Declining rDNA Stability in Aging Yeast Figure 2. LOH rates at MET15 in aging cells present a constant ratio of reciprocal/non-reciprocal events. A diagram of UCC5185 colony color markers located on Chromosome XII, with expected results of reciprocal and non-reciprocal LOH events. Non-reciprocal events can also lead to black/white half-sectored colonies. (Note: The normal chromosomal copies of ADE2 have been deleted.) B) LOH rates for UCC5185 at MET15 reported as total LOH events per cell division. Error bars indicate SEM. The sample size at each time point ranges from 21 to 28. LOH rates are significantly increased at 45, 70 and 95 hours, (unpaired t-test; P values are 0.0003, ,0.0001, and ,0.0001 respectively). C) LOH events from panel B segregated into reciprocal and non-reciprocal LOH rates. Error bars indicate SEM. doi:10.1371/journal.pgen.1002015.g002 DNA damage within the rDNA array, rather than an age- associated defect in a particular repair pathway. Age-associated rDNA recombination events are FOB1-dependent In haploid cells, double stranded breaks (DSBs) within the rDNA are often initiated by the DNA replication fork-blocking (RFB) activity of Fob1 [22–23]. Replication forks traveling opposite to the direction of transcription of the 35S rRNA are blocked by the specific interaction of Fob1 with sequences within the non-transcribed region 1 (NTS1) [24]. This source of DNA replication stress can cause fork collapse to generate DSBs, which can be repaired by homologous recombination to yield a variety of products [25–26]. We tested whether Fob1 was required for age-associated LOH at the MET15 locus. In young cells, fob1D led to a reduction in the MET15 LOH rate of ,2-fold (Figure 3), but this rate was still significantly higher than the LOH rate on chromosome IV, suggesting that other mechanisms must contribute to the hotspot activity affecting MET15. When we measured MET15 LOH in aging fob1D cells we found the age-associated increase in rate of LOH was completely suppressed (Figure 3). This result indicated that Fob1 activity is required for this aging phenotype and suggests that Fob1-mediated DSBs are a critical intermediate for these LOH events. A FOB1-independent aging process acts cooperatively with Fob1 to generate LOH events in aged cells We offer two simple models to explain the requirement for FOB1 in the age-associated increase in LOH at MET15. Fob1 activity may be required to generate an aging factor or process that leads to increased LOH. Alternatively, an aging process may occur independent of Fob1, while Fob1 remains necessary for this process to manifest as LOH events in old cells. To distinguish between these modes of action, we generated a diploid MEP strain in which FOB1 is expressed from a tetracycline-repressible promoter [27]. When the TET-FOB1 strain is aged over a 70- hour time course in the absence of repressor, cells exhibit a normal PLoS Genetics | www.plosgenetics.org 4 March 2011 | Volume 7 | Issue 3 | e1002015