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J. Biol. Chem., Vol. 279, Issue 33, 34763-34769, August 13, 2004

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Structure-based Analysis of the Metal-dependent Mechanism of H-N-H Endonucleases^*

María J. Maté and Colin Kleanthous

From the Department of Biology, Area 10, P. O. Box 373, University of York, Heslington YO10 5YW, United Kingdom

Controversy surrounds the metal-dependent mechanism of H-N-H endonucleases, enzymes involved in a variety of biological functions, including intron homing and DNA repair. To address this issue we determined the crystal structures for complexes of the H-N-H motif containing bacterial toxin colicin E9 with Zn²⁺, Zn²⁺·DNA, and Mg²⁺·DNA. The structures show that the rigid V-shaped architecture of the active site does not undergo any major conformational changes on binding to the minor groove of DNA and that the same interactions are made to the nucleic acid regardless of which metal ion is bound to the enzyme. The scissile phosphate contacts the single metal ion of the motif through distortion of the DNA brought about by the insertion of the Arg-96-Glu-100 salt bridge into the minor groove and a network of contacts to the DNA phosphate backbone that straddle the metal site. The Mg²⁺-bound structure reveals an unusual coordination scheme involving two H-N-H histidine residues, His-102 and His-127. The mechanism of DNA cleavage is likely related to that of other single metal ion-dependent endonucleases, such as I-PpoI and Vvn, although in these enzymes the single alkaline earth metal ion is coordinated by oxygen-bearing amino acids. The structures also provide a rationale as to why H-N-H endonucleases are inactive in the presence of Zn²⁺ but active with other transition metal ions such as Ni²⁺. This is because of coordination of the Zn²⁺ ion through a third histidine, His-131. "Active" transition metal ions are those that bind more weakly to the H-N-H motif because of the disengagement of His-131, which we suggest allows a water molecule to complete the catalytic cycle.

Received for publication, April 5, 2004 , and in revised form, June 8, 2004.

The atomic coordinates and structure factors (code 1V13, 1V14, and 1V15) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* This work was funded by the Biotechnology and Biological Sciences Research Council of the UK. 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.

To whom correspondence should be addressed. Tel.: 44-1904-328820; Fax: 44-1904-328825; E-mail: ck11{at}york.ac.uk.

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