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Molecular and Cellular Biology, January 2009, p. 448-457, Vol. 29, No. 2
0270-7306/09/$08.00+0     doi:10.1128/MCB.01006-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Crystal Structure of CRN-4: Implications for Domain Function in Apoptotic DNA Degradation{triangledown}

Yu-Yuan Hsiao,1,2 Akihisa Nakagawa,3 Zhonghao Shi,2 Shohei Mitani,4 Ding Xue,3 and Hanna S. Yuan2,5*

Institute of Bioinformatics and Structural Biology, National Tsing Hua University, Hsinchu, Taiwan, Republic of China,1 Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan, Republic of China,2 Department of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder, Colorado,3 Department of Physiology, Tokyo Women's Medical University, School of Medicine, and CREST, JST, Tokyo 162-8666, Japan,4 Graduate Institute of Biochemistry and Molecular Biology, National Taiwan University, Taipei, Taiwan, Republic of China5

Received 26 June 2008/ Returned for modification 4 September 2008/ Accepted 23 October 2008

Cell death related nuclease 4 (CRN-4) is one of the apoptotic nucleases involved in DNA degradation in Caenorhabditis elegans. To understand how CRN-4 is involved in apoptotic DNA fragmentation, we analyzed CRN-4's biochemical properties, in vivo cell functions, and the crystal structures of CRN-4 in apo-form, Mn2+-bound active form, and Er3+-bound inactive form. CRN-4 is a dimeric nuclease with the optimal enzyme activity in cleaving double-stranded DNA in apoptotic salt conditions. Both mutational studies and the structures of the Mn2+-bound CRN-4 revealed the geometry of the functional nuclease active site in the N-terminal DEDDh domain. The C-terminal domain, termed the Zn-domain, contains basic surface residues ideal for nucleic acid recognition and is involved in DNA binding, as confirmed by deletion assays. Cell death analysis in C. elegans further demonstrated that both the nuclease active site and the Zn-domain are required for crn-4's function in apoptosis. Combining all of the data, we suggest a structural model where chromosomal DNA is bound at the Zn-domain and cleaved at the DEDDh nuclease domain in CRN-4 when the cell is undergoing apoptosis.

* Corresponding author. Mailing address: Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan 11529. Phone: 886-2-27884151. Fax: 886-2-27826085. E-mail: hanna{at}sinica.edu.tw

{triangledown} Published ahead of print on 3 November 2008.

Molecular and Cellular Biology, January 2009, p. 448-457, Vol. 29, No. 2
0270-7306/09/$08.00+0     doi:10.1128/MCB.01006-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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