Divalent Ion-Induced Switch in DNA Cleavage of KpnI Endonuclease Probed through Surface-Enhanced Raman Spectroscopy

• Published in: The journal of physical chemistry. B 2021-03, Vol.125 (9), p.2241-2250
• Main Authors: Aggarwal, Shantanu, Mondal, Sayan, Siddhanta, Soumik, Bharat, Engleng, Nagamalleswari, Easa, Nagaraja, Valakunja, Narayana, Chandrabhas
• Format: Article
• Language: English
• Subjects: B: Biophysical and Biochemical Systems and Processes; Cations, Divalent; Deoxyribonucleases, Type II Site-Specific - genetics; DNA Cleavage; Ions; Spectrum Analysis, Raman
• ISSN: 1520-6106; 1520-5207
• DOI: 10.1021/acs.jpcb.0c10667

We demonstrate the remarkable ability of surface-enhanced Raman spectroscopy (SERS) to track the allosteric changes in restriction endonuclease KpnI (R.KpnI) caused by metal ions. R.KpnI binds and promiscuously cleaves DNA upon activation by Mg2+ ions. However, the divalent ion Ca2+ induces high fidelity cleavage, which can be overcome by higher concentrations of Mg2+ ions. In the absence of any 3D crystal structure, for the first time, we have elucidated the structural underpinnings of such a differential effect of divalent ions on the endonuclease activity. A combined SERS and molecular dynamics (MD) approach showed that Ca2+ ion activates an enzymatic switch in the active site, which is responsible for the high fidelity activity of the enzyme. Thus, SERS in combination with MD simulations provides a powerful tool for probing the link between the structure and activity of enzyme molecules that play vital roles in DNA transactions.