where ,25% of the original mother population retained viability, and continued to increase as the viability of the population declined below 10%. Thus, the increase in LOH rate at MET15 affects cells that have exceeded the median life span potential of the population. In contrast, while there was a trend towards increasing LOH rates with increasing replicative age for the first 72 hours at the chromosome IV locus, it did not reach the level of significance at any age (p.0.05, Fisher’s exact test using contingency tables) (Figure 1B). These Declining rDNA Stability in Aging Yeast results suggested that this age-associated increase in LOH was locus-specific, affecting MET15 on chromosome XII. LOH events at MET15 originate within the rDNA array The MET15 locus lies ,250 kbp distal to the rDNA array, a series of ,150–200 tandemly repeated copies of the genes encoding the structural RNA components of the ribosome [21]. To determine if the rDNA array represented a ‘‘hotspot’’ that was responsible for elevated LOH rates at MET15, we constructed a diploid strain that carried heterozygous markers immediately adjacent to each end of the rDNA array and at MET15 (Figure 1C). In aged populations, we isolated half-sectored colonies based on LOH at MET15 and scored each sector for the presence of the rDNA-linked markers on chromosome XII. We found that 90% of MET15 LOH events were linked to LOH events at the TRP1 marker distal to the rDNA array, but not the KANMX marker proximal to the array. This indicates that most MET15 LOH events originate within the rDNA array, with homozygosity extending ,250 kbp from the rDNA array to the MET15 locus, and presumably the remaining ,625 kbp to the telomere (Figure 1D). This LOH pattern is indicative of mitotic recombi- nation events between homologous chromosomes. The fraction of mitotic recombination events originating within the rDNA array was significantly higher than expected based on the sequence distance represented by the rDNA array (based on a 95% confidence interval, binomial distribution), consistent with previ- ous examinations of LOH at MET15 [11,17]. Thus, MET15 LOH events are primarily serving as a read out for an age-associated increase in mitotic recombination that affects the rDNA array. Aging populations maintain a constant ratio of reciprocal to non-reciprocal LOH events on chromosome XII LOH events in S. cerevisiae generating half sectors can be further classified as either reciprocal (LOH occurs in both cells) or nonreciprocal (one cell undergoes LOH while the other remains heterozygous) (see Figure 2A and reviewed in [5]). This classification has facilitated a better mechanistic understanding of LOH events. For instance, in a previous examination of mutations that increase LOH rates in young cells, we found that the ratio of reciprocal to non-reciprocal LOH events reflected the type of defect [17]: Mutations that affect specific DNA repair pathways bias LOH events away from the wild type ratio. By contrast, mutations that increase LOH rates but do not alter the ratio were consistent with a general increase in DNA damage that did not alter normal repair pathways. To examine reciprocal and non-reciprocal events in aging cells, we used the diploid MEP strain UCC5185 carrying ADE2 and MET15 in opposition at the MET15 locus. By examining the color of both half sectors, it is possible to distinguish reciprocal from non-reciprocal events (Figure 2A). Consistent with the first MEP strain examined above, we observed a significant increase in total LOH events at MET15 in populations of aging UCC5185 cells (Figure 2B). By 45 hours, when populations have passed their median viability, median LOH rates were increased 2-fold compared to the rate observed in the young population (Figure 2B) (p,0.0001, Fisher’s exact test based on contingency tables). Between 70 and 95 hours, when population viability had fallen below ,10%, median LOH rates were increased by four to eight-fold (p,0.0001, Fisher’s exact test based on contingency tables). We found both reciprocal and non- reciprocal events increased with similar kinetics in aging populations (Figure 2C), indicating that cells maintain a stable ratio of these events throughout the aging process. This result suggests that aging cells may experience an increased frequency of PLoS Genetics | www.plosgenetics.org 3 March 2011 | Volume 7 | Issue 3 | e1002015