Major groove interactions of vaccinia Topo I provide specificity by optimally positioning the covalent phosphotyrosine linkage.

Vaccinia virus topoisomerase I (vTopo) and other eukaryotic type IB enzymes maintain DNA topology by relaxing superhelical tension in DNA. The Topo reaction is comprised of multiple steps as shown in Figure 1: i) non-covalent binding of the enzyme to duplex DNA with a dissociation constant KD, ii) cleavage of a single DNA strand by an active site tyrosine nucleophile leading to formation of a covalent 3′-phosphotyrosyl linkage and a free 5′ -hydroxyl (kcl), iii) release of superhelical tension by a strand rotation mechanism (ksup), and iv) religation of the DNA break by attack of the 5′-hydroxyl nucleophile (kr). Vaccinia topoisomerase is unique among these enzymes in that it exhibits high specificity for cleavage at the consensus pentapyrimidine sequence 5′-(C/T)CCTT↓ (1) (2) (3) (4) (5) (6) (7) (8). However, the remarkable site-specificity of vTopo is not understood at the molecular level because the holoenzyme has proven refractory to crystallization, necessitating the application of other approaches for elucidation of this question. Fig. 1 Topoisomerase reaction cycle. A) The three steps in the vTopo reaction involve DNA binding, reversible DNA backbone cleavage described by the rate constants kcl and kr, and for supercoiled DNA substrates, supercoil relaxation while the enzyme is covalently ... One way to define specificity for vTopo is to compare the efficiency of forming the covalent adduct at the consensus site as compared to another site that differs from the consensus sequence. Sequence specific interactions within the consensus sequence are used to kinetically or thermodynamically facilitate the formation of the enzyme-DNA covalent adduct, whereas alternative sequences cannot realize the same benefit, resulting in specificity. This is a useful definition because the lifetime (or concentration) of the covalent phosphotyrosine adduct largely determines how efficiently the enzyme removes supercoils from DNA using a strand rotation mechanism as defined in the legend to Figure 1A (9, 10). Using this definition, specificity can be achieved at one of three stages of the reaction (Fig. 1B). If strong sequence-specific enzyme-DNA interactions are only used to discriminate at the noncovalent binding step (ES, Fig. 1B), then the relative amount of covalent adduct at the specific sequence would be increased relative to the nonspecific sequence because the specific site has higher occupancy. This follows because the relative amount of covalent adduct is determined by the ratio Kcl/KD. (This analysis makes the limiting assumption that Kcl is the same for the specific and nonspecific substrates.) Alternatively, enzyme interactions with the consensus site could be weak in the noncovalent complex, and then become strong at the transition state for cleavage (ES‡, Fig. 1B). In this case, the activation barrier for cleavage is lowered in the presence of the consensus interactions, thereby increasing the rate of formation of the covalent adduct (E-S, Fig. 1). In this regard, many of the direct enzyme catalytic interactions with the phosphoryl group have been found to act solely by transition state stabilization (11). However, transition state stabilization by itself is unsatisfactory for specificity because it would also give rise to a decrease in the lifetime of the covalent intermediate at the specific site. This follows because stabilization of ES‡ has an equal effect on enhancing the rates of both cleavage and ligation: as the religation rate increases, fewer supercoils are removed each time the covalent complex is formed, resulting in decreased specificity by our definition. Finally, if sequence specific interactions are used to stabilize the covalent complex (E-S, Fig. 1), then its relative concentration would be greater at consensus sites, generating specificity (Fig. 1B). It is clear from this analysis that an understanding of specificity requires measurements of the relative strength of interactions in the ES, ES‡ and E-S complexes as a function of discrete changes in the consensus DNA sequence. Previous footprinting, chemical protection/interference and UV cross-linking studies have indicated that vTopo makes contacts within the major groove of the GGGAA sequence of the nonscissile strand (4, 7, 12-14). For instance, site-specific modification with benzo-[c]-phenanthrene at N2 and N6 of G can reduce the cleavage rate by a factor of 1000 (6), and site-specific modification of the exocylic amino groups of the +1 and +2 adenines with benzo-[a]-pyrene decreased the rate of cleavage by approximately 103-fold (15). The rate effects on strand cleavage arising from the more subtle major groove modifications of 8-oxoguanine, 8-oxoadenine and 2-aminopurine have also been explored, where deleterious effects as large as 35-fold at the +3G were observed (15). In addition, stereospecific methylphosphonate substitutions of the nonbridging phosphate oxygens in the DNA backbone of this sequence have highlighted the significance of major and minor groove electrostatic interactions towards sequence specific recognition (8). Although these previous findings highlight the significance of major groove interactions, the discrete energetic role of the hydrogen bonding groups that are presented within the major groove of the GGGAA sequence has never been evaluated at each step along the reaction coordinate. In this paper, we describe a study in which the N7 hydrogen bond acceptor groups of each guanine and adenine within the consensus sequence are replaced with unnatural 7-deaza bases. In separate experiments, the individual guanine O6 and adenine N6 groups are removed to investigate hydrogen bonding at these major groove positions (Fig. 2). Systematic dissection of the energetics of these changes at each step along the reaction pathway shows that major groove hydrogen bonds are in general used to increase the stability of the covalent E-S complex, without having any energetic effect on the noncovalent or transition state complexes (Fig. 1B). A reasonable physical interpretation is that these interactions are used to optimally position the covalent phosphotyrosine linkage relative to the attacking 5′ -hydroxyl nucleophile. Fig. 2 DNA substrate used in this study. The normal G/C and A/T base pairs of the consensus sequence were replaced with unnatural bases substitutions in the GGGAA sequence of the consensus site are shown. The arrows indicate potential contact points that are ...