HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Doudeva, L. G. Articles by Yuan, H. S. Search for Related Content PubMed PubMed Citation Articles by Doudeva, L. G. Articles by Yuan, H. S. Social Bookmarking                   What's this?

Crystal structural analysis and metal-dependent stability and activity studies of the ColE7 endonuclease domain in complex with DNA/Zn²⁺ or inhibitor/Ni²⁺

¹ Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan, Republic of China
² Institute of Biochemistry, National Yang-Ming University, Taipei, Taiwan, Republic of China
³ Institute of Biochemistry and Molecular Biology, College of Medicine, National Taiwan University, Taipei, Taiwan, Republic of China

(RECEIVED October 10, 2005; FINAL REVISION November 10, 2005; ACCEPTED November 14, 2005)

The nuclease domain of ColE7 (N-ColE7) contains an H-N-H motif that folds in a -metal topology. Here we report the crystal structures of a Zn²⁺-bound N-ColE7 (H545E mutant) in complex with a 12-bp duplex DNA and a Ni²⁺-bound N-ColE7 in complex with the inhibitor Im7 at a resolution of 2.5 Å and 2.0 Å, respectively. Metal-dependent cleavage assays showed that N-ColE7 cleaves double-stranded DNA with a single metal ion cofactor, Ni²⁺, Mg²⁺, Mn²⁺, and Zn²⁺. ColE7 purified from Escherichia coli contains an endogenous zinc ion that was not replaced by Mg²⁺ atconcentrations of <25mM, indicating thatzincisthe physiologically relevant metal ion in N-ColE7 in host E. coli. In the crystal structure of N-ColE7/DNA complex, the zinc ion is directly coordinated to three histidines and the DNA scissile phosphate in a tetrahedral geometry. In contrast, Ni²⁺ is bound in N-ColE7 in two different modes, to four ligands (three histidines and one phosphate ion), or to five ligands with an additional water molecule. These data suggest that the divalent metal ion in the His-metal finger motif can be coordinated to six ligands, such as Mg²⁺ in I-PpoI, Serratia nuclease and Vvn, five ligands or four ligands, such as Ni²⁺ or Zn²⁺ in ColE7. Universally, the metal ion in the His-metal finger motif is bound to the DNA scissile phosphate and serves three roles during hydrolysis: polarization of the P–O bond for nucleophilic attack, stabilization of the phosphoanion transition state and stabilization of the cleaved product.

Keywords: Protein nucleic acid interactions; nonspecific nuclease; DNase; DNA hydrolysis mechanism; H-N-H motif; -metal motif; colicin E7

Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.051903406.

Reprint requests to: Hanna S. Yuan, Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan 11529, Republic of China; e-mail: hanna{at}sinica.edu.tw; fax: 886-2-27826085.

CiteULike    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

				J. B. Robbins, M. Stapleton, M. J. Stanger, D. Smith, J. T. Dansereau, V. Derbyshire, and M. Belfort Homing endonuclease I-TevIII: dimerization as a means to a double-strand break Nucleic Acids Res., March 12, 2007; 35(5): 1589 - 1600. [Abstract] [Full Text] [PDF]

				E. Cascales, S. K. Buchanan, D. Duche, C. Kleanthous, R. Lloubes, K. Postle, M. Riley, S. Slatin, and D. Cavard Colicin Biology Microbiol. Mol. Biol. Rev., March 1, 2007; 71(1): 158 - 229. [Abstract] [Full Text] [PDF]

				Y.-T. Wang, W.-J. Yang, C.-L. Li, L. G. Doudeva, and H. S. Yuan Structural basis for sequence-dependent DNA cleavage by nonspecific endonucleases Nucleic Acids Res., January 28, 2007; 35(2): 584 - 594. [Abstract] [Full Text] [PDF]