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J. Biol. Chem., Vol. 279, Issue 38, 39718-39726, September 17, 2004

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Individual Nucleotide Bases, Not Base Pairs, Are Critical for Triggering Site-specific DNA Cleavage by Vaccinia Topoisomerase^*

Ligeng Tian, Jane M. Sayer, Donald M. Jerina, and Stewart Shuman¶

From the Molecular Biology Program, Sloan-Kettering Institute, New York, New York 10021 and the Laboratory of Bioorganic Chemistry, NIDDK, National Institutes of Health, DHHS, Bethesda, Maryland 20892

Vaccinia DNA topoisomerase forms a covalent DNA-(3'-phosphotyrosyl)-enzyme intermediate at a specific target site 5'-C⁺⁵C⁺⁴C⁺³T⁺²T⁺¹pN^-1 in duplex DNA. Here we study the effects of abasic lesions at individual positions of the scissile and nonscissile strands on the rate of single-turnover DNA transesterification and the cleavage-religation equilibrium. The rate of DNA incision was reduced by factors of 350, 250, 60, and 10 when abasic sites replaced the -1N, +1T, +2T, and +4C bases of the scissile strand, but abasic lesions at +5C and +3C had little or no effect. Abasic lesions in the nonscissile strand in lieu of +4G, +3G, +2A, and +1A reduced the rate of cleavage by factors of 130, 150, 10, and 5, whereas abasic lesions at +5G and -1N had no effect. The striking positional asymmetry of abasic interference on the scissile and nonscissile strands highlights the importance of individual bases, not base pairs, in promoting DNA cleavage. The rate of single-turnover DNA religation by the covalent topoisomerase-DNA complex was insensitive to abasic sites within the CCCTT sequence of the scissile strand, but an abasic lesion at the 5'-OH nucleoside (-1N) of the attacking DNA strand slowed the rate of religation by a factor of 600. Nonscissile strand abasic lesions at +1A and -1N slowed the rate of religation by factors of 140 and 20, respectively, and strongly skewed the cleavage-religation equilibrium toward the covalent complex. Thus, abasic lesions immediately flanking the cleavage site act as topoisomerase poisons.

Received for publication, July 1, 2004

^* This work was supported by National Institutes of Health Grants AI053471 and GM46330 (to S. S.). 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. Tel.: 212-639-7145; Fax: 212-717-3623; E-mail: s-shuman{at}ski.mskcc.org.

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