Declining rDNA Stability in Aging Yeast Figure 8. ERC accumulation in aging diploid cells. A) Southern blot of total genomic DNA isolated from aging populations and digested with BamHI and RecBCD. Genotypes and replicative age in hours indicated at top. ERC species indicated at right: ERC Concatamers- Con.; Dimers- Di.; Monomers- Mono. Mean bud scar counts at 26 hours: WT = 15.3 fob1D = 15.6, bud6D = 12.9. Lower panel: Southern blot of total genomic DNA isolated from aging cell populations, digested with BamHI and probed for NPR2. B) ERC levels normalized to NPR2 (panel A). For individual ERC species, wild type 5-hour lanes are set as 1x fold. Total ERCs were calculated as the sum of the integrated density of each ERC species, with the wild type 5-hour lane set as 1x fold. C) Total LOH rates at MET15 in aging cultures of the bud6D strain UCC8904. Error bars indicate SEM. doi:10.1371/journal.pgen.1002015.g008 Discussion Age-associated genomic instability in respiration-competent cells Previously we discovered an age-associated, genome-wide increase in LOH in yeast that results from the loss of mtDNA and respiratory function in the progeny of aging cells [11–12]. Here we used our recently developed MEP [16] to examine LOH in cells that retain respiratory function throughout their life span. Although the MEP strains are in the same S288C strain background as strains used in previous studies, they display a greater capacity to maintain mitochondrial function both in logarithmically growing cultures and during aging [11,46]. This greater stability of mitochondrial function in the MEP strain is similar to that found for most natural isolates of S. cerevisiae [14]. The faithful maintenance of functional mitochondria depends on over 100 genetic loci [15] and we have not determined the precise genetic basis for the phenotypic differences we observe. Using the MEP, we have discovered a distinct age-associated LOH phenotype in cells that retain respiration competence. This new LOH phenotype is distinguished by an apparent specificity for the rDNA array and dependence on the replication fork-block protein Fob1. As cells pass their median life span, they experience a significant increase in homologous recombination within the rDNA array which leads to LOH along a ,875 kbp span from the rDNA to the telomere of the right arm of chromosome XII. These genomic alterations are mechanistically equivalent to events that generate partial uniparental disomy in mammalian cells, which has recently been found to occur at high frequency in many human cancers [6,47–48]. We observed a significant increase in both reciprocal and non- reciprocal recombination events, which likely report two different routes of DSB repair. Reciprocal LOH events likely result from strand invasion leading to Holliday junction formation and resolution with crossing over [49], while the non-reciprocal events we observed are consistent with break-induced replication (BIR) initiated within the rDNA and propagated to the telomere of chromosome XII [17,49]. These recombination events also differ from the age-associated genomic instability described in cells that have lost respiration competence [11], where LOH events were PLoS Genetics | www.plosgenetics.org 8 March 2011 | Volume 7 | Issue 3 | e1002015