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J. Biol. Chem., Vol. 283, Issue 23, 16093-16103, June 6, 2008

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Chemical and Traditional Mutagenesis of Vaccinia DNA Topoisomerase Provides Insights to Cleavage Site Recognition and Transesterification Chemistry^*

Lyudmila Yakovleva, Shengxi Chen, Sidney M. Hecht, and Stewart Shuman¹

From the Molecular Biology Program, Sloan-Kettering Institute, New York, New York 10065 and the Departments of Chemistry and Biology, University of Virginia, Charlottesville, Virginia 22901

Vaccinia DNA topoisomerase IB (TopIB) relaxes supercoils by forming and resealing a covalent DNA-(3'-phosphotyrosyl)-enzyme intermediate. Here we gained new insights to the TopIB mechanism through "chemical mutagenesis." Meta-substituted analogs of Tyr²⁷⁴ were introduced by in vitro translation in the presence of a chemically misacylated tRNA. We report that a meta-OH reduced the rate of DNA cleavage 130-fold without affecting the rate of religation. By contrast, meta-OCH₃ and NO₂ groups elicited only a 6-fold decrement in cleavage rate. We propose that the meta-OH uniquely suppresses deprotonation of the para-OH nucleophile during the cleavage step. Assembly of the vaccinia TopIB active site is triggered by protein contacts with a specific DNA sequence 5'-C⁺⁵C⁺⁴C⁺³T⁺²T⁺¹pN (where denotes the cleavage site). A signature -helix of the poxvirus TopIB (¹³²GKMKYLKENETVG¹⁴⁴) engages the target site in the major groove and thereby recruits catalytic residue Arg¹³⁰ to the active site. The effects of 11 missense mutations at Tyr¹³⁶ highlight the importance of van der Waals interactions with the 3'-G⁺⁴pG⁺³p dinucleotide of the nonscissile strand for DNA cleavage and supercoil relaxation. Asn¹⁴⁰ and Thr¹⁴² donate hydrogen bonds to the pro-(S_p)-oxygen of the G⁺³pA⁺² phosphodiester of the nonscissile strand. Lys¹³³ and Lys¹³⁵ interact with purine nucleobases in the major groove. Whereas none of these side chains is essential per se, an N140A/T142A double mutation reduces the rate of supercoil relaxation and DNA cleavage by 120- and 30-fold, respectively, and a K133A/K135A double mutation slows relaxation and cleavage by 120- and 35-fold, respectively. These results underscore functional redundancy at the TopIB-DNA interface.

Received for publication, February 27, 2008 , and in revised form, March 20, 2008.

^* This work was supported, in whole or in part, by National Institutes of Health Grants GM46330 (to S. S.) and CA077359 (to S. M. H.). 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.

¹ An American Cancer Society Research Professor. To whom correspondence should be addressed. Tel.: 212-639-7145; Fax: 212-772-8410; E-mail: s-shuman{at}ski.mskcc.org.

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