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J. Biol. Chem., Vol. 282, Issue 13, 9941-9951, March 30, 2007

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Probing the Roles of Active Site Residues in the 3'-5' Exonuclease of the Werner Syndrome Protein^*

Jung Min Choi, Sung Yun Kang¹, Won Jin Bae¹, Kyeong Sik Jin, Moonhor Ree, and Yunje Cho²

From the National Creative Research Center for Structural Biology and Department of Life Science, Pohang University of Science and Technology, Hyo-ja dong, San31, Pohang, KyungBook 790-784, South Korea and the Department of Chemistry, National Research Laboratory for Polymer Synthesis & Physics, Pohang Accelerator Laboratory, Center for Integrated Molecular Systems, Polymer Research Institute, and BK School of Molecular Science, Pohang University of Science and Technology, Pohang, KyungBook 790-784, South Korea

Werner syndrome is a premature aging disease caused by mutations in the WS gene and a deficiency in the function of Werner protein (WRN). The lack of WRN results in a cellular phenotype of genomic instability. WRN belongs to the RecQ DNA helicase family, but unlike other RecQ family members it possesses a functional exonuclease domain. We determined the crystal structure of mWRNexo (residues 31–238) bound to Zn²⁺ and the sulfate ion. Compared with the structure of human WRNexo (hWRNexo), notable conformational changes were observed in several active site residues in an H5–H6 loop and in helices H6 and H7 of mWRNexo, presumably because of the presence of sulfate, which mimics the phosphate of substrate DNA. In particular, the side chains of Lys¹⁸⁵ and Tyr²⁰⁶ were reoriented toward the Zn²⁺ ion, whereas the side chain of Arg¹⁹⁰ pointed away from the active site center. Mutational analysis of these conserved residues abolished WRN exonuclease activity, suggesting that these residues play a critical role in the WRNexo activity. Based on substrate modeling and mutational analyses, we propose a mechanism by which WRNexo becomes activated upon substrate DNA binding. We also describe the low resolution trimeric structure of mouse WRNexoL (mWRNexoL, residues 31–330), as elucidated by small angle x-ray scattering (SAXS) analyses.

Received for publication, October 13, 2006 , and in revised form, December 8, 2006.

The atomic coordinates and structure factors (code 2E6L and 2E6M) 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 funds from the National Creative Research Initiatives (Ministry of Science and Technology). 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.

¹ These authors contributed equally to this work.

² To whom correspondence should be addressed: Dept. of Life Science, Pohang University of Science and Technology, Pohang, KyungBook, South Korea. Tel.: 8254-279-2288; Fax: 8254-279-8111; E-mail: yunje{at}postech.ac.kr.

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