Abstract #64

Dissecting the Mechanism of Age-Induced Loss of Heterozygosity. MP Andersen, LL Carr, DE Gottschling, ED Hetrick, DL Lindstrom, ZW Nelson. FHCRC, Seattle, WA.
   We recently discovered that there is a dramatic increase in loss of heterozygosity (LOH) in the progeny of old S. cerevisiae cells [Science 301:1908 (2003)]. Once a first LOH event occurs in a progenitor cell, the cell remains in a hyper-LOH state as all subsequent progeny cells have a higher frequency of genomic instability. The age-induced LOH is qualitatively different than LOH in young cells; LOH proceeds predominantly by reciprocal recombination in young cells, while in old cells it occurs primarily via the non-reciprocal pathway of break-induced replication (BIR). We speculate that this phenomenon is the result of an age-dependent loss-of-function within a gene product that is critical for normal genome maintenance [Curr Opin Microbiol 7:673 (2004)]. In order to identify gene products that might be damaged in old cells, we carried a genome-wide screen of the non-essential gene deletion strains identifying those with elevated levels of LOH. We found 72 mutants that have increased LOH on three different chromosomes (III, IV and XII). From this set of mutants, we have identified six that phenocopy the age-induced LOH; they have elevated rates of LOH that occur primarily via BIR. We speculate that these mutants represent candidate genes whose gene products become defective in old cells, thus leading to increased LOH. We also developed a genetic method for routinely and easily enriching for old yeast mother cells. In these strains all daughter cells from a mother are killed in a regulated way, thus permitting us to stage an entire population of cells to be the same age, without contaminating young cells. We have tagged several of the candidate gene products identified in the LOH screen with GFP and combined them with the new technology to examine their behavior in young and old cells. One of our candidates, which in young cells is nuclear localized and participates in DNA repair, was found to be mis-localized in old cells. This exciting finding suggests that mislocalization of genome maintenance proteins is responsible for age-induced LOH.

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