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J. Biol. Chem., Vol. 279, Issue 45, 47132-47138, November 5, 2004

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Identification of a Zinc Finger Domain in the Human NEIL2 (Nei-like-2) Protein^*

Aditi Das, Lavanya Rajagopalan, Venkatarajan S. Mathura, Samuel J. Rigby¶, Sankar Mitra, and Tapas K. Hazra||

From the Sealy Center for Molecular Science and Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston, Texas 77555-1079, Roskamp Institute, Sarasota, Florida 34243, and ^¶Department of Geology, Scripps Institution of Oceanography, La Jolla, California 92093

The recently identified human NEIL2 (Nei-like-2) protein, a DNA glycosylase/AP lyase specific for oxidatively damaged bases, shares structural features and reaction mechanism with the Escherichia coli DNA glycosylases, Nei and Fpg. Amino acid sequence analysis of NEIL2 suggested it to have a zinc finger-like Nei/Fpg. However, the Cys-X₂-His-X₁₆-Cys-X₂-Cys (CHCC) motif present near the C terminus of NEIL2 is distinct from the zinc finger motifs of Nei/Fpg, which are of the C4 type. Here we show the presence of an equimolar amount of zinc in NEIL2 by inductively coupled plasma mass spectrometry. Individual mutations of Cys-291, His-295, Cys-315, and Cys-318, candidate residues for coordinating zinc, inactivated the enzyme by abolishing its DNA binding activity. H295A and C318S mutants were also shown to lack bound zinc, and a significant change in their secondary structure was revealed by CD spectra analysis. Molecular modeling revealed Arg-310 of NEIL2 to be a critical residue in its zinc binding pocket, which is highly conserved throughout the Fpg/Nei family. A R310Q mutation significantly reduced the activity of NEIL2. We thereby conclude that the zinc finger motif in NEIL2 is essential for its structural integrity and enzyme activity.

Received for publication, June 4, 2004 , and in revised form, August 25, 2004.

The atomic coordinates and structure factors (code 1vzp) 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 supported by U. S. Public Health Service Grants RO1 CA81063 and PO1 CA92584. 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: Sealy Center for Molecular Science, University of Texas Medical Branch, 6.136 Medical Research BLDG., Rte. 1079, Galveston, TX. Tel.: 409-772-2156; Fax: 409-747-8607; E-mail: tkhazra{at}utmb.edu.

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