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Related Experiment Videos

DNA supercoiling by DNA gyrase. A static head analysis.

H V Westerhoff1, M H O'Dea, A Maxwell

  • 1Laboratory of Molecular Biology, National Institute of Diabetes, and Digestive and Kidney Diseases, Bethesda, MD 20892.

Cell Biophysics
|January 1, 1988
PubMed
Summary

DNA gyrase supercoiling is regulated by ATP and ADP concentrations, with spermidine influencing the extent of DNA linking. The study reveals a kinetic scheme explaining these DNA gyrase behaviors.

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Area of Science:

  • Molecular Biology
  • Biochemistry
  • Enzymology

Background:

  • DNA gyrase is a crucial enzyme for managing DNA topology in bacteria.
  • Understanding DNA gyrase's mechanism is vital for developing novel antibiotics.

Purpose of the Study:

  • To investigate the influence of ATP and ADP concentrations on DNA gyrase supercoiling activity.
  • To determine the effect of spermidine on DNA gyrase's steady-state linking number.

Main Methods:

  • Purified DNA gyrase was used to supercoil plasmid pBR322 DNA.
  • Linking number was measured at steady state under varying ATP, ADP, and spermidine concentrations.

Main Results:

  • In the absence of spermidine, the linking number depended on the ATP-to-ADP ratio, not their sum.

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  • Spermidine decreased the linking number but maintained dependence on the ATP-to-ADP ratio.
  • The steady state was reached regardless of the direction of the reaction.

    • Conclusions:

    • A kinetic scheme for DNA gyrase was proposed that explains the experimental findings.
    • Significant DNA gyrase slip occurs without spermidine, which may be regulated by spermidine's effect on enzyme coupling.