We use ESR spectroscopy and MD simulations to investigate the role of Cu2+ in the catalytic activity of endonuclease EcoRI. EcoRI is a homodimer, which recognizes and cleaves a specific DNA site in the presence of Mg2+. Unexpectedly, Cu2+ strongly inhibits this cleavage. ESR data establish that Cu2+ coordinates to His114 in each monomer of EcoRI. MD simulations reveal that Cu2+ disrupts hydrogen bonding between His114 and DNA. Lacking this interaction, the DNA rotates to a conformation that cannot support a critical water network, leading to inhibition. Interestingly, Mg2+ catalyzed DNA cleavage kinetics shows a positive cooperativity. Electrostatic potential calculations show that the binding of Mg2+ in one catalytic site causes the electrostatic potential to become more negative in the metal-free site, which enhances binding of the second Mg2+. These findings help us gain a better understanding of the role of metal ions in the catalytic activity of this endonuclease.

Insights on Cu<sup>2+</sup> inhibition of endonuclease catalysis by ESR spectroscopy and MD simulations

244th National Meeting (2012)

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Insights on Cu2+ inhibition of endonuclease catalysis by ESR spectroscopy and MD simulations

Insights on Cu²⁺ inhibition of endonuclease catalysis by ESR spectroscopy and MD simulations