One of the greatest risk factors associated with carcinogenesis is age. Cancer risk increases exponentially toward the end of life in humans and other mammalian species 
. Somatic genetic changes contribute significantly to the development of most tumors. However, the rate at which spontaneous mutations arise in normal adult cells has been hypothesized to be too low to generate all the genetic changes necessary to produce tumors at the observed rates 
. Consequently, Loeb et al.
developed the mutator hypothesis, which postulates an increased mutation rate in precancerous cells 
. A variety of mechanisms could lead to such an increase, but a favored model is that sporadic mutations in, or epigenetic silencing of, genes responsible for maintaining genome integrity lead to increased rates of mutation. Once acquired, this mutator phenotype may serve as the driving force toward carcinogenesis as individuals age.
When genomes of tumors are examined, loss of heterozygosity (LOH) is observed as a common mechanism in which the sole functional allele of a tumor suppressor gene is inactivated by somatic mutation 
. LOH can be generated by many different mutational events, including point mutations, small deletions or inversions, mitotic recombination or chromosome loss. Recent advances in high-resolution single-nucleotide polymorphism arrays (SNP arrays) have revealed that a surprising number of tumors contain long tracts of homozygosity that are not accompanied by a change in gene copy number. This type of LOH arises from somatic mitotic recombination events and is referred to as partial (or acquired) uniparental disomy (UPD) 
. Importantly, UPD can alter the genotype for hundreds of genes following a single event, thereby amplifying its potential to contribute to cancer development 
Within any genome, there are regions that exhibit higher rates of mitotic recombination than the genomic average as the result of their proximity to hotspots or common fragile sites 
. These regions typically represent slow-replicating sequences, and agents that generate DNA replication stress often reveal their fragility. These regions are also often associated with non-histone protein complexes that inhibit DNA replication fork progression. This generates DNA replication stress that may lead to an increased frequency of DNA damage due to replication fork collapse 
. While conservative repair of this DNA damage would have no genetic consequence, the higher incidence of damage results in a greater chance that an alternative repair pathway will be utilized that does confer a genotypic change.
Previously, we found evidence for a mutator phenotype associated with advancing replicative age in a common lab strain of the budding yeast, Saccharomyces cerevisiae
. Pedigree analysis revealed that old cells begin to produce offspring that have dramatically higher incidences of genomic instability, which is manifest as an apparent ~100-fold increase in LOH on at least two different chromosomes 
. Virtually all the LOH occurred via mitotic recombination and gave rise to UPD genotypes. Subsequent analysis revealed that these LOH events were a consequence of loss of mitochondrial DNA in daughter cells, which led to a transient “crisis” state characterized by cell cycle arrest and high mortality 
. Cells that survived this crisis showed a high frequency of LOH events in their nuclear genome.
While yeast cells lacking mitochondrial DNA cannot perform oxidative phosphorylation (respiration), they remain viable by relying on aerobic glycolysis (fermentation). In contrast, most eukaryotic cells retain, and even require, respiration. Thus we were interested in determining whether S. cerevisiae
cells that retain respiratory competence throughout their replicative life span (RLS) also exhibit a mutator phenotype. The frequency at which functional mitochondria are successfully segregated during cell division varies widely between strains of S. cerevisiae
; alleles in over 100 genes can affect mitochondrial DNA transmission frequency 
. Therefore, in this study we sought to use a strain in which respiration competence was faithfully transmitted with increasing replicative age.
We recently developed the Mother Enrichment Program (MEP), a genetic program to facilitate replicative aging studies in yeast 
. The MEP provides an efficient and inducible selection against newborn daughter cells. When the MEP is active, mother cells continue to divide and age normally, while the division of newborn daughter cells is arrested. The MEP provides the opportunity to follow a cohort of mother cells in liquid culture throughout their entire RLS without any requirement for removing progeny cells. Once cells have reached a desired age, the MEP can be switched off and aged mothers will resume production of viable daughters, allowing for colony-based phenotypic analysis. Compared to pedigree analysis, which is done by single-cell micromanipulation, the MEP can dramatically improve the sensitivity of the LOH assay by increasing the sample number of aged cells by over three orders of magnitude. Here we report our finding that replicative age is accompanied by a progressive decline in rDNA array stability, leading to higher incidence of LOH affecting the right arm of chromosome XII.