Site-specific DNA Transesterification by Vaccinia Topoisomerase

• Published in: The Journal of biological chemistry Vol. 278; no. 43; pp. 42170 - 42177
• Main Authors: Yakovleva, Lyudmila, Tian, Ligeng, Sayer, Jane M., Kalena, Govind P., Kroth, Heiko, Jerina, Donald M., Shuman, Stewart
• Format: Journal Article
• Language: English
• Published: Elsevier Inc 24.10.2003; American Society for Biochemistry and Molecular Biology
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M308079200

Vaccinia DNA topoisomerase forms a covalent DNA-(3′-phosphotyrosyl)-enzyme intermediate at a specific target site 5′-C+5C+4C+3T+2T+1p↓N-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.