Ca2+ Binding to the ExDxD Motif Regulates the DNA Cleavage Specificity of a Promiscuous Endonuclease

Nagamalleswari, Easa and Vasu, Kommireddy and Nagaraja, Valakunja (2012) Ca2+ Binding to the ExDxD Motif Regulates the DNA Cleavage Specificity of a Promiscuous Endonuclease. In: BIOCHEMISTRY, 51 (44). pp. 8939-8949.

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Abstract

Most of the restriction endonucleases (REases) are dependent on Mg2+ for DNA cleavage, and in general, Ca2+ inhibits their activity. RKpnI, an HNH active site containing beta beta alpha-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 Ca-2+ binding motif, E(132)xD(134)xD(136), 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.

Item Type:	Journal Article
Publication:	BIOCHEMISTRY
Publisher:	American Chemical Society
Additional Information:	Copyright for this article belongs to AMER CHEMICAL SOC, WASHINGTON,
Department/Centre:	Division of Biological Sciences > Microbiology & Cell Biology
Date Deposited:	28 Dec 2012 10:17
Last Modified:	28 Dec 2012 10:17
URI:	http://eprints.iisc.ac.in/id/eprint/45468

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