Ca²⁺ binding to the ExDxD motif regulates the DNA cleavage specificity of a promiscuous endonuclease

Abstract

Most of the restriction endonucleases (REases) are dependent on Mg²⁺ for DNA cleavage, and in general, Ca²⁺ inhibits their activity. R.KpnI, an HNH active site containing ββα-Me finger nuclease, is an exception. In presence of Ca²⁺, the enzyme exhibits high-fidelity DNA cleavage and complete suppression of Mg²⁺-induced promiscuous activity. To elucidate the mechanism of unusual Ca²⁺-mediated activity, we generated alanine variants in the putative Ca²⁺ binding motif, E₁₃₂xD₁₃₄xD₁₃₆, of the enzyme. Mutants showed decreased levels of DNA cleavage in the presence of Ca²⁺. We demonstrate that ExDxD residues are involved in Ca²⁺ coordination; however, the invariant His of the catalytic HNH motif acts as a general base for nucleophile activation, and the other two active site residues, D148 and Q175, also participate in Ca²⁺-mediated cleavage. Insertion of a 10-amino acid linker to disrupt the spatial organization of the ExDxD and HNH motifs impairs Ca²⁺ binding and affects DNA cleavage by the enzyme. Although ExDxD mutant enzymes retained efficient cleavage at the canonical sites in the presence of Mg²⁺, the promiscuous activity was greatly reduced, indicating that the carboxyl residues of the acidic triad play an important role in sequence recognition by the enzyme. Thus, the distinct Ca²⁺ binding motif that confers site specific cleavage upon Ca²⁺ binding is also critical for the promiscuous activity of the Mg²⁺-bound enzyme, revealing its role in metal ion-mediated modulation of DNA cleavage.