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    DNA Polymerization in Microgravity and the Future of Human Space Travel

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    Rosenstein, Aaron
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    Abstract
    The coming decades will represent a quantum leap in the field of crewed space travel, with planned missions back to the Moon, forward to Mars, and possibly beyond. The substantial biological threats of long-term space exploration are still key barriers to enacting these goals. Microgravity and radiation encountered in space are cellular stressors that could severely compromise the health of future astronauts leaving earth’s orbit. Of specific concern is the impact of these stresses on DNA. The Polymerase Error Rate in Space (PolERIS) experiment was devised to identify whether DNA polymerase enzymes, essential for both replication and repair of the genome, are more prone to errors in microgravity. This research necessitated the development of both novel genetics and engineering-based approaches to conduct experiments in microgravityaboard a parabolic flight plane. It was determined that Klenow fragment (exo+), an E. coli DNA polymerase I derivative with a 3’→5’ exonuclease proofreading domain, was 1.1-fold more prone to both substitution (p = 0.02) and deletion (p = 1.51E-7) errors in microgravity when compared to earth-like gravity. This effect was more pronounced in Klenow fragment (exo-), which does not posess proofreading capability, where substitutions and deletions respectively occurred 2.6-fold (p = 1.98E-11) and 1.5-fold (p = 8.74E-13 ) more frequently in microgravity than earthlike gravity.

    In addition to ensuring genomic protection in space, the ability for cells to remain viable on long-term space missions may require innovative solutions, such as inducing cellular biostasis for both preservation of cellular isolates and possibly humans in the far future. The Biostasis project of the Defense Advanced Research Projects Agency, USA requires a comprehensive set of assays to quantify various modes of DNA damage potentially incurred during cellular stasis. A research exchange program facilitated early-stage efforts to develop DNA repair pathway-specific cell-based fluorescent biosensors. Thus, together with the PolERIS experiment, this thesis contributes to our current understanding of how polymerases exhibit altered functionality in microgravity, and strategies to detect specific modes of DNA damage with novel cell-based biosensors.
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    http://hdl.handle.net/1974/28576
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