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J. Biol. Chem., Vol. 282, Issue 44, 32320-32326, November 2, 2007

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Dual Role for Zn²⁺ in Maintaining Structural Integrity and Inducing DNA Sequence Specificity in a Promiscuous Endonuclease^*

Matheshwaran Saravanan¹, Kommireddy Vasu, Soumitra Ghosh, and Valakunja Nagaraja²

From the Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560 012, India and the Jawaharlal Nehru Centre for Advanced Scientific Research, Bangalore 560 064, India

We describe two uncommon roles for Zn²⁺ in enzyme KpnI restriction endonuclease (REase). Among all of the REases studied, KpnI REase is unique in its DNA binding and cleavage characteristics. The enzyme is a poor discriminator of DNA sequences, cleaving DNA in a promiscuous manner in the presence of Mg²⁺. Unlike most Type II REases, the active site of the enzyme comprises an HNH motif, which can accommodate Mg²⁺, Mn²⁺, or Ca²⁺. Among these metal ions, Mg²⁺ and Mn²⁺ induce promiscuous cleavage by the enzyme, whereas Ca²⁺-bound enzyme exhibits site-specific cleavage. Examination of the sequence of the protein revealed the presence of a zinc finger CCCH motif rarely found in proteins of prokaryotic origin. The zinc binding motif tightly coordinates zinc to provide a rigid structural framework for the enzyme needed for its function. In addition to this structural scaffold, another atom of zinc binds to the active site to induce high fidelity cleavage and suppress the Mg²⁺- and Mn²⁺-mediated promiscuous behavior of the enzyme. This is the first demonstration of distinct structural and catalytic roles for zinc in an enzyme, suggesting the distinct origin of KpnI REase.

Received for publication, July 19, 2007 , and in revised form, August 31, 2007.

^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. 1 and Table 1.

¹ Senior research fellow of the Council of Scientific and Industrial Research, Government of India.

² To whom correspondence should be addressed. Tel.: 91-80-23600668; Fax: 91-80-23602697; E-mail: vraj{at}mcbl.iisc.ernet.in.

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