Stereochemical Outcome and Kinetic Effects of Rp- and Sp-Phosphorothioate Substitutions at the Cleavage Site of Vaccinia Type I DNA Topoisomerase † James T. Stivers,* ,‡ Barbara Nawrot, § G. Jayashree Jagadeesh, ‡ Wojciech J. Stec, § and Stewart Shuman | Center for AdVanced Research in Biotechnology of the UniVersity of Maryland and the National Institute for Standards and Technology, 9600 Gudelsky DriVe, RockVille, Maryland 20850, Department of Bioorganic Chemistry, Centre of Molecular and Macromolecular Studies, Polish Academy of Sciences, Lodz, Poland, and Molecular Biology Program, Sloan-Kettering Institute, New York, New York 10021 ReceiVed October 20, 1999; ReVised Manuscript ReceiVed February 1, 2000 ABSTRACT: To probe the mechanism of the reversible DNA phosphodiester bond cleavage and religation mechanism of the type I topoisomerase from vaccinia virus, we have synthesized DNA substrates carrying a single nonbridging Rp- or Sp-phosphorothioate (Ps) modification at the scissile phosphodiester (Pd) bond. Analysis of the stereochemical outcome of the net cleavage and rejoining reaction established that the reaction proceeds with retention of configuration, as expected for a double-displacement mechanism. Single-turnover kinetic studies on irreversible strand cleavage using 18/24 mer suicide substrates showed thio effects (k Pd /k Ps ) of 340- and 30-fold for the Rp-Ps and Sp-Ps stereoisomers, respectively, but ≈10- fold smaller thio effects for the reverse single-turnover religation reaction (Rp-Ps ) 30 and Sp-Ps ) 3). As compared to the smaller suicide cleavage substrates, approach-to-equilibrium cleavage studies using 32/32 mer substrates showed 7-9-fold smaller thio effects on cleavage, similar effects on religation, and the same ratio of the Rp to Sp thio effect as the suicide cleavage reaction (≈10). In general, thio effects of 2.4-7.2-fold on the cleavage equilibrium are observed for the wild-type and H265A enzymes, suggesting differences in the interactions of the enzyme with the nonbridging sulfur in the noncovalent and covalent complexes. Studies of the cleavage, religation, and approach-to-equilibrium reactions catalyzed by the H265A active site mutant revealed a stereoselective, 11-fold decrease in the Rp-thio effect on cleavage and religation as compared to the wild-type enzyme. This result suggests that His-265 interacts with the nonbridging pro-Rp oxygen in the transition state for cleavage and religation, consistent with the arrangement of this conserved residue in the crystal structure of the human topoisomerase-DNA complex. In general, the greatest effect of thio substitution and the H265A mutation is to destabilize the transition state, with smaller effects on substrate binding. The interaction of His-265 with the pro-Rp nonbridging oxygen is inconsistent with the proposal that this conserved residue acts as a general acid in the strand cleavage reaction. The vaccinia virus type IB topoisomerase catalyzes revers- ible site-specific cleavage and religation of the phosphodi- ester backbone of duplex DNA at CCCTTV sites using an active site tyrosyl residue as the nucleophile and the 5′-deoxyribose hydroxyl as the leaving group (Figure 1) (1). Type IB topoisomerases play an essential role in eukaryotic and mammalian DNA metabolism by allowing supercoiled DNA to “relax” around the swivel point generated by enzyme when it is covalently attached to a single strand of duplex DNA (2). In addition, the potentially powerful anticancer drug, camptothecin, selectively targets cellular type 1B enzymes by blocking the strand religation reaction (3). Thus, studies that identify and quantify the enzyme-substrate interactions that are involved in binding and catalysis are essential to fully elucidate the mechanism of these reactions. The phosphodiester cleavage reactions of type I topo- isomerases are unique in that the reaction is freely reversible and the energy of the initial DNA linkage is stored in the phosphotyrosyl covalent intermediate. Thus, single-turnover kinetic studies of cleavage and religation may be performed using reversible kinetic conditions or, alternatively, irrevers- ible conditions (4). Using such approaches, kinetic studies on the strand cleavage and religation reactions of the vaccinia enzyme have been performed using small duplex oligonucle- otide substrates (4, 5). These studies have indicated that the chemical step of cleavage was the slowest step in a single- turnover of the enzyme and that a simple two-state mecha- nism for cleavage and religation was sufficient to account for the observed kinetic results (Figure 1). 1 More recent † This work was supported by the National Institute of Standards and Technology, NIH Grant GM46330 to S.S., and by Committee of Scientific Research Grant 4 P05F 006 17 to W.J.S. * To whom correspondence and reprint requests should be addressed. Tel: 301-738-6264. FAX: 301-738-6255. E-mail: stivers@carb.nist.gov. ‡ Center for Advanced Research in Biotechnology of the University of Maryland and the National Institute for Standards and Technology. § Polish Academy of Sciences. | Sloan-Kettering Institute. 1 In previous kinetic, pH-rate, and thio effect studies using a different set of substrates than used here, we inferred the existence of another conformational state of the covalent enzyme-DNA complex (4). However, this state was calculated to comprise less than 1% of the total enzyme bound DNA, and thus, the previous data were also compatible with a two-state equilibrium. 5561 Biochemistry 2000, 39, 5561-5572 10.1021/bi992429c CCC: $19.00 © 2000 American Chemical Society Published on Web 04/13/2000