The Fidelity of DNA Replication in Microgravity

摘要

Long-term space missions will expose crew members, their cells as well as their microbiomes to prolonged periods of microgravity and ionizing radiation, environmental stressors for which almost no earth-based organisms has evolved to survive. Despite the potential health hazards, the impact of these stresses on DNA replication and repair machinery has not been fully explored. DNA polymerases are central to both these processes, and are indispensable to the maintenance of genomic integrity. The "Polymerase Error-Rate in Space (PolERIS) experiment" was designed to assess whether a differential in DNA polymerase fidelity and replication rates exist under conditions of microgravity generated by parabolic flight when compared to earth-like gravity. Testing required the development of a modular payload, which was used as a platform for conducting DNA polymerization reactions. Primed synthetic single-stranded DNA was used as a template by solutions containing one of two enzymes (Klenow fragment exonuclease+/-; with and without proofreading exonuclease activity respectively) upon commencement of a parabolic arc. Following completion of a 20 second parabola, reactions were quenched using a divalent cation chelator and heat inactivated. When coupled with a novel unique mutation identifier algorithm developed for this experiment, an extremely accurate determination of any difference in DNA polymerase error rate was achieved. Initial results indicated a significant increase in replication fidelity in microgravity when compared to earth-like gravity in Klenow exonuclease-, but not with Klenow exonuclease+. While this result may not be biologically relevant, this experiment indicates that enzymatic activity can indeed be impacted by gravitational force. As such, the PolERIS experiment represents a new approach to conducting enzymology in microgravity, and introduces a flexible semi-autonomous payload suitable for testing other life-science related experiments. Furthermore, the de