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Mr. McMurray’s previous work identified an apparent epigenetic state into which aging Saccharomyces cerevisiae mother cells enter after approximately 25 mitotic cell divisions, characterized by a dramatic increase in the rate of loss of heterozygosity (LOH) at multiple loci. Additionally, the mechanism of LOH is different than that of young cells, occurring primarily by apparent break-induced replication (BIR) with a strong daughter cell bias. These characteristics of age-induced LOH led us to develop a model for the defect arising in old mother cells: we proposed that the DNA damage checkpoint becomes defective in aging cells, leading to broken chromosomes persisting after mitosis. The daughter bias of age-induced LOH could then be explained by a segregation bias of chromosome fragments lacking a centromere (“acentric” fragments), which would provide daughter cells with substrates for BIR while allowing mother cells to repair by mechanisms that do not result in LOH. In order to test this model, Mr. McMurray chose to examine LOH in young cells with engineered DNA damage checkpoint dysfunction. First, he measured the rate and mechanism of spontaneous LOH in young checkpoint-deficient cells, and found that no change relative to wild type. From this result, Mr. McMurray can conclude that a checkpoint defect alone cannot explain the increased rate nor altered mechanism of LOH in the progeny of old cells, and that repair of spontaneous DNA double-strand breaks (DSBs) in ways that suppress LOH does not require checkpoint function. Next, Mr. McMurray asked whether increasing amounts of DNA damage affected LOH. Treating cells with the alklyating agent methylmethane sulfonate (MMS) induced a dramatic increase in the rate of LOH. However, in checkpoint-proficient cells, these LOH events rarely occurred by BIR, indicating that age-induced LOH events are not simply the result of an increase in DSBs in old mother cells. In checkpoint-deficient MMS-treated cells, on the other hand, nearly half of the observed LOH events occurred by BIR. To ask whether these BIR events displayed the same daughter bias as age-induced BIR events, Mr. McMurray separated single daughter cells from young, MMS-treated checkpoint-deficient cells and identified in which cell LOH occurred. Mr. McMurray found a modest but reproducible 2.5:1 daughter:mother bias, superficially consistent with our model for age-induced LOH. Finally, by activating the spindle checkpoint with the drug nocodazole, Mr. McMurray was able to suppress these LOH events by forcing cells lacking the DNA damage checkpoint to arrest prior to mitosis, providing time for repair of broken chromosomes. These results strongly suggest that separating the two fragments of a broken chromosome by unrestrained mitosis results in situations of loss of genetic information in daughter cells. Mr. McMurray is now creating strains to directly visualize the acentric fragment of a broken chromosome in DNA-damaged young cells or undamaged old cells to confirm that biased segregation underlies the daughter bias of age-induced LOH.
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