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J. Biol. Chem., Vol. 279, Issue 22, 23335-23342, May 28, 2004

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Benzo[c]phenanthrene Adducts and Nogalamycin Inhibit DNA Transesterification by Vaccinia Topoisomerase^*

Lyudmila Yakovleva, Christopher J. Handy, Jane M. Sayer, Michael Pirrung¶, Donald M. Jerina, and Stewart Shuman||

From the Molecular Biology Program, Sloan-Kettering Institute, New York, New York 10021, Laboratory of Bioorganic Chemistry, NIDDK/National Institutes of Health, Bethesda, Maryland 20892, and ^¶Department of Chemistry, Duke University, Durham, North Carolina 27708

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 position-specific DNA intercalators on the rate and extent of single-turnover DNA transesterification. Chiral C-1 R and S trans-opened 3,4-diol 1,2-epoxide adducts of benzo[c]phenanthrene (BcPh) were introduced at single N²-deoxyguanosine and N⁶-deoxyadenosine positions within the 3'-G⁺⁵G⁺⁴G⁺³A⁺²A⁺¹T^-1A^-2 sequence of the nonscissile DNA strand. Transesterification was unaffected by BcPh intercalation between the +6 and +5 base pairs, slowed 4-fold by intercalation between the +5 and +4 base pairs, and virtually abolished by BcPh intercalation between the +4 and +3 base pairs and the +3 and +2 base pairs. Intercalation between the +2 and +1 base pairs by the +2R BcPh dA adduct abolished transesterification, whereas the overlapping +1S BcPh dA adduct slowed the rate of transesterification by a factor of 2700, with little effect upon the extent of the reaction. Intercalation at the scissile phosphodiester (between the +1 and -1 base pairs) slowed transesterification by a factor of 450. BcPh intercalation between the -1 and -2 base pairs slowed cleavage by two orders of magnitude, but intercalation between the -2 and -3 base pairs had little effect. The anthracycline drug nogalamycin, a non-covalent intercalator with preference for 5'-TG dinucleotides, inhibited the single-turnover DNA cleavage reaction of vaccinia topoisomerase with an IC₅₀ of 0.7 µM. Nogalamycin was most effective when the drug was pre-incubated with DNA and when the cleavage target site was 5'-CCCTTG instead of 5'-CCCTTA. These findings demarcate upstream and downstream boundaries of the functional interface of vaccinia topoisomerase with its DNA target site.

Received for publication, February 3, 2004 , and in revised form, March 22, 2004.

^* 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.

The on-line version of this article (available at http://www.jbc.org contains Table S1.

^|| 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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