Figure 3. Age-associated LOH events depend on FOB1. Total LOH rates at MET15 from individual aging cultures of UCC526 (fob1D). Error bars indicate SEM. doi:10.1371/journal.pgen.1002015.g003 increase in LOH (Figure 4; Fob1 ON). In contrast, when the TET- FOB1 strain is aged over a 70-hour time course in the presence of doxycycline (a tetracycline analog), it behaves as a fob1 allele and age-associated LOH is suppressed (Figure 4; Fob1 Off). Critically, after the TET-FOB1 strain is aged over a 65-hour time course in the presence of doxycycline, removal of the repressor results in a rapid ,5-fold increase in LOH levels within 5 hours (Figure 4; Fob1 Off R On). This result suggests that the process responsible for increased LOH rates in aging cells occurs independently of Fob1, but that Fob1 activity is required in old cells for this aging process to generate LOH events. We hypothesize that the aging process may involve attenuation of an activity that normally keeps the rate of homologous recombination relatively low within the rDNA array. By extension, elimination (e.g. by gene mutation) of such an activity in young cells should phenocopy the age-associated increase in LOH. Our characterization of the age-associated LOH phenotype has delineated two criteria such candidate activities must possess: Deletion of a candidate activity must show a FOB1-dependent increase in LOH at the MET15 locus, and the ratio of reciprocal- to-nonreciprocal LOH events should be the same as in wild type cells. Declining rDNA Stability in Aging Yeast Disruption of cohesion increases LOH rates independent of Fob1 One mechanism by which LOH could increase in old cells is through an age-associated defect in sister chromatid cohesion, which normally restricts template selection during DSB repair to favor sister chromatids [28–31]. Such a defect would allow more recombination to occur between homologs during repair of Fob1- mediated DSBs, thus giving rise to increased LOH. The effect of disrupting sister chromatid cohesion was examined by deleting the genes encoding the cohibin complex proteins Csm1 and Lrs4. The cohibins physically interact with both Fob1 and cohesins, anchoring cohesin rings within the rDNA array and inhibiting both unequal sister chromatid exchange (USCE) in haploid cells and homologous recombination in diploids [17,31]. Consistent with these reports, we found that csm1D or lrs4D alleles resulted in high rates of MET15 LOH in young cells (Figure 5). But importantly, when csm1D or lrs4D were combined with fob1D, rDNA recombination rates in young cells remained high (Figure 5). Thus, loss of cohesion leads to Fob1-independent rDNA recombination, which strongly suggests that this is unlikely to be the mechanism underlying the increased rDNA recombination observed in aging cells. A decline in Sir2 protein levels in aging cells does not correlate with increased LOH rates The protein deacetylase Sir2 has been implicated in the yeast aging process and life span determination [32]. Furthermore, loss of Sir2 function leads to high rates of USCE in haploid cells, resulting in dramatic copy-number instability within the rDNA array [33– 35], and increased rates of homologous recombination between rDNA arrays in diploid cells [11]. Therefore, we examined the potential role of Sir2 in age-associated LOH in detail. It was recently shown that Sir2 protein levels decline in aging haploid cells [36]. To determine if Sir2 levels are reduced in aging diploid cells, we purified aged mother cells from liquid MEP cultures and prepared total protein extracts. When the MEP is activated, the time a mother cell spends in culture corresponds to her replicative lifetime [16]. Hence we can age-match different strains by aging these MEP cultures for equivalent periods of time (the strains analyzed here divide at the same approximate rate; data not shown). To confirm age matching between strains, purified 26- Figure 4. An inducible allele of FOB1 reveals the accumulation of aging factors in the absence of Fob1. Total MET15 LOH rates in aging UCC8912 cells exposed to no doxycycline (FOB1 On; filled squares) or 20 mg/mL doxycycline (FOB1 Off; open squares). At 65 hours, a portion of the Fob1 Off culture was harvested and transferred to media with no doxycycline (FOB1 Off R On; triangles). doi:10.1371/journal.pgen.1002015.g004 Figure 5. Double mutant analysis of fob1 and cohibin mutants. Scatter plots of total LOH rates at the MET15 locus in young cells with genotypes as indicated. doi:10.1371/journal.pgen.1002015.g005 PLoS Genetics | www.plosgenetics.org 5 March 2011 | Volume 7 | Issue 3 | e1002015