Individual Nucleotide Bases, Not Base Pairs, are Critical For Triggering Site-Specific DNA Cleavage by Vaccinia Topoisomerase

Vaccinia DNA topoisomerase forms a covalent DNA-(3'-phosphotyrosyl)-enzyme intermediate at a specific target site 5'-C +5 C +4 C +3 T +2 T +1 p ↓ N -1 in duplex DNA. Here we study the effects of abasic lesions at individual positions of the scissile and nonscissile strands on the rate of single-turnover DNA transesterification and the cleavage-religation equilibrium. The rate of DNA incision was reduced by factors of 350, 250, 60, and 10 when abasic sites replaced the –1N, +1T, +2T and +4C bases of the scissile strand, but abasic lesions at +5C and +3C had little or no effect. Abasic lesions in the nonscissile strand in lieu of +4G, +3G, +2A, and +1A reduced the rate of cleavage by factors of 130, 150, 10, and 5, whereas abasic lesions at +5G and –1N had no effect. The striking positional asymmetry of abasic interference on the scissile and nonscissile strands highlights the importance of individual bases, not base pairs, in promoting DNA cleavage. The rate of single-turnover DNA religation by the covalent topoisomerase-DNA complex was insensitive to abasic sites within the CCCTT sequence of the scissile strand, but an abasic lesion at the 5’-OH nucleoside (–1N) of the attacking DNA strand slowed the rate of religation by a factor of 600. Nonscissile strand abasic lesions at +1A and –1N slowed the rate of religation by factors of ~140 and 20, respectively, and strongly skewed the cleavage-religation equilibrium toward the covalent complex. Thus, abasic lesions immediately flanking the cleavage site act as topoisomerase poisons. Here we conduct a systematic analysis of the effects of abasic lesions at six positions of the scissile strand and six positions of the nonscissile strand within the target sequence 5’-CCCTTA/3’-GGGAAT. The key instructive findings are that individual bases, not the base pairs, are critical determinants of the rate of DNA cleavage. We also find that abasic lesions at the +1 and –1 positions flanking the scissile phosphodiester slow the rate of religation and the topoisomerase reaction equilibrium. endpoint values (defined as 100%), and the cleavage rate constants ( k cl ) were calculated by fitting the normalized data to the equation 100-%Cleavage (norm) = 100e -kt . Single-turnover religation . Cleavage reaction mixtures containing (per 20 µ l) 0.3 pmol of 32 P-labeled 18-mer/30-mer DNA (unmodified or abasic) and 2, 4 or 8 pmol of topoisomerase were incubated at 37°C for 10 to 60 min to form the suicide intermediate. Religation was initiated by the simultaneous addition of NaCl to 0.5 M and a 5'-OH 18-mer acceptor strand d(GTTCCGATAGTGACTACA) to a concentration of 15 pmol/22 µ l (i.e., a 50-fold molar excess over the input DNA substrate). Aliquots were withdrawn at various times and quenched immediately with 1% SDS. A time zero sample was withdrawn prior to addition of the acceptor strand. The samples were digested for 60 min at 37°C with 10 µ g of proteinase K, then mixed with an equal volume of 95% formamide 20 mM EDTA, heat-denatured, and analyzed by electrophoresis through a 17% polyacrylamide gel containing 7 M urea in TBE. Religation of the covalently bound 12-mer strand to the 18-mer acceptor DNA yielded a 5’- 32 P-labeled 30-mer strand transfer product. The extent of religation (expressed as the percent of the covalent intermediate converted into 30-mer) was plotted as a function of reaction time. The data were normalized to the endpoint values and k rel was determined by fitting the data to the equation 100-%Religated (norm) = 100 e -kt . Tris-HCl (pH 7.5), 0.3 pmol of 34-mer/30-mer DNA, and 9, 18, 37, 75, 150, or 300 ng of topoisomerase were incubated at 37°C for 10 min. The reactions were initiated by the addition of topoisomerase to prewarmed reaction mixtures. The reaction was quenched by adding SDS to 0.5%. The samples were digested for 60 min at 37°C with 10 µ g of proteinase K, mixed with an equal volume of formamide/EDTA, and then analyzed by electrophoresis through a 17% polyacrylamide gel containing 7 M urea in TBE. The cleavage product, a 32 P-labeled 12-mer bound to a short peptide, was well resolved from the input 34-mer The These results indicate that: (i) vaccinia topoisomerase does not rely on contacts to the +5C or +3C bases during the forward transesterification reaction and (ii) the most important contributions of the scissile strand bases are made by +2T, +1T and –1N. strand DNA cleavage. impact on religation. These results imply that the interaction of vaccinia topoisomerase with the +1T:A base pair is functionally important, but significantly different in atomic detail, during the cleavage and religation reactions.


INTRODUCTION
Poxvirus topoisomerases are exemplary type IB topoisomerase family members; they cleave and rejoin one strand of the DNA duplex through a transient DNA-(3'-phosphotyrosyl)enzyme intermediate. Vaccinia topoisomerase cleaves duplex DNA at a pentapyrimidine target sequence, 5'-(T/C)CCTTp↓ (1). (The Tp↓ nucleotide is defined as the +1 nucleotide.) Topoisomerases encoded by other genera of poxviruses recognize the same DNA target sequence (2)(3)(4)(5)(6), despite the large variations in overall G/C contents of the genomes of the different poxvirus genera. Available structural and biochemical studies suggest that the assembly of a catalytically competent topoisomerase active site is triggered by recognition of the 5'-CCCTT/3'-GGGAA target sequence (7,8).
Early studies using nuclease footprinting, modification interference, modification protection, analog substitution, and UV crosslinking techniques suggested that vaccinia topoisomerase makes contact with several nucleotide bases and the sugar-phosphate backbone of DNA within and immediately flanking the CCCTT element (9)(10)(11)(12)(13)(14)(15). Recent studies have focused on delineating the features of the DNA interface that affect the kinetics of transesterification. For example, position-specific covalent polycyclic aromatic hydrocarbon diol epoxide-DNA adducts have been exploited to probe the minor groove interface (16) and the effects of intercalation at all of the dinucleotides steps spanning the target site (17,18). The aromatic hydrocarbon adduct studies delineated the margins of the functional DNA interface at atomic resolution, but did not reveal the nature of the DNA contacts within the essential zone of DNA.
Modifications of the nonbridging and 5'-bridging oxygens of the DNA phosphodiester backbone have been especially informative in that regard. Phosphorothioate and methylphosphonate modifications at the scissile phosphodiester have illuminated the chemical mechanism of topoisomerase IB, the roles of the individual amino acids in either transition-state 4 atomic-resolution map of the DNA backbone contacts required for active site assembly (8).
Whereas sterically subtle modifications of nonbridging phosphate oxygens flanking the cleavage site can have drastic effects on transesterification chemistry (8), phosphorothiolate substitutions for the 5' bridging oxygens of the scissile strand have no significant effect on the rate of DNA cleavage by vaccinia topoisomerase (6).
A major outstanding question is how the poxvirus topoisomerase reads the nucleotide sequence at its cleavage site. Available evidence suggests that most of the site-specificity is achieved at the level of transesterification chemistry rather than at the noncovalent DNA binding step (24). Whereas the affinity for the target site, the rate of cleavage, and the cleavage equilibrium constant are affected by the nucleotide sequence context surrounding the 5'-(C/T)CCTT target site (1,15) in ways that are not well understood in structural terms, the dominant factor triggering the DNA incision reaction is the pentamer 5'-CCCTT/3'-GGGAA. We have begun to systematically address the features of the individual bases that affect the kinetics of DNA cleavage, via position-specific base modifications entailing relatively small additions to, or subtractions from, the standard base structures (11,17) as well as modification by more bulky adducts (16,18). The addition of new substituents to the purine and pyrimidine rings provides a means of mapping functionally relevant sites of protein-DNA contact. The caveat to the new substituent approach is that a particular site of modification interference (presumably arising via steric hindrance) cannot be equated with a specific atomic interaction with the DNA. Rather, the power of the new substituent approach resides mainly in its ability to identify DNA structural elements that are not functionally relevant. Moreover, the sensitivity of the base modification method necessarily depends on the extent to which the particular modification alters the size, shape and hydrogen-bonding potential of the base or base-pair. In general, the most informative modification interference effects will be those elicited by the subtlest modifications.
A corollary of this proposition is that the most straightforward approach to assessing the relevance of a given base to topoisomerase catalysis is to remove the base rather than add new substituents to the base. Missing base analysis has been facilitated by the availability of by guest on July 9, 2020 http://www.jbc.org/ Downloaded from 5 synthetic DNAs containing position-specific tetrahydrofuran (THF) abasic sites (Fig. 1 (25,26).
Such studies have broad physiological relevance insofar as: (i) abasic lesions arise in vivo with high frequency as a consequence of base-excision repair by DNA glycosidases and (ii) topoisomerases may reinforce the cytotoxicity of DNA lesions (27).
Here we conduct a systematic analysis of the effects of abasic lesions at six positions of the scissile strand and six positions of the nonscissile strand within the target sequence 5'-CCCTTA/3'-GGGAAT. The key instructive findings are that individual bases, not the base pairs, are critical determinants of the rate of DNA cleavage. We also find that abasic lesions at the +1 and -1 positions flanking the scissile phosphodiester slow the rate of religation and thereby poison the topoisomerase reaction equilibrium.

Position-specific scissile strand abasic interference effects on DNA cleavage.
A series of oligodeoxynucleotide 18-mer scissile strands containing a single THF abasic site within the 5'-C +5 C +4 C +3 T +2 T +1 A -1 sequence were 5' 32 P-labeled and then annealed to an unlabeled 30-mer strand to form "suicide" cleavage substrates for vaccinia topoisomerase (Fig.   1B). The presence of the abasic lesions at the correct positions was assessed by limited digestion of the suicide substrates with E. coli exonuclease III, which possesses 3' exonuclease and abasic endonuclease activities. Whereas exonuclease III digests the unmodified suicide substrate exclusively in the 3' exonuclease mode to generate a ladder of partially shortened 5' 32 P-labeled products (21), the abasic suicide substrates were also cleaved endonucleolytically at the 5'-phosphodiester flanking the abasic nucleoside to yield a discrete 5' 32 P-labeled product ( Fig. 2). Digestion in parallel of the 6 position-specific abasic substrates yielded a series of endonucleolytic cleavage products of the expected size and differing by 1-nucleotide spacing.
The cleavage transesterification reaction of vaccinia topoisomerase results in covalent attachment of the 32 P-labeled 12-mer 5'-pCGTGTCGCCCTTp to the enzyme via Tyr274. The unlabeled 6-mer 5'-OH leaving strand ATTCCC dissociates spontaneously from the protein-DNA complex. Loss of the leaving strand drives the reaction toward the covalent state, so that the reaction can be treated kinetically as a first-order unidirectional process. The reaction of excess topoisomerase with the unmodified control substrate attained an endpoint at which 95% of the DNA was converted to covalent topoisomerase-DNA complex; the reaction was complete within 20 s. The extent of transesterification after 5 s was 89% of the endpoint value. From this datum, we calculated a single-turnover cleavage rate constant (k cl ) of 0.46 s -1 (Fig. 1B).
Single abasic lesions spanning positions +5 to -1 on the scissile strand had no significant effect on the extents of DNA cleavage (90-98%), but they exerted disparate position-specific effects on the rate of the reaction. The k cl values for the +5 abasic (0.25 s -1 ) and +3 abasic substrates (0.18 s -1 ) were within a factor of 2 or 3 of the value for unmodified control DNA. In contrast, abasic lesions at +2, +1 and -1 slowed k cl by factors of 60 (to 0.0073 s -1 ), 260 (0.0018 by guest on July 9, 2020 http://www.jbc.org/ Downloaded from s -1 ) and 350 (0.0013 s -1 ), respectively. The +4 abasic lesion caused a 10-fold decrement in k cl (0.043 s -1 ). These results indicate that: (i) vaccinia topoisomerase does not rely on contacts to the +5C or +3C bases during the forward transesterification reaction and (ii) the most important contributions of the scissile strand bases are made by +2T, +1T and -1N.
Nonscissile strand abasic effects on DNA cleavage.
An unmodified 5' 32 P-labeled 18-mer scissile strand was annealed to a series of 30-mer nonscissile strands containing a single THF abasic site within the 3'-G +5 G +4 G +3 A +2 A +1 T -1 element (Fig. 3). Single abasic lesions spanning positions +5 to -1 on the nonscissile strand had no significant effect on the extents of DNA cleavage (80-97%), but they elicited position-specific effects on the rate of cleavage that, with the exception of the +5 abasic site, were drastically different than the effects exerted by the loss of the complementary base on the scissile strand.
The first instructive finding was that elimination of the +5G base at the "upstream" margin of the target site had no significant impact on k cl (0.33 s -1 ). Thus, neither component base of the +5 C:G base pair was functionally important for the forward cleavage reaction. A second notable finding was that loss of the -1T base at the "downstream" margin on the nonscissile strand also had no effect on k cl (0.46 s -1 ), but in this case, there was a huge disparity between the benign effect of an abasic lesion on the nonscissile strand compared with the 350-fold decrement in k cl that occurred when the complementary -1A base was missing from the scissile strand.
The k cl values for the +1 abasic (0.085 s -1 ), +2 abasic (0.046 s -1 ), +3 abasic (0.003 s -1 ), and +4 abasic (0.0035 s -1 ) substrates were slowed by factors of 5, 10, 150, and 130 relative to the k cl for unmodified DNA. A gradient of increasing severity of abasic interference was evident as the nonscissile strand lesion was phased away from the cleavage site; this contrasts with a severity gradient of opposite directionality for cleavage interference by abasic lesions on the scissile strand. The strand-selectivity can be quantified as the ratio of the cleavage rate constants for the missing scissile strand and nonscissile strand bases of each base-pair. These SS/NS abasic ratios are as follows: 0.75 for +5C:G, 12 for +4C:G, 60 for +3C:G, 0.15 for +2T:A, 0.02 for Effects of missing base pairs on DNA cleavage. 5' 32 P-labeled 18-mer scissile strands containing single abasic sites were annealed to abasic 30-mer nonscissile strands to form a series of suicide cleavage substrates lacking both complementary bases of each base-pair within the CCCTTA element (Fig. 4). The effects of missing base pairs ranged from modest (e.g., +5, +3 and -1) to severe (+4, +2 and +1). The missing base pair interference effects we classify as modest were those that had little effect on the extent of cleavage (78-98%) and for which the rate decrement incurred by deleting both bases were either no worse than, or only modestly worse than, the interference caused by a single abasic lesion. We can quantify the missing base pair effect as the inverse ratio of the cleavage rate constant for the missing pair substrate to the slower of the two rate constants for a single abasic substrate lacking either the scissile or nonscissile strand base. For example, the missing base pair effect at position -1A:T was 1.1 (= 0.0013/0.0011), which means that complete elimination of the -1 base pair was no worse than deleting just the -1A base on the scissile strand. The missing base pair effect at +5C:G was 4 (= 0.25/0.059), but the notable finding was that the rate of cleavage of a DNA substrate lacking the +5C:G pair was slowed by only a factor of 8 compared to an unmodified DNA. The missing pair effect at +3C:G was 7 (= 0.003/0.00043).
The +4, +2 and +1 missing pair interference effects we classify as severe affected the cleavage endpoint (11-62%) and elicited strongly synergistic effects on k cl compared to the single abasic lesions (Fig. 4). The missing base pair effects at +4C:G, +2T:A and +1T:A were 440, 1300, and 240, respectively. It is likely that these severe effects of deleting both bases of the pair are caused by secondary structural changes to the DNA target site, especially to the phosphodiester backbone with which the topoisomerase makes electrostatic contacts that are essential for the cleavage reaction (8).

Position-specific abasic interference with DNA religation.
The observed abasic interference effects on the rate of DNA cleavage could reflect a requirement for specific bases for either: (i) chemical catalysis of transesterification or (ii) DNA-12 assisted assembly of a catalytically competent active site. The observed rate of covalent complex formation by vaccinia topoisomerase with unmodified DNA is believed to be limited by the chemical step itself rather than by requisite pre-cleavage conformational steps (29). The topoisomerase catalytic cycle entails two transesterification reactions, cleavage and religation.
Religation occurs via the attack of the DNA 5'-OH on the covalent intermediate, leading to expulsion of the Tyr274 leaving group and restoration of the DNA phosphodiester backbone (30). Religation is believed to be the microscopic reversal of the cleavage reaction. Thus, changes in the structure of the topoisomerase or the DNA target site that inhibit the chemical step directly will likely slow both the forward cleavage reaction and the DNA religation reaction.
However, changes that impede active site assembly to the point that it becomes limiting for cleavage will affect the cleavage reaction selectively; they would have less impact on the rate of containing +5, +4, or +3 abasic sites on the scissile strand was virtually indistinguishable from that of unmodified DNA, as gauged by the completeness of the reactions after 5 s (Fig. 5B). The religation results are consistent with the minimal/mild effects of the +5, +4 and +3 abasic lesions on the rate of the forward cleavage reaction. Note that the religation rate constant of vaccinia topoisomerase (k rel ~1.0-1.2 s -1 ) is too fast to measure manually, which means that the religation rate would have to be slowed at least several fold to be detectable in our assays.
The religation of covalent complexes containing +2 or +1 abasic lesions was effectively complete in 10 s; the reactions attained 56% and 52% of the endpoint values after 5 s, from which we estimated k rel values of 0.16 s -1 and 0.15 s -1 , respectively. We surmise that the +2 and +1 T bases contribute no more a ~6-fold enhancement of the rate of religation, which contrasts with their 60-fold and 250-fold contributions to the rate of cleavage. We infer that the +2T and +1T bases play a role in the assembly of a catalytically competent active site.
The role of the -1 scissile strand base in DNA religation was assessed by forming the suicide intermediate on an unmodified DNA substrate and then initiating religation by adding a 5'-OH 18-mer strand containing a 5'-terminal THF abasic moiety in lieu of a 5'-terminal deoxyadenosine nucleoside (Fig. 6). Religation to the -1 abasic acceptor was efficient but slow; the reaction was complete after 30 min and the observed single-turnover religation rate constant of 0.0017 s -1 was slower by a factor of 600 than religation to an unmodified DNA strand. Thus, the -1 base on the scissile strand is crucial for both the cleavage and religation transesterification steps.
We also assayed religation by preformed topoisomerase-DNA complexes containing abasic lesions on the nonscissile strand (Fig. 5C). The +5 abasic lesion, which had no significant effect on cleavage, also had no apparent impact on religation, which was complete in 5 s. The +4 abasic lesion had no apparent effect on religation, but this was in contrast to its 130-fold reduction in the rate of cleavage. We infer that the +4G base is critical for assembly of the topoisomerase active site. Loss of the +2A base did not slow religation appreciably, whereas it caused a 10-fold reduction in the rate of cleavage. Religation by the +3 abasic topoisomerase-DNA complex was not complete until 60 s; we derived a single-turnover religation rate constant of 0.09 s -1 . The ~10-fold slowing effect of the +3 abasic site on k rel was less severe than its 150fold decrement in k cl .
Religation was severely and selectively affected by abasic lesions at nonscissile strand positions +1 and -1 (Fig. 5C). The religation rate constant of the +1 abasic topoisomerase-DNA complex (0.0072 s -1 ) was slowed by a factor of ~140 (Fig. 5C). This finding contrasts with the 5fold slowing effect of the +1 abasic site on the rate of the forward cleavage reaction. The rate of religation by the -1 abasic complex (0.044 s -1 ) was about 4% of the normal religation rate; the same -1 abasic lesion had no effect on the forward cleavage rate.

Abasic effects on the cleavage-religation equilibrium.
A synthetic 5' 32 P-labeled CCCTT-containing duplex containing 12-bp of DNA upstream of the cleavage site and 18-bp of DNA downstream of the cleavage site was employed to assay transesterification under equilibrium conditions (Fig. 7). This DNA is an equilibrium substrate because the 5'-OH leaving strand generated upon cleavage at CCCTT remains stably associated with the topoisomerase-DNA complex via base-pairing to the nonscissile strand. We determined by enzyme titration that vaccinia topoisomerase cleaved 25% of the unmodified substrate at saturation. The cleavage-religation equilibrium constant (K cl = covalent complex/noncovalent complex = k cl /k rel ) was thus 0.33 for the unmodified DNA.
The yield of covalent intermediate as a function of input topoisomerase was determined for a series of modified equilibrium substrates containing single abasic lesions in the nonscissile strand. The +5 abasic substrate was cleaved to an extent of 40% of the input DNA. The observed K cl of 0.66 indicated that the +5 abasic site exerted a 2-fold greater effect on ligation than cleavage. The +4, +3 and +2 abasic lesions reduced the yield of covalent adduct at equilibrium to 1%, 5% and 9% of input DNA, respectively. The measured K cl value of 0.01 for the +4 abasic substrate was in good agreement with the ratio of the separately determined single-turnover cleavage and religation rate constants (k cl /k rel = 0.016). Similarly the measured

Abasic effects on site-specific DNA cleavage
We systematically probed the contributions of individual bases and base-pairs to sitespecific DNA transesterification by vaccinia virus DNA topoisomerase, by replacing each nucleoside of the 5'-CCCTT↓A/3'-GGGAAT target site with an abasic THF nucleoside. The rate of DNA cleavage was reduced by factors of 350, 250, 60, and 10 when abasic sites replaced the -1A, +1T, +2T and +4C bases of the scissile strand, but abasic lesions at +5C and +3C had little or no effect. Abasic lesions in the nonscissile strand in lieu of +4G, +3G, +2T, and +1T reduced the rate of cleavage by factors of 130, 150, 10, and 5, whereas abasic lesions at +5G and -1T had no effect. The reduced rates of cleavage of abasic DNAs were not caused by interference with noncovalent binding, insofar as the observed rate constants did not increase when the concentration of topoisomerase was doubled or quadrupled (data not shown). The simplest interpretation of these findings is that abasic interference is a consequence of the loss of base-specific contacts between the topoisomerase and the 5'-CCTT/3'-GGAA element.
The striking positional asymmetry of abasic interference on the scissile and nonscissile strands highlights the importance of individual bases, not base pairs, in promoting DNA cleavage. This result surprised us, as classical models of protein binding to specific sequences in double-stranded DNA highlight hydrogen bonding interactions between amino acid side chains and the DNA base-pairs as determinants of sequence specificity (32,33). Fig. 8  We suggest that protein contacts to the +1 and +2T bases of the scissile strand and the +3 and +4G bases of the nonscissile strand trigger assembly of a catalytically competent active site subsequent to initial noncovalent binding of topoisomerase to the CCCTT target site. Whereas the proposed precleavage conformational step required for active site assembly is not normally rate-limiting when topoisomerase cleaves unmodified DNA, we suggest that it becomes ratelimiting when topoisomerase cleaves these four abasic DNAs. Because the +4G, +3G +2T and +1T abasic lesions exclusively or selectively interfere with the cleavage reaction, but not the religation step, we infer that the putative base contacts needed to promote active site assembly prior to cleavage are not required to maintain the active site once the covalent intermediate is formed.
The abasic effects tell us that neither the +5C:G base pair, nor the individual +2A, +1A, +3C or +4C bases are essential for the DNA cleavage step. Thus, the essential contributions of +4G, +3G, +2T and +1T are not contingent on hydrogen bonding to the base on the opposing strand.
Extensive additional interference studies, entailing subtle modifications of the individual atoms of the pyrimidine and purine bases, will be required to delineate which atomic contacts to the bases are functionally relevant. However, we can speculate a bit in light of available structural and functional studies. For example, it is likely that some interaction of vaccinia topoisomerase with the +3G base of the nonscissile strand takes place in the major groove, because 8-oxo modification of +3G resulted in a 35-fold decrement in k cl (17). On the other hand, it is unlikely that the topoisomerase makes essential contacts in the major groove to the O6 atom of the +4G or +3G bases, insofar as replacing these guanines with 2-aminopurine had no effect on k cl (17).
Contacts to the +2T and +1T are also likely to occur in the major groove, because early studies showed that replacing the +2 T:A or +1 T:A pair with a cytosine:inosine base pair strongly suppressed the yield of the covalent topo-DNA intermediate (10). Changing T :A to C:I alters the surface of the major groove, but the minor groove is identical in both cases.
Although the minor groove affords a narrower ingress for protein functional groups to contact the DNA bases, it is evident from multiple DNA co-crystal structures of topoisomerase IB and tyrosine recombinase enzymes that there is a conserved contact between an invariant lysine side chain of the topoisomerase/recombinase (corresponding to Lys167 in vaccinia topoisomerase) and the minor groove face of the +1 base immediately 5' of the scissile phosphodiester (34)(35)(36)(37)(38). This lysine, which is an essential general acid catalyst of the transesterification reaction (19), is located atop a conformationally mobile beta-hairpin loop. The contact between the Lys Nζ and the O2 of the +1 thymine is the only base-specific contact seen in the human topoisomerase IB-DNA co-crystal structure (38). In light of our present demonstration that the +1T base is important for cleavage, but not religation, we posit that an equivalent contact of Lys167 with the +1T base helps recruit the beta-hairpin loop and Lys167 from its ground-state position outside the circumference of the DNA double helix (7) to a catalytically competent position within the minor groove adjacent to the scissile phosphodiester.
Whereas the recruitment of Lys167 to the active site might be rate-limiting for cleavage of the +1 abasic substrate, the +1 thymine is apparently not critical for transesterification chemistry once an active site has been assembled in the covalent intermediate.

Role of the -1 base in DNA cleavage
Vaccinia topoisomerase displays no preference for a particular base pair immediately 3' of the scissile phosphodiester (1,18). Here we found that an abasic lesion at -1 on the scissile strand slowed k cl by a factor of 350, whereas a -1 abasic lesion on the nonscissile strand had no effect at all. How can we rationalize the apparent requirement for a -1 base, when neither the base pair, not the identity of the -1 base, is important? We propose that the severe abasic interference at -1 reflects a requirement for continuous base stacking in the scissile strand to ensure optimal orientation of the scissile phosphodiester for coordination by the catalytic residues (Arg130, Lys157, Arg220 and His265) or nucleophilic attack by Tyr274 or proper orientation of the 5'-O leaving group of the -1 nucleoside sugar.

Abasic effects on religation
The Elimination of the +1A base of the nonscissile strand strongly impedes religation by vaccinia topoisomerase, but largely spares the forward cleavage reaction. This abasic lesion slowed k rel by more than two orders of magnitude and increased K eq by a factor of 35. In contrast, elimination of the opposing +1T base on the scissile strand slowed the cleavage reaction by a factor of 250 and had relatively little impact on religation. These results imply that the interaction of vaccinia topoisomerase with the +1T:A base pair is functionally important, but significantly different in atomic detail, during the cleavage and religation reactions.