Investigating the Effects of DNA Packaging on Natural Mutation Frequency

DNA gyrase causes negative supercoiling in DNA and plays an important role in DNA packaging. We hypothesized that DNA treated with a gyrase inhibitor would be less supercoiled, and thus more accessible to repair enzymes. This would lead to a lower natural mutation rate in treated cells compared to untreated cells. To test this hypothesis, Escherichia coli DH5 α pUC19 containing a plasmid which carried ampicillin resistance was treated with a gyrase inhibitor and its plasmid analyzed for evidence of elimination of supercoiling. The antibiotic novobiocin was used as a gyrase inhibitor and ampicillin resistance was used as an indication of mutation, and hence formed the basis of calculating mutation rates. The methods of experimentation were: (a) Dilution susceptibility test to determine what concentration of novobiocin to use; (b) Gram stain analysis to confirm the effects of novobiocin on cell size; and (c) Luria-Delbruck fluctuation assay to determine the natural mutation rates of treated and untreated cells. The results obtained were not as expected. Treated cells had a higher mutation rate than untreated cells. This result is contrary to our hypothesis. Inhibition of DNA gyrase might actually inhibit, not enhance the accessibility of DNA to repair enzymes. Alternatively, these results could be affected by other factors not taken into account, such as the possibility of inhibition of transcription in unwound DNA, and thus the inhibition of the synthesis of repair enzymes, or the concentrations of novobiocin used might have been too high.