hour populations were stained with calcofluor white to count bud scars (see Figure 6 legend). Western blotting with an anti-Sir2 antibody confirmed that Sir2 protein levels were dramatically reduced in aging diploid cells when normalized to total protein (Figure 6A). Sir2 levels in aging populations had fallen 10-fold by 26 hours, and were further reduced by 50 hours. This same decline was also observed with a polyclonal antiserum raised to a different region of the Sir2 protein (data not show). However, this behavior was not a universal feature of proteins in aging cells: Western blotting against the vacuolar protein Vma2 (Figure 6A) and the kinase Pkc1 (data not shown) both showed no decline in aging cells. Therefore, Sir2 protein levels specifically decline in aging cells, however this decline precedes any significant change in LOH rates by 20 hours, or approximately 12 generations. Modulation of Sir2 protein levels in aging cells does not affect age-associated LOH The observation that Sir2 protein levels decline precipitously 20 hours before rDNA recombination rates increase significantly led us to further examine the relationship between Sir2 depletion Figure 6. Declining Sir2 levels do not correlate with the onset of age-associated LOH events. A) Western blotting against Sir2 and Vma2 in total protein extracts prepared from cells aged for hours as indicated at top. Sir2 levels were normalized to Vma2 and quantified by densitometry, with wild type Sir2 levels in log cultures set to 1x Fold. Mean bud scar counts at 26 hours: WT = 15.5, SIR2OE = 18.0. B) Total LOH rates at MET15 of wild type UCC5185 (filled squares) compared to the SIR2OE strain UCC8910 (open squares). Error bars indicate SEM. C) Western blotting against Sir2 and Vma2 in total protein extracts prepared from aged cells. Genotype and hours of aging indicated at top. Mean bud scar counts at 26 hours: sas2D = 16.8, fob1D = 15.7. doi:10.1371/journal.pgen.1002015.g006 Declining rDNA Stability in Aging Yeast and age-associated LOH. We tested the effect of SIR2 gene copy number on rDNA recombination using diploid strains homozygous for a tandem repeat of SIR2 under control of its native promoter (SIR2OE). Previous studies indicated that this modest level of over- expression is sufficient to increase silencing of RNA Polymerase II- dependent transcription within the rDNA array [34,37] and extend RLS [32], while higher expression of Sir2 is toxic [38]. Sir2 over- expression in young (log-phase) diploid cells was verified by Western blot (Figure 6A). The decline in Sir2 protein levels was partially suppressed in the SIR2OE strain: Sir2 levels in SIR2OE populations were elevated at 26 hours compared to wild type, but declined precipitously to wild type levels by 50 hours (Figure 6A). Next, we examined LOH rates at MET15 in the SIR2OE strain to determine if increased Sir2 levels suppressed the age-associated change in LOH rates. Although MET15 LOH rates in young SIR2OE cells were significantly lower than the wild type strain (p,0.0001, Fisher’s exact test based on contingency tables), we observed no difference in the timing or magnitude of the age- associated increase in MET15 LOH rates in SIR2OE cells compared to wild type (Figure 6B). This result offers further support that Sir2 protein levels are not the determining factor regulating rDNA recombination in aging cells. To confirm this result, we attempted to stabilize Sir2 protein levels by a second method. In a previous study, Dang, et al. found that deletion of SAS2, the gene encoding the catalytic histone acetyltransferase subunit of the SAS complex that acts antagonis- tically to Sir2 for modification of histone H4 at lysine 16, stabilized Sir2 levels in aging cells [36]. When we constructed a sas2 deletion strain in the MEP background and examined Sir2 protein levels, we saw no evidence of Sir2 stabilization in this mutant background (Figure 6C). Finally, we asked whether deletion of fob1, which does suppress age-associated rDNA recombination, might also stabilize Sir2 protein levels in aging cells. Again, we observed no stabilization of Sir2 protein levels (Figure 6C). Thus, we found no evidence of a correlation between Sir2 protein levels and LOH rates in aging populations. Following the same logic behind our examination of cohesion, we characterized the pattern of LOH events observed in young cells when sir2 was deleted. We found that elevated MET15 LOH rates in young sir2D cells were dependent on FOB1 (Figure 7A). In this regard it shows similarity to the age-associated LOH phenotype. However, the ratios of reciprocal to non-reciprocal events in sir2D cells was significantly shifted to favor non-reciprocal events (Figure 7B), indicating that loss of Sir2 function does not accurately phenocopy the age-associated LOH phenotype. Lastly, we examined LOH rates in aging sir2D cells to determine if there is an age-associated increase in the absence of Sir2. Consistent with our earlier results [11], there was a very high level of LOH in young cells. Nevertheless, after 70 hours of replicative aging, LOH rates at MET15 increased significantly (p = 0.0003, Fisher’s exact test based on contingency tables; Figure 7C). It is worth noting that sir2D cells have a short RLS [32]; thus at 70 hours only the longest-lived 1% of the total population are represented. When taken together, these data - the lack of correlation between Sir2 protein levels and rDNA recombination rates in aging cells, the difference in reciprocal/non-reciprocal LOH events between aging wild type and sir2D cells, and the Sir2- independent increase in LOH in aging cells - indicate that declining Sir2 levels are insufficient to explain the age-associated rDNA recombination phenotype. ERCs accumulate to high levels in fob1D cells Intrachromosomal recombination between the rDNA repeats can produce extrachromosomal rDNA circles (ERCs) that have PLoS Genetics | www.plosgenetics.org 6 March 2011 | Volume 7 | Issue 3 | e1002015