Kinetics of E. coli Topoisomerase I and Energetic Studies of DNA Supercoiling by Isothermal Titration Calorimetry Thesis

thesis or dissertation chair

fiu authors

  • Xu, Xiaozhou

abstract

  • In this thesis, on the basis of the asymmetrical charge distribution of E. coli topoisomerase I, I developed a new rapid procedure to purify E. coli DNA topoismoerase I in the milligram range. The new procedure includes using both cation- and anion-exchange columns, i.e., SP-sepharose FF and Q-sepharose FF columns. E. coli topoisomerase I purified here is free of nuclease contamination. The kinetic constants of the DNA relaxation reaction of E. coli DNA topoisomerase I were determined as well. I also used isothermal titration calorimetry to investigate the energetics of DNA supercoiling by using the unwinding properties of DNA intercalators, ethidium and daunomycin. After comparing the enthalpy changes of these DNA intercalators binding to supercoiled and nicked or relaxed plasmid DNA pXXZ06, I determined the DNA supercoiling enthalpy is about 12 kcal/mol per turn of DNA supercoil, which is in good agreement with the previously published results.

publication date

  • October 28, 2010

keywords

  • DNA Supercoiling
  • E. coli Topoisomerase I
  • enzyme purification
  • isothermal titration calorimetry

Digital Object Identifier (DOI)