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J. Biol. Chem., Vol. 278, Issue 43, 42170-42177, October 24, 2003

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Site-specific DNA Transesterification by Vaccinia Topoisomerase

EFFECTS OF BENZO[]PYRENE-dA, 8-OXOGUANINE, 8-OXOADENINE, AND 2-AMINOPURINE MODIFICATIONS^*

Lyudmila Yakovleva, Ligeng Tian, Jane M. Sayer, Govind P. Kalena, Heiko Kroth, Donald M. Jerina, and Stewart Shuman¶

From the Molecular Biology Program, Sloan-Kettering Institute, New York, New York 10021 and Laboratory of Bioorganic Chemistry, NIDDK, National Institutes of Health, Department of Health and Human Services, 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 base modifications on the rate and extent of single-turnover DNA transesterification. Chiral trans opened C-10 R and S adducts of benzo[a]pyrene (BP) 7,8-diol 9,10-epoxide were introduced at single N⁶-deoxyadenosine (dA) positions within the 3'-G⁺⁵G⁺⁴G⁺³A⁺²A⁺¹T^-1A^-2 sequence of the nonscissile DNA strand. The R and S BPdA adducts intercalate from the major groove on the 5' and 3' sides of the modified base, respectively, and perturb local base stacking. We found that R and S BPdA modifications at +1A reduced the transesterification rate by a factor of 700–1000 without affecting the yield of the covalent topoisomerase-DNA complex. BPdA modifications at +2A reduced the extent of transesterification and elicited rate decrements of 200- and 7000-fold for the S and R diastereomers, respectively. In contrast, BPdA adducts at the -2 position had no effect on the extent of the reaction and relatively little impact on the rate of cleavage. A more subtle probe of major groove contacts entailed substituting each of the purines of the nonscissile strand with its 8-oxo analog. The +3 oxoG modification slowed transesterification 35-fold, whereas other 8-oxo modifications were benign. 8-Oxo substitutions at the -1 position in the scissile strand slowed single-turnover cleavage by a factor of six but had an even greater slowing effect on religation, which resulted in an increase in the cleavage equilibrium constant. 2-Aminopurine at positions +3, +4, or +5 in the nonscissile strand had no effect on transesterification per se but had synergistic effects when combined with 8-oxoA at position -1 in the scissile strand. These findings illuminate the functional interface of vaccinia topoisomerase with the DNA major groove.

Received for publication, July 24, 2003

^* 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.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Tables S1 and S2.

^¶ 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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