Instead, a reversible aging process that impacts rDNA stability, and can generate LOH events at some frequency, may limit RLS. Curiously, we find that the highest rates of age-associated LOH are observed at the latest time points, where the population consists of the longest-lived survivors (,10% of the population remains viable), and when the hazard rate of death has declined from its peak near the median life span [16]. This lack of correlation between LOH rate and hazard rate of death suggests that rDNA recombination is not limiting for life span of most cells. Nevertheless, the longest-lived individuals in the population no longer experience the same hazard rate of death, and thus their life span may be limited by a different mechanism (e.g. rDNA recombination) than the average population. DNA replication stress and age-associated LOH If pathways that have been previously identified to modulate rDNA recombination do not adequately account for the age- associated increase in LOH rates, can this phenotype be a response to a change in a more general biological process that is most readily manifested at the rDNA? Since DSBs within the rDNA array normally arise through the interaction between DNA replication forks and Fob1 [57], we speculate that DSB rates in the rDNA are modulated by DNA replication stress. This model is supported by several lines of evidence: A hypomorphic allele of the essential DNA replication helicase encoded by DNA2 results in an increased frequency of DSBs within the rDNA, which can be suppressed by deletion of FOB1 [57–58]. Similarly, mutations in DNA polymerase a and d subunits can also lead to increased DSBs within the rDNA array [26,59]. Deletion of RRM3, a helicase that functions to remove non-histone protein barriers from DNA, also affects rDNA recombination [60–61]. Further evidence comes from a screen for deletion mutants that increase LOH in young cells [17], which classified mutations based on locus specificity, magnitude and ratio of reciprocal/non-reciprocal events. The group of deletion mutants that showed a bias toward increasing LOH at MET15 on chromosome XII (and presumably originate in the rDNA) included five genes implicated in the regulation of nucleotide pools, which can be a source of DNA replication stress [62]. While the effects of DNA replication stress on the rDNA may be dependent on Fob1, the mode of action may not necessarily be specific to the rDNA. Ivessa, et al. identified genomic regions prone to DNA replication fork pausing in an rrm3D mutant which included centromeric regions, tRNAs and sub-telomeric sequences in addition to the rDNA array [9]. Similarly, RRM3 was originally identified in a genetic screen for mutations that induce gene duplication at the tandemly repeated CUP1 locus [60], which suggests that other regions of the genome that combine replication fork blocks with repetitive sequence elements could also generate age-associated LOH events. While repetitive elements are uncommon in the S. cerevisiae genome, the ubiquitous nature of these features in mammalian genomes suggests great potential for age-associated genomic instability generated by a similar mech- anism. Materials and Methods Subcloning and strain construction Genotypes of all yeast strains used in this study are provided in Table S1, oligonucleotide sequences are provided in Table S2, and plasmids are listed in Table S3. The two-chromosome LOH reporter strain UCC8918 was generated by mating UCC8917 x UCC5181. UCC8917 was derived from UCC5179 by one-step integration of a PCR Declining rDNA Stability in Aging Yeast fragment carrying ADE2 into an intergenic region of chromosome IV (coordinates 1,515,634–1,515,738), which was generated using oligos MarthaN/H2L and MarthaN/H2R with pRS402 [63] as a template. The diploid strain with multiple heterozygous markers on chromosome XII (UCC8915) was generated by mating UCC8914 x UCC5179. UCC8914 was derived from UCC8913 by one-step integration of a PCR fragment carrying TRP1 into an intergenic region distal to the right end of the rDNA array (coordinates 486062-486189), which was generated using oligos RDNRF and RDNRR with pRS304 [64] as a template. Integration was verified by PCR from genomic DNA using oligos RDNRconF and RDNRconR. UCC8913 was derived from UCC5181 by one-step integration of a PCR fragment carrying KANMX into an intergenic region proximal to the left end of the rDNA array (coordinates 450191-450372), which was generated using oligos RDNLF and RDNLR with pRS400 [63] as a template. Integration was verified by PCR from genomic DNA using oligos RDNLconF and RDNLconR. MEP deletion strains for bud6D, csm1D, lrs4D, sir2D and sas2D were generated by one-step gene disruption and verified by PCR using the following oligonucleotides and DNA templates: BUD6: deletion- Bud6delF/bud6delR, template- pRS306, confirmation- Bud6delcheck/Bud6delchkDN. CSM1: deletion- CSM1A/ CSM1D, template- UCC7629-1 genomic DNA, confirmation- CSM1A/CSM1B. LRS4: deletion- LRS4A/LRS4D, template- UCC7598-1 genomic DNA, confirmation- LRS4A/LRS4B. SIR2: deletion- SIR2KO1/SIR2KO2, template- pRS400 [63], confir- mation- 59SIR2/39SIR2. SAS2: deletion- SAS2KOF/SAS2KOR, template- pRS400 [63], confirmation- SAS2conF/SAS2conR. The sir2D fob1D double mutant was constructed by transforming UCC8832 with plasmid pRS314-SIR2 [65] to complement the mating defect of sir2D, followed by mating to UCC524 and sporulation. Because both deletions are marked with KANMX, PCR was used to identify double mutant spore products to generate UCC8839 and UCC8840. These haploids were mated to generate UCC8844, which was subsequently cured of the pRS314-SIR2 plasmid. Standard mating and sporulation, fol- lowed by PCR to identify double mutant spore products, was used to generate csm1D fob1D and lrs4D fob1D double mutant strains. To generate SIR2OE strains, a genomic clone of SIR2 along with ,500 bp of upstream sequence and ,250 bp of downstream sequence was subcloned by PCR amplification with SIR2ecoriF and SIR2ecoriR primers, digested with EcoRI, and ligated into pRS306 cut with EcoRI to create the integration plasmid pRS306- SIR2. The plasmid was cut with BglII and transformed into UCC5179 and UCC5181 to generate strains UCC8908 and UCC8909. Correct integration was confirmed by PCR. These haploids were mated together to generate the diploid strain UCC8910. The SIR2hemi diploid was generated by transforming UCC8832 with pRS314-SIR2, mating to UCC5179, and subsequently curing the diploid of the plasmid. TET-FOB1 construction The TET-OP2 promoter was inserted upstream of the endogenous FOB1 locus by one-step integration of a PCR fragment generated using primers FOB1tetF and FOB1tetR with pKAN-TETO2 as a template. The TETR’-SSN6 cassette was inserted by one-step integration into the met15D0 locus by digesting plasmid pLMI-tetR’S with PacI. The LEU2 marker was subsequently exchanged for ADE2 or MET15 using the primers LEU2swapF and LEU2swapR with the appropriate plasmid templates (pRS402 and pRS401, respectively [63]). The VP16 activation domain from the tTA activator was replaced with PLoS Genetics | www.plosgenetics.org 10 March 2011 | Volume 7 | Issue 3 | e1002015