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J. Biol. Chem., Vol. 283, Issue 29, 20361-20371, July 18, 2008

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A Novel Family of Sequence-specific Endoribonucleases Associated with the Clustered Regularly Interspaced Short Palindromic Repeats^*

Natalia Beloglazova, Greg Brown, Matthew D. Zimmerman^¶||, Michael Proudfoot, Kira S. Makarova^**, Marina Kudritska^||, Samvel Kochinyan, Shuren Wang^¶||, Maksymilian Chruszcz^¶||, Wladek Minor^¶||, Eugene V. Koonin^**1, Aled M. Edwards^||, Alexei Savchenko^||, and Alexander F. Yakunin²

From the Banting and Best Department of Medical Research, University of Toronto, Toronto, Ontario M5G 1L6, Canada, Structural Proteomics in Toronto, Ontario Cancer Institute, Max Bell Research Centre 5R407, Toronto, Ontario M5G 2C4, Canada, the ^¶Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, Virginia 22908, the ^||Midwest Center for Structural Genomics, Biosciences Division, Argonne National Laboratory, Argonne, Illinois 60439, and the ^**National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, Maryland 20894

Clustered regularly interspaced short palindromic repeats (CRISPRs) together with the associated CAS proteins protect microbial cells from invasion by foreign genetic elements using presently unknown molecular mechanisms. All CRISPR systems contain proteins of the CAS2 family, suggesting that these uncharacterized proteins play a central role in this process. Here we show that the CAS2 proteins represent a novel family of endoribonucleases. Six purified CAS2 proteins from diverse organisms cleaved single-stranded RNAs preferentially within U-rich regions. A representative CAS2 enzyme, SSO1404 from Sulfolobus solfataricus, cleaved the phosphodiester linkage on the 3'-side and generated 5'-phosphate- and 3'-hydroxyl-terminated oligonucleotides. The crystal structure of SSO1404 was solved at 1.6Å resolution revealing the first ribonuclease with a ferredoxin-like fold. Mutagenesis of SSO1404 identified six residues (Tyr-9, Asp-10, Arg-17, Arg-19, Arg-31, and Phe-37) that are important for enzymatic activity and suggested that Asp-10 might be the principal catalytic residue. Thus, CAS2 proteins are sequence-specific endoribonucleases, and we propose that their role in the CRISPR-mediated anti-phage defense might involve degradation of phage or cellular mRNAs.

Received for publication, April 28, 2008 , and in revised form, May 14, 2008.

The atomic coordinates and structure factors (code 2i8e) 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, in whole or in part, by National Institutes of Health Grants GM62414 and GM074942 from the Protein Structure Initiative (Midwest Center for Structural Genomics). This work was also supported by Genome Canada (through the Ontario Genomics Institute), the Ontario Research and Development Fund, and the Canadian Foundation for Innovation. 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.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Table 1, Fig. 1, and additional references.

¹ Supported by the Intramural Research Program of the National Institutes of Health (National Library of Medicine, National Center for Biotechnology Information).

² To whom correspondence should be addressed. Tel.: 416-978-4013; Fax: 416-978-8528; E-mail: a.iakounine{at}utoronto.ca.

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