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J. Biol. Chem., Vol. 280, Issue 25, 23484-23489, June 24, 2005

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Dual Mechanisms whereby a Broken RNA End Assists the Catalysis of Its Repair by T4 RNA Ligase 2^*

Jayakrishnan Nandakumar and Stewart Shuman

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

T4 RNA ligase 2 (Rnl2) efficiently seals 3'-OH/5'-PO₄RNA nicks via three nucleotidyl transfer steps. Here we show that the terminal 3'-OH at the nick accelerates the second step of the ligase pathway (adenylylation of the 5'-PO₄ strand) by a factor of 1000, even though the 3'-OH is not chemically transformed during the reaction. Also, the terminal 2'-OH at the nick accelerates the third step (attack of the 3'-OH on the 5'-adenylated strand to form a phosphodiester) by a factor of 25–35, even though the 2'-OH is not chemically reactive. His-37 of Rnl2 is uniquely required for step 3, providing a 10² rate acceleration. Biochemical epistasis experiments show that His-37 and the RNA 2'-OH act independently. We conclude that the broken RNA end promotes catalysis of its own repair by Rnl2 via two mechanisms, one of which (enhancement of step 3 by the 2'-OH) is specific to RNA ligation. Substrate-assisted catalysis provides a potential biochemical checkpoint during nucleic acid repair.

Received for publication, January 24, 2005 , and in revised form, March 28, 2005.

^* This work was supported in part by Grant GM63611 from the National Institutes of Health. 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.

American Cancer Society Research Professor. To whom correspondence should be addressed. Fax: 212-717-3623; E-mail: s-shuman{at}ski.mskcc.org.

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