A surface plasmon resonance study of the intermolecular interaction between Escherichia coli topoisomerase i and pBAD/Thio supercoiled plasmid DNA

Purushottam Babu Tiwari, Thirunavukkarasu Annamalai, Bokun Cheng, Gagandeep Narula, Xuewen Wang, Yuk Ching Tse-Dinh, Jin He, Yesim Darici

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Abstract

To date, the bacterial DNA topoisomerases are one of the major target biomolecules for the discovery of new antibacterial drugs. DNA topoisomerase regulates the topological state of DNA, which is very important for replication, transcription and recombination. The relaxation of negatively supercoiled DNA is catalyzed by bacterial DNA topoisomerase I (topoI) and this reaction requires Mg2+. In this report, we first quantitatively studied the intermolecular interactions between Escherichia coli topoisomerase I (EctopoI) and pBAD/Thio supercoiled plasmid DNA using surface plasmon resonance (SPR) technique. The equilibrium dissociation constant (Kd) for EctopoI-pBAD/Thio interactions was determined to be about 8 nM. We then studied the effect of Mg2+ on the catalysis of EctopoI-pBAD/Thio reaction. A slightly higher equilibrium dissociation constant (∼15 nM) was obtained for Mg2+ coordinated EctopoI (Mg2+EctopoI)-pBAD/Thio interactions. In addition, we observed a larger dissociation rate constant (kd) for Mg2+EctopoI-pBAD/Thio interactions (∼0.043 s-1), compared to EctopoI-pBAD/Thio interactions (∼0.017 s -1). These results suggest that enzyme turnover during plasmid DNA relaxation is enhanced due to the presence of Mg2+ and furthers the understanding of importance of the Mg2+ ion for bacterial topoisomerase I catalytic activity.

Original languageEnglish (US)
Pages (from-to)445-450
Number of pages6
JournalBiochemical and Biophysical Research Communications
Volume445
Issue number2
DOIs
Publication statusPublished - Mar 7 2014
Externally publishedYes

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Keywords

  • Bacterial topoisomerase I
  • Capture covalent method
  • Equilibrium dissociation constant
  • Supercoiled plasmid DNA
  • Surface plasmon resonance

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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