Site-specific DNA transesterification by vaccinia topoisomerase: effects of benzo[alpha]pyrene-dA, 8-oxoguanine, 8-oxoadenine and 2-aminopurine modifications.
Vaccinia DNA topoisomerase forms a covalent DNA-(3'-phosphotyrosyl)-enzyme intermediate at a specific target site 5'-C+5C+4C+3T+2T+1p downward arrow 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.[1]References
- Site-specific DNA transesterification by vaccinia topoisomerase: effects of benzo[alpha]pyrene-dA, 8-oxoguanine, 8-oxoadenine and 2-aminopurine modifications. Yakovleva, L., Tian, L., Sayer, J.M., Kalena, G.P., Kroth, H., Jerina, D.M., Shuman, S. J. Biol. Chem. (2003) [Pubmed]
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