RUI: Mechanisms of Aging in Saccharomyces cerevisiae

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In research funded by an NSF POWRE grant awarded to the investigator, the yeast DNA2 gene was found to be essential for normal life span. DNA2 encodes a replicative and post replication repair helicase distantly related to SGS1 and Werner syndrome helicases. Four dna2 mutants have average life spans from 4.9 to 8.9 generations, while wild type is 23.8 generations. The sgs1 and dna2 mutants do not appear to be epistatic. Other replication mutants also have short life spans. Two strains with all rDNA deleted and rDNA supplied by plasmids have shortened life spans. These results led to the replication block model of DNA aging in yeast, suggesting that replicative senescence can result from accumulation of blocked replication forks. In addition, Y55 diploids were recently found to reset their aging clocks in meiosis. This project tests the model of replicative aging resulting from blocked replication forks and will begin a search for genes involved in resetting the yeast aging clock. The replication fork block hypothesis is being tested by observing the aging phenotypes of size increases, sterility, nucleolar fragmentation, and rDNA amplification in replication and post replication repair mutants other than dna2 and in ribosomal deletion strains, to see whether or not aspects of normal aging are accelerated in these mutants. If so, the particular process may be related to aging pacesetting. If the rDNA deletions appear to age faster, then epistasis with mutants of the silent information regulators and helicases will be tested. The structures of the DNA, with particular emphasis on structures that would result from stalled replication forks, will be examined using fiber-FISH, 2D gels, and pulsed field and normal electrophoresis combined with nuclease digestion. In addition, resetting of the aging clock is being examined in mutants affecting budding asymmetry. Whether meiosis affects clock resetting in a different meiosis strain, BR3604, will be determined. If the clock is reset in meiosis, sporulation mutants will be used to test the involvement of specific steps in meiosis in the resetting.This novel research promises to further our understanding at the molecular level of the process of aging. The use of the yeast system enhances the promise of the project because of the many molecular tools available for yeast and the extensive existing body of knowledge about the system. This research will be carried out at Pomona College, an undergraduate institution. The active involvement of students there in this research is an especially attractive feature of the project.