almost uniformly non-reciprocal. The phenotypic differences between the earlier studies and those reported here support the conclusion that a different mechanism leads to age-associated LOH events in cells that maintain respiration competence. Because most eukaryotic cells cannot tolerate the loss of mtDNA, it is likely that the findings we report here about genomic instability may be relevant to the aging process in other organisms. Loss of cohesion or Sir2 protein does not appear to lead to increased age-associated LOH rates Taking advantage of the requirement for Fob1, we investigated potential mechanisms leading to the age-associated LOH phenotype. Disruption of cohesion, via the deletion of the cohibins LRS4 and CSM1, was eliminated as a potential mechanism because it leads to Fob1-independent LOH events. While SIR2 deletion generated Fob1-dependent LOH events in young cells, the LOH events observed in this strain were significantly biased towards a non-reciprocal pathway compared to the LOH events generated in young or old wild type cells. Additionally, when sir2D cells were aged they also displayed an age-associated increase in LOH in the longest-lived fraction of the population. Both the difference in repair bias and presence of an age-associated increase in LOH independent of SIR2 suggests that loss of Sir2 function in aging cells is likely not the driver of age-associated LOH. Using the MEP for biochemical analysis of populations at their median RLS Aging is typically accompanied by an exponentially increasing hazard rate of death – i.e. the probability that an individual within a population will die within a given time interval. Previous biochemical analyses of aging cells focused on cells aged for only ,10 generations due to technical limitations [50]. At this age, a diploid cell population in the common laboratory S288C strain is still relatively young: It retains .95% viability and has yet to experience the dramatic increase in hazard rate of death that affects cells near their median RLS [16,51]. Using the MEP, we developed an effective purification method to isolate age-matched populations near their median RLS which allowed us to make new observations that change our understanding of age-associated processes. Earlier reports concluded that fob1D effectively suppressed the accumulation of ERCs [32,42]. By examining populations at their median life span we found that ERC levels in fob1D cells actually increase significantly as cells approach their median RLS, although their accumulation was reduced/delayed compared to wild type cells. The extension of our ability to observe age-associated genetic and biochemical changes with the MEP allows us to begin to develop an understanding of the order of events that affect an aging population. Declining Sir2 protein levels in aging cells It was recently reported that Sir2 protein levels decline in aging haploid cells after only 7-9 generations, a relatively early point in their life span [36]. Our findings are consistent with this earlier work: Sir2 protein levels in aging diploid cells show a precipitous decline by 24 hours. However, this occurs at a time in which LOH rates have not yet increased and ,95% of cells remain viable. Furthermore, when Sir2 was over expressed, no suppression of age-associated LOH rates was observed. Taking all these results together, we interpret these data to indicate that declining Sir2 protein levels do not correlate with the increase in age-associated LOH and offer further evidence that Sir2 is unlikely to be responsible for the age-associated increase in LOH. One potential caveat to our interpretation is that if there is significant cell-to-cell Declining rDNA Stability in Aging Yeast heterogeneity in levels of Sir2 over-expression, then LOH may occur in a subpopulation of cells with lower Sir2 levels. Why might reduced Sir2 protein levels in aging cells fail to alter rDNA recombination rates in a fashion similar to young cells? One possible explanation is a phenotypic lag of the increased rDNA recombination that lasts for .10 generations after the reduction of Sir2 protein. For instance, if Sir2 is completely absent in most cells, then one of it substrates (e.g. K16 of histone H4) may not immediately become acetylated [52]. In fact, newly synthesized histones start out in an unacetylated state, and how a heritable switch between acetylated and unacetylated states of histones occurs remains mysterious [53]. Another possibility is that a very low level of Sir2 protein may be sufficient to suppress rDNA recombination. Relevant to this idea, different regions of silent chromatin can compete for recruitment of Sir2 [37,54]. If the rDNA array is dominant in such a competition, the effect of declining Sir2 levels could be masked. Are ERCs responsible for age-associated LOH? If ERC accumulation is the aging process that leads to age- associated LOH, their influence on LOH must be Fob1- dependent. Indeed, it has previously been shown that plasmids containing the RFB sites from an rDNA repeat can integrate into the chromosomal rDNA array in a Fob1-dependent manner [55]. Thus, ERCs may be capable of initiating recombination events in a Fob1-dependent manner that somehow increases the frequency of homologous recombination in diploid cells. Alternatively, accumulating ERCs could simply titrate away factors that affect the frequency or stability of stalled DNA replication forks and thus increase their conversion to DSBs within the rDNA array [26]. In order to determine whether ERCs play a role in age- associated LOH, it is necessary to eliminate ERC formation and/ or accumulation in aging cells. Using the bud6D strain we reduced ERC levels at a median RLS by approximately 50%, down to a level that was equivalent to a younger wild type population (26 hours of replicative aging; Figure 8B). It is significant that at this younger age, wild type cells show no increase in LOH rate at the MET15 locus. Despite this reduction in ERC levels, the bud6D strain showed no suppression of age-associated LOH with age. While we interpret this as evidence that ERC accumulation does not drive age-associated LOH at MET15, it remains formally possible that this reduced level of ERCs is still above a threshold required to increase LOH, or that a subpopulation of cells contain higher ERC levels that drive age-associated LOH events. A clearer understanding of the contribution of ERCs to age-associated LOH will require more refined control of ERC initiation and accumulation. ERCs, LOH, and life span Previously, life span extension by fob1D was interpreted as a result of reduced ERC accumulation to a degree at which RLS becomes limited by an alternative mechanism [32,42]. However, we found that ERCs accumulate to high levels even in fob1D cells (Figure 8). This result could be interpreted in two ways that support opposing models for how life span is limited in fob1D cells. First, the delayed accumulation of ERCs early in life could extend, but still limit, RLS in fob1D cells. Alternatively, the life span- limiting function of ERCs may be Fob1-dependent, suggesting that RLS in fob1D cells is limited by an alternative mechanism. Recently it has been argued that rDNA stability, rather than ERC accumulation, limits life span [41]. However, because life span potential is ‘re-set’ in daughter cells [56], we cannot conclude that an LOH event (which would be heritable) directly limits RLS: PLoS Genetics | www.plosgenetics.org 9 March 2011 | Volume 7 | Issue 3 | e1002015