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J. Biol. Chem., Vol. 279, Issue 18, 18220-18231, April 30, 2004

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Characterization of a Baculovirus Enzyme with RNA Ligase, Polynucleotide 5'-Kinase, and Polynucleotide 3'-Phosphatase Activities^*

Alexandra Martins and Stewart Shuman

From the Molecular Biology Program, Sloan-Kettering Institute, New York, New York 10021

The end-healing and end-sealing steps of the phage T4-induced RNA restriction-repair pathway are performed by two separate enzymes, a bifunctional polynucleotide 5'-kinase/3'-phosphatase and an ATP-dependent RNA ligase. Here we show that a single trifunctional baculovirus enzyme, RNA ligase 1 (Rnl1), catalyzes the identical set of RNA repair reactions. Three enzymatic activities of baculovirus Rnl1 are organized in a modular fashion within a 694-amino acid polypeptide consisting of an autonomous N-terminal RNA-specific ligase domain, Rnl1-(1–385), and a C-terminal kinase-phosphatase domain, Rnl1-(394–694). The ligase domain is itself composed of two functional units. The N-terminal module Rnl1-(1–270) contains essential nucleotidyltransferase motifs I, IV, and V and suffices for both enzyme adenylylation (step 1 of the ligation pathway) and phosphodiester bond formation at a preactivated RNA-adenylate end (step 3). The downstream module extending to residue 385 is required for ligation of a phosphorylated RNA substrate, suggesting that it is involved specifically in the second step of the end-joining pathway, the transfer of AMP from the ligase to the 5'-PO₄ end to form RNA-adenylate. The end-healing domain Rnl1-(394–694) consists of a proximal 5'-kinase module with an essential P-loop motif (⁴⁰⁴GSGKS⁴⁰⁸) and a distal 3'-phosphatase module with an essential acylphosphatase motif (⁵⁶⁰DLDGT⁵⁶⁴). Our findings have implications for the evolution of RNA repair systems and their potential roles in virus-host dynamics.

Received for publication, December 8, 2003 , and in revised form, January 12, 2004.

^* This work was supported by National Institutes of Health Grant GM42498. 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. E-mail: s-shuman{at}ski.mskcc.org.

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