Microorganisms and PCR in Astrobiology
This study used a yeast mutant to elucidate the aspects of DNA repair in microgravity, which will be an important parameter in future space missions. They simulated microgravity from a Canadian airport:
Toronto/Kingston, Ontario, Canada: Fidelity of a Bacterial DNA Polymerase in Microgravity: A Model for Health in Space
Long-term space missions will expose crew members, their cells as well as their microbiomes to prolonged periods of microgravity and ionizing radiation, envi...
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'....Klenow fragment....Rad54-3 yeast mutants....temperature-conditional for DSB (double-strand-breaks) repair, had decreased DNA repair capabilities in orbit.
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Space-based microgravity, as well as microgravvity for ~20-second intervals during parabolic flight is termed "real microgravity"....substitutions may occur in the keto-enol tautomerization or oxidation of DNA bases, resulting in non-canonical base-pairing....Klenow (exo-) has neither 3'---->5' or 5'---->3' exonuclease activity due to inactivating mutations (D355A & E357A) in the proof-reading domain....considered a model polymerase.
Klenow fragment enters its steady-state phase within 20 seconds, as pre-steady-state elongation occurs within the first 100 msec from reaction initiation. These experiments are timely as astronauts are currently preparing to undertake prolonged exploratory missions where robust polymerase repair activities are essential for survival.
The results of this experiment also have applicability in biotechnology. Taq polymerase, the most commonly used enzyme for conducting PCR, is a 3'--->5' proofreading exonuclease-deficient DNA polymerase I derived from Thermus aquaticus. Therefore, the increase in the error rate of Klenow (exo-) in microgravity observed here may be applicable to the fidelity of Taq polymerase-mediated PCR in microgravity, and is therefore relevant for future biological research in space.
Even the relatively small decrease in polymerase fidelity observed in microgravity of Klenow (exo-), when adjusted based on the fidelity of Taq polymeriase and propagated over 30 cycles for a 1KB template, yields a concerning ~335% increase in incorrect templates in solution.'