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. 2011 Mar;7(3):e1002015.
doi: 10.1371/journal.pgen.1002015. Epub 2011 Mar 17.

Replicative age induces mitotic recombination in the ribosomal RNA gene cluster of Saccharomyces cerevisiae

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Replicative age induces mitotic recombination in the ribosomal RNA gene cluster of Saccharomyces cerevisiae

Derek L Lindstrom et al. PLoS Genet. 2011 Mar.

Abstract

Somatic mutations contribute to the development of age-associated disease. In earlier work, we found that, at high frequency, aging Saccharomyces cerevisiae diploid cells produce daughters without mitochondrial DNA, leading to loss of respiration competence and increased loss of heterozygosity (LOH) in the nuclear genome. Here we used the recently developed Mother Enrichment Program to ask whether aging cells that maintain the ability to produce respiration-competent daughters also experience increased genomic instability. We discovered that this population exhibits a distinct genomic instability phenotype that primarily affects the repeated ribosomal RNA gene array (rDNA array). As diploid cells passed their median replicative life span, recombination rates between rDNA arrays on homologous chromosomes progressively increased, resulting in mutational events that generated LOH at >300 contiguous open reading frames on the right arm of chromosome XII. We show that, while these recombination events were dependent on the replication fork block protein Fob1, the aging process that underlies this phenotype is Fob1-independent. Furthermore, we provide evidence that this aging process is not driven by mechanisms that modulate rDNA recombination in young cells, including loss of cohesion within the rDNA array or loss of Sir2 function. Instead, we suggest that the age-associated increase in rDNA recombination is a response to increasing DNA replication stress generated in aging cells.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Age-associated LOH events are a result of recombination within the rDNA array.
A) Right y-axis: LOH rates at MET15 (open boxes) reported as total LOH events per cell division. Error bars indicate Standard Error of the Mean (SEM). Left y-axis: Percent viability of mother cells in the aging culture (grey line). B) Right y-axis: LOH rates at Chromosome IV (black line) reported as total LOH events per cell division. Error bars indicate SEM. Left y-axis: Percent viability of mother cells in the aging culture (closed circles). C) A schematic of marker placement on Chromosome XII used to determine homologous recombination break points. D) Table indicating the relative sizes of intervals between markers and the proportion of LOH events that originate within each interval.
Figure 2
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.
Figure 3
Figure 3. Age-associated LOH events depend on FOB1.
Total LOH rates at MET15 from individual aging cultures of UCC526 (fob1Δ). Error bars indicate SEM.
Figure 4
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 µg/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 → On; triangles).
Figure 5
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.
Figure 6
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: sas2Δ = 16.8, fob1Δ = 15.7.
Figure 7
Figure 7. Characterization of LOH events in young sir2Δ cells.
A) Scatter plots of total LOH rates at the MET15 locus in young cells with genotypes as indicated. B) Rates of total LOH events at MET15 in young cells for wild type (UCC5185) and sir2Δ (UCC8836) strains. Rates of reciprocal/non-reciprocal events were significantly different in the sir2Δ strain but not in wild type (Fisher's exact test by contingency tables, p values indicated above columns). C) Rates of total LOH events at MET15 in replicatively aging sir2Δ cultures. Right y-axis: LOH rates at MET15 (open boxes) reported as total LOH events per cell division. Error bars indicate SEM. Left y-axis: Percent viability of mother cells in the aging culture (grey line).
Figure 8
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 fob1Δ  = 15.6, bud6Δ  = 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 bud6Δ strain UCC8904. Error bars indicate SEM.

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