PROJECT SUMMARY Age is the greatest carcinogen, yet the mechanism by which the incidence of cancer dramatically increases as humans age is unclear. In an effort to better understand this phenomenon, we developed a system in the yeast Saccharomyces cerevisiae that recapitulates one of the hallmarks of cancer - genomic instability - as a function of cellular age. Specifically, we examined the relationship between aging and genomic instability by monitoring loss of heterozygosity (LOH) on different chromosomes during replicative aging in yeast. We discovered that aging yeast mother cells underwent a switch to a nearly 100-fold increase in LOH in their progeny. Recently, we gained a better understanding of this phenomenon and have developed a working hypothesis that breaks the age-associated LOH into a series of connected events. It begins with a loss of, or damage to, the mtDNA. This leads to a reduction in the inner mitochondrial membrane potential ( ), which in turn leads to a defect in iron-sulfur cluster (ISC) biosynthesis and/or assembly of the ISCs into proteins required for maintenance of genome integrity. The reduced function of these genome integrity proteins then leads to increased levels of LOH. This hypothesis serves as a working model to formulate our questions and approaches. We now propose to test the steps in this hypothesis using the powerful collection of resources and tools available in S. cerevisiae. As part of this approach, we developed an inducible genetic system, the Mother Enrichment Program (MEP), which permits for the first time the facile isolation of replicatively aged S. cerevisiae cells for biochemical, cell biological and genetic analysis. This will permit us to gain further understanding of the earliest events in aging that ultimately lead to age-associated LOH. Because each of the processes in this progression to age-associated LOH are conserved, we believe that what we learn in the proposed plan will ultimately be translatable to identifying candidate genes and processes in humans that are similarly affected by aging and lead to age-related oncogenesis.