Figure 7. Characterization of LOH events in young sir2D cells. A) Scatter plots of total LOH rates at the MET15 locus in young cells with genotypes as indicated. B) Rates of total LOH events at MET15 in young cells for wild type (UCC5185) and sir2D (UCC8836) strains. Rates of reciprocal/non-reciprocal events were significantly different in the sir2D strain but not in wild type (Fisher’s exact test by contingency tables, p values indicated above columns). C) Rates of total LOH events at MET15 in replicatively aging sir2D cultures. Right y-axis: LOH rates at MET15 (open boxes) reported as total LOH events per cell division. Error bars indicate SEM. Left y-axis: Percent viability of mother cells in the aging culture (grey line). doi:10.1371/journal.pgen.1002015.g007 been postulated to induce senescence in aging mothers by an undefined mechanism [39]. Each repeat contains an origin of DNA replication, allowing ERCs to replicate independently, but lacks a centromeric sequence to ensure equal partitioning of ERCs between mother and daughter cells during mitosis [39–40]. Consequently, ERCs preferentially accumulate within aging Declining rDNA Stability in Aging Yeast mother cells. It has been suggested recently that ERCs can influence rDNA array stability in haploid cells [41]. Given these findings, we explored whether ERC levels play a role in age- associated LOH in diploid cells. In order to examine this issue, we found it necessary to further develop methods employed to examine ERC levels with age. Previous analyses of ERC levels have been technically limited to the examination of relatively young cells (,10 generations) [42], whereas we observe age-associated increases in LOH after the median life span of our diploid strains (36 generations for UCC5185) [16]. To better understand the kinetics of ERC accumulation, we purified aged mother cells from MEP cultures and quantified ERC levels by Southern blotting. To improve ERC quantitation, we removed the linear rDNA array by digestion with RecBCD exonuclease, a highly processive exonuclease that does not degrade intact or nicked circular DNA [43] (Figure S2). Monomeric, dimeric, and multimeric ERC species were identified based on their migration rates [44] (Figure 8A) and quantified by densitometry (Figure 8B). First, we compared ERC levels in wild type and fob1D cells. A modest increase in all three species of ERCs was evident in wild type cells after aging replicatively for 26 hours, a relatively young age when the population maintains ,90% viability (Figure 1A and [16]). As the population approaches its median RLS by 50 hours, ERC levels continued to increase dramatically. Similar to previous reports, in relatively young cells, ERC levels in a fob1D diploid were low compared to wild type cells (Figure 8B and [42]), but their accumulation was not completely prevented. After 26 hours of replicative aging, total ERC levels in the fob1D strain had increased ,5-fold above young wild type levels, but were still reduced ,50% compared to the age-matched wild type population. Interestingly, as the fob1D population continued to age, ERC accumulation continued: Total ERC levels in fob1D cells were still ,50% lower than wild type cells after aging replicatively for 50 hours, but by this age ERC levels in the fob1D strain had increased ,17-fold above young wild type levels. These results indicate that ERC accumulation is an aging process that is not strictly dependent on Fob1 activity. The delayed accumulation of ERCs could reflect a reduction in the rate of formation of ERCs from the rDNA array in the fob1D strain, while amplification of ERCs by replication is unaffected [39]. Age-associated LOH is not suppressed by reduced ERC accumulation While the fob1D strain permitted us to determine the utility of our ERC measurements with age, it was not useful for examining the effect of ERC accumulation on LOH because, as shown above, Fob1 is required for the age-associated LOH events. For this, we examined a genetic background that could reduce ERC accumulation independently of Fob1. Deletion of BUD6 has been reported to disrupt the asymmetric segregation of ERCs, resulting in extended life span and a prediction of reduced ERC accumulation in mother cells [45]. Consistent with this prediction, we found that deletion of bud6D reduced the level of ERCs throughout the life span of a population. The bud6D cells showed a 50% reduction in ERCs after 50 hours of replicative aging: A level that was achieved in wild type populations aged for only 26 hours (Figure 8B). Remarkably, we saw no change in the age-associated increase in LOH rate at MET15 in the bud6D strain (Figure 8C). This indicated that in the presence of Fob1 activity, a 50% reduction in ERC accumulation was not sufficient to suppress age- associated LOH. PLoS Genetics | www.plosgenetics.org 7 March 2011 | Volume 7 | Issue 3 | e1002015