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

作者：Tiwari Purushottam Babu; Annamalai Thirunavukkarasu; Cheng Bokun; Narula Gagandeep; Wang Xuewen; Tse Dinh Yuk Ching^*; He Jin; Darici Yesim

来源：Biochemical and Biophysical Research Communications, 2014, 445(2): 445-450.

DOI：10.1016/j.bbrc.2014.02.015

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

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 supercolied 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 (Ectopol) and pBAD/Thio supercolied plasmid DNA using surface plasmon resonance (SPR) technique. The equilibrium dissociation constant (K-d) 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 (similar to 15 nM) was obtained for Mg2+ coordinated Ectopol (Mg(2+)EctopoI)-pBAD/Thio interactions. In addition, we observed a larger dissociation rate constant (k(d)) for Mg(2+)Ectopol-pBAD/Thio interactions (similar to 0.043 s(-1)), compared to EctopoI-pBAD/Thio interactions (similar to 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.

• 出版日期2014-3-7