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J. Biol. Chem., Vol. 283, Issue 6, 3059-3066, February 8, 2008

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Biochemical Activities of Arabidopsis RNA-dependent RNA Polymerase 6^*

From the Department of Botany and Plant Sciences, University of California, Riverside, California 92521

In Arabidopsis, genetic evidence demonstrates that RNA-dependent RNA polymerase 6 (RDR6) plays a fundamental role in at least four RNA silencing pathways whose functions range from defense against transgenes or viruses to endogene regulation in development and in stress responses. Despite its critical role in RNA silencing, the biochemical activities of RDR6 have yet to be characterized. In this study, we transiently expressed Arabidopsis RDR6 in Nicotiana benthamiana and investigated the biochemical activities of immunopurified RDR6 in vitro. We showed that RDR6 possesses terminal nucleotidyltransferase activity as well as primer-independent RNA polymerase activity on single-stranded RNAs. We found that RDR6 cannot distinguish RNAs with or without a cap or poly(A) tail. We also demonstrated that RDR6 has strong polymerase activity on single-stranded DNA. All these activities require the conserved catalytic Asp⁸⁶⁷ residue. Our findings have important implications on the processes involving RDR6 in vivo and provide new biochemical insights into the mechanisms of RNA silencing in Arabidopsis.

Received for publication, November 1, 2007 , and in revised form, December 3, 2007.

^* This work was supported by National Science Foundation Grant MCB-0718029 (to X. C.). 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 Figs. S1–S6.

¹ To whom correspondence should be addressed. Tel.: 951-827-3988; Fax: 951-827-4437; E-mail: xuemei.chen{at}ucr.edu.

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