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J. Biol. Chem., Vol. 278, Issue 11, 9905-9911, March 14, 2003

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Benzo[a]pyrene-dG Adduct Interference Illuminates the Interface of Vaccinia Topoisomerase with the DNA Minor Groove^*

Ligeng Tian, Jane M. Sayer^§, Heiko Kroth^§, Govind Kalena^§, Donald M. Jerina^§, and Stewart Shuman^¶

From the  Molecular Biology Program, Sloan-Kettering Institute, New York, New York 10021 and the ^§ Laboratory of Bioorganic Chemistry, NIDDK, National Institutes of Health, DHHS, Bethesda, Maryland 20892

Received for publication, December 6, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Vaccinia DNA topoisomerase forms a covalent DNA-(3'-phosphotyrosyl)-enzyme intermediate at a pentapyrimidine target site 5'-C⁺⁵C⁺⁴C⁺³T⁺²T⁺¹p in duplex DNA. The enzyme engages the target site within a C-shaped protein clamp. Here we mapped the interface of topoisomerase with the DNA minor groove by introducing chiral C-10 R and S 7,8-diol 9,10-epoxide adducts of benzo[a]pyrene (BP) at single N²-deoxyguanosine (dG) positions within the nonscissile DNA strand. These trans opened BPdG adducts fit into the minor groove without perturbing helix conformation or base pairing, and the R and S diastereomers are oriented in opposite directions within the minor groove. We measured the effects of the BPdG adducts on the rate and extent of single-turnover DNA transesterification. We observed a sharp margin of interference effects, whereby +5 and 2 BPdG modifications were well tolerated but +4, +3, and 1 BPdG adducts were severely deleterious. Stereoselective effects at the 1 nucleoside (the R isomer interfered, whereas the S isomer did not) delineated at high resolution the downstream border of the minor groove interface. BPdG inhibition of transesterification is likely caused by steric exclusion of constituents of the topoisomerase from the minor groove. We also applied the BPdG interference method to probe the interactions of exonuclease III with the minor groove. DNAs containing these BPdG adducts were protected from digestion by exonuclease III, which was consistently arrested at positions 2-4 nucleotides prior to the BP-modified guanosine.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Type IB topoisomerases modulate the topological state of DNA by cleaving and rejoining one strand of the DNA duplex. Cleavage is a transesterification reaction in which the scissile phosphodiester is attacked by a tyrosine of the enzyme, resulting in the formation of a DNA-(3'-phosphotyrosyl)-enzyme intermediate and the expulsion of a 5'-OH DNA strand. In the religation step, the DNA 5'-OH group attacks the covalent intermediate resulting in expulsion of the active site tyrosine and restoration of the DNA phosphodiester backbone. Vaccinia topoisomerase is a prototype of the type IB topoisomerase family, which includes eukaryotic nuclear and mitochondrial topoisomerase IB, the poxvirus topoisomerases, and poxvirus-like topoisomerases encoded by bacteria (1-4). The vaccinia enzyme is distinguished from the nuclear topoisomerase I by its compact size (314 amino acids) and its site specificity in DNA transesterification (5). Vaccinia topoisomerase binds and cleaves duplex DNA at a pentapyrimidine target sequence 5'-(T/C)CCTTp. The Tp nucleotide (defined as the +1 nucleotide) is linked to Tyr-274 of the enzyme.

The DNA features that contribute to target site recognition and catalysis have been examined using synthetic substrates containing a single CCCTT site. The DNase I footprint of vaccinia topoisomerase covers ~13 nucleotides upstream (5') and ~9-13 nucleotides downstream (3') of the scissile bond (6). Exonuclease III footprinting suggests a two-part interaction of topoisomerase with the DNA 5' of the site of covalent adduct formation (7). A transient margin of protection from exonuclease III digestion, which extends to positions +13 to +14 on the scissile strand, gives way to a long-lived margin of protection at positions +7 to +9. The tightly protected segment includes the entire CCCTT element that directs site-specific transesterification. Modification interference, modification protection, analog substitution, and UV cross-linking experiments indicate that vaccinia topoisomerase makes contact with several nucleotide bases and the sugar-phosphate backbone of DNA in the vicinity of the CCCTT recognition site. For example, dimethyl sulfate protection and interference experiments revealed interactions in the major groove with the three guanine bases of the pentamer motif complementary strand (3'-GGGAA) (8).

The contributions of individual phosphates to binding specificity were initially inferred from the effects of phosphate ethylation on protein binding (9). Ethylation of four phosphates on the scissile strand (positions CpCpCpTpTp within the pentamer motif) and three phosphates on the nonscissile strand (3'-GpGpGpApA) interfered with topoisomerase-DNA complex formation. In a B-form structure of the CCCTT-containing DNA substrate, the relevant topoisomerase-phosphate contacts are arrayed across the minor groove of the double helix (9). The major groove base-specific contacts that comprise the topoisomerase-DNA interface are situated on the opposite face of the DNA helix from the specific phosphate contacts and from the scissile phosphate. These results suggested that topoisomerase binds circumferentially to its target site in duplex DNA (9).

Subsequent structural analyses of the human and vaccinia topoisomerases revealed that the type IB enzymes do indeed form a C-shaped protein clamp around the DNA duplex (10, 11). The carboxyl catalytic domain interacts with the minor groove face of the DNA at the cleavage site, while the amino domain is positioned on the major groove side of the target site. The crystal structures, together with functional studies of the vaccinia enzyme (12, 13), imply that several of the catalytic amino acids either coordinate the scissile phosphodiester from the minor groove side or penetrate directly into the minor groove.

In the present study, we use a minor groove-specific interference approach to delineate the dimensions of the minor groove interface between vaccinia topoisomerase and its cleavage site. We introduce 7,8-diol 9,10-epoxide adducts of benzo[a]pyrene (BP)¹ at the exocyclic N²-amino group of single deoxyguanosine (dG) positions within the nonscissile strand of a suicide cleavage substrate for vaccinia topoisomerase. These adducts are derived from trans opening with inversion at C-10 of the (+)-(7R,8S,9S,10R)- and ()-(7S,8R,9R,10S)- enantiomers of the diol epoxides in which the benzylic 7-hydroxyl group and the epoxide oxygen are trans (see Fig. 1). NMR structures have established that these BPdG adducts fit into the minor groove with no significant perturbations of Watson-Crick base pairing or B-form helix conformation (14, 33, 34). Moreover, the S and R adducts have opposite orientations in the minor groove, such that the S adduct points toward the 5' end of the modified strand, whereas the R adduct points toward the 3' end of the modified strand (see Fig. 1). Thus, trans opened BPdG adducts provide an elegant means to gauge the effects of space occupancy within the minor groove.

This technique has been applied previously to human topoisomerase IB, whereby a BPdG adduct was placed on the scissile strand at the nucleoside immediately 3' of the phosphodiester that is preferentially cleaved by the human enzyme in unmodified DNA (15, 16). The BPdG adduct suppressed cleavage at the normal site but promoted alternative cleavages elsewhere on both strands of the DNA substrate. This response to interfering lesions is typical for human topoisomerase I, which lacks stringent sequence specificity for DNA transesterification and simply finds another site when confronted with an impediment (17). Vaccinia topoisomerase is more amenable to quantitatively informative interference studies because the pre-steady-state kinetic parameters for transesterification are known (18-20) and the enzyme does not relinquish its site specificity in response to a DNA lesion (21).

Here we introduce BPdG lesions into the G-rich nonscissile strand at the three guanines within the 3'-GGGAA complement of the 5'-CCCTT cleavage site (these are defined as the +5, +4, and +3 G residues) and also at the two nucleosides immediately downstream of the cleavage site (the 1 and 2 nucleosides). We observe a sharp margin of interference effects, whereby +5 and 2 BPdG modifications are well tolerated but +4, +3, and 1 BPdG adducts are severely deleterious. The stereoselective effects at the 1 nucleoside (the R diastereomer interferes, whereas the S diastereomer does not) delineate at high resolution the downstream border of the minor groove interface. We also apply the BPdG interference method to probe the interactions of Escherichia coli exonuclease III with the DNA minor groove.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

DNA Substrates-- Modified oligonucleotides containing single chiral BPdG adducts (Fig. 1) were synthesized and purified on a 1.5-µmol scale as described previously (35). The modified dG was introduced by manual coupling using the diastereomeric mixture of suitably protected R and S C-10 adducted phosphoramidates (36). After completion of the synthesis and removal of protecting groups, the resultant diasteromeric oligonucleotides were purified and separated from each other by high performance liquid chromatography (Table I). Absolute configurations of the separated diastereomers were determined from their circular dichroism spectra. The pyrene chromophore produces a positive band in the 320-360-nm range for the R diastereomer and a negative band for the S diastereomer (37). Unmodified oligonucleotides were purchased from BIOSOURCE International. DNA concentrations were determined by UV absorbance at 260 nM. Unmodified 34-mer scissile strands were 5' ³²P-labeled by enzymatic phosphorylation in the presence of [-³²P]ATP and T4 polynucleotide kinase. The labeled oligonucleotides were gel-purified and hybridized to standard or BPdG-modified 18-mer oligonucleotides at a 1:4 molar ratio of 34-mer to 18-mer. Annealing reaction mixtures containing 0.2 M NaCl and oligonucleotides as specified were heated to 80 °C and then slow-cooled to 22 °C. The hybridized DNAs were stored at 4 °C. The structures of the annealed duplexes are depicted in Figs. 2 and 5.

View larger version (48K): [in this window] [in a new window]   Fig. 1.   Opposite orientation of trans S and R BPdG adducts in the DNA minor groove. Structures of the C-10 trans S and R BPdG adducts and their diol epoxide precursors are shown in the bottom panel beneath space-filling views of the NMR structures of the chiral BP adducts (colored light blue) in the minor groove of an 11-bp B-form duplex DNA (14). BP is covalently attached to the DNA strand to the right side of the BP moiety. The S diastereomer is oriented toward the 5' end of the strand to which it is attached, and the R diastereomer points toward the 3' end.

View larger version (25K): [in this window] [in a new window]   Fig. 2.   Effects of BPdG adducts at positions 1 and 2 of the nonscissile strand on the rate of DNA transesterification by vaccinia topoisomerase. The 34-mer/18-mer CCCTT-containing substrate is shown at the bottom of the figure. The site of cleavage is indicated by a vertical arrow, and the 5' 32P-label on the scissile strand is denoted by an asterisk. The unmodified control substrates containing a 1C:G base pair (panel A) or a 2C:G base pair (panel B) are depicted in greater detail with the phosphodiester backbones drawn as horizontal lines, and the base pairs as vertical lines. The numerical coordinates of the nucleotides are indicated above the control sequence in panel A. The S and R diastereomers of the BPdG adducts are depicted as horizontal bars in their respective orientations from the site of covalent attachment to guanine on the nonscissile strand. Cleavage rate constants and reaction endpoints for transesterification by vaccinia topoisomerase are indicated to the right of each structure.

Vaccinia Topoisomerase-- Recombinant vaccinia topoisomerase was produced in E. coli (BL21) by infection with bacteriophage CE6 (22) and then purified to apparent homogeneity from the soluble bacterial lysate by phosphocellulose and Source S-15 chromatography steps. Protein concentration was determined by using the dye-binding method (Bio-Rad) with bovine serum albumin as the standard.

DNA Transesterification-- Reaction mixtures containing (per 20 µl) 50 mM Tris-HCl (pH 7.5), 0.3 pmol 34-mer/18-mer DNA, and 75 or 150 ng (2 or 4 pmol) of vaccinia topoisomerase were incubated at 37 °C. Aliquots (20 µl) were withdrawn at the times specified and quenched immediately with SDS (1% final concentration). The products were analyzed by electrophoresis through a 10% polyacrylamide gel containing 0.1% SDS. Free DNA migrated near the dye front. Covalent complex formation was revealed by transfer of radiolabeled DNA to the topoisomerase polypeptide. The extent of covalent complex formation was quantified by scanning the dried gel using a Fujifilm BAS-2500 PhosphorImager. A plot of the percentage of input DNA cleaved versus time established the end point values for cleavage. The data were then normalized to the end point values (defined as 100%), and the cleavage rate constants (k_cl) were calculated by fitting the normalized data to the equation 100  % cleavage_(norm) = 100 e^kt. The values of k_obs at the saturating level of input topoisomerase (75 ng) and the actual end point cleavage values are listed in Figs. 2 and 5.

Cleavage site-Specificity-- Reaction mixtures (20 µl) containing 50 mM Tris-HCl (pH 7.5), 0.3 pmol of standard or BPdG-modified 34-mer/18-mer DNA, and 75 ng of vaccinia topoisomerase were incubated at 37 °C for either 30 s (standard, 2R, 2S, and +5R BPdG), 3 min (+5S and 1S BPdG), 4 h (1R BPdG), or 24 h (+4R and +4S BPdG). The reactions were quenched with 1% SDS. Half of the sample was digested for 2 h at 37 °C with 10 µg of proteinase K, and the other half was not digested. The mixtures were adjusted to 47% formamide, heat denatured, and analyzed by electrophoreses through a 17% denaturing polyacrylamide gel containing 7 M urea in TBE (90 mM Tris borate, 2.5 mM EDTA). The reaction products were visualized by autoradiographic exposure of the gel.

Exonuclease III Digestion-- 5' ³²P-labeled BPdG-modified 18-mer oligonucleotides were hybridized to complementary 34-mer DNAs at a 1:4 molar ratio of labeled strand to 34-mer. Exonuclease III reaction mixtures (60 µl) containing 66 mM Tris-HCl (pH 8.0). 0.66 mM MgCl₂, 1.8 pmol of 18-mer/34-mer, and 1.0 unit of E. coli exonuclease III (New England Biolabs) were incubated at 22 °C. Aliquots (10 µl) were withdrawn at the times specified and quenched by adding EDTA to 30 mM final concentration. The samples were adjusted to 47% formamide, heat denatured, and analyzed by electrophoresis through a 17% denaturing polyacrylamide gel containing 7 M urea in TBE. The reaction products were visualized by autoradiographic exposure of the gel.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Stereospecific Minor Groove Interference at the Nucleoside Flanking the Cleavage Site-- A pair of 18-mer strands containing a single S or R BPdG adduct at position 1 of the nonscissile strand were synthesized and then annealed to 5' ³²P-labeled 34-mer scissile strands to form "suicide" substrates for vaccinia topoisomerase (Fig. 2A). Transesterification results in covalent attachment of a 5' ³²P-labeled 12-mer (5'-pCGTGTCGCCCTTp) to the enzyme via Tyr-274. The unlabeled 22-mer 5'-OH leaving strand 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 (18-20). The reaction of excess topoisomerase with the unmodified control substrate attained an end point at which 87% of the DNA was converted to covalent topoisomerase-DNA complex, and the reaction was complete within 20 s. The extent of transesterification after 5 s was 85% of the end point value. From this datum, we calculated a single-turnover cleavage rate constant (k_cl) of 0.4 s¹ (Fig. 2). Introduction of a chiral S BPdG at position 1 had only a modest (4-fold) effect on the cleavage rate constant (k_cl = 0.1 s¹) and no effect on the reaction end point (Figs. 2A and 3.). In contrast, the R diastereomer at position 1 reduced the rate of transesterification by a factor of 200 (k_cl = 0.002 s¹) without influencing the end point (Figs. 2A and 3). The k_obs for cleavage of either of the 1 BPdG-modified substrates did not increase when the concentration of topoisomerase in the reaction mixture was increased 2-fold (not shown). This indicated that the slowed cleavage rate was not caused by a defect in the initial binding of topoisomerase to the substrate. The results define a stereospecific interference effect at the 1 nucleoside, whereby the R diastereomer elicits 50-fold greater inhibition of transesterification than the S diastereomer. Note that the interfering 1R BPdG adduct is oriented toward the scissile phosphodiester in the minor groove, whereas the non-interfering 1S adduct is pointing away from the cleavage site (Fig. 2A).

View larger version (14K): [in this window] [in a new window]   Fig. 3.   Kinetic analysis of cleavage of 1 BPdG-modified substrates. Transesterification reaction mixtures contained 0.3 pmol of the 1S or 1R BPdG substrates and 75 ng (2 pmol) of topoisomerase. The extents of covalent adduct formation are plotted as a function of reaction time.

To address whether the 1 BPdG substitutions altered the site of cleavage within the 34-mer scissile strand, the reaction products were digested with proteinase K in the presence of SDS to remove the covalently linked topoisomerase. The radiolabeled DNA reaction products were then analyzed by denaturing polyacrylamide gel electrophoresis (Fig. 4). Reaction of topoisomerase with the unmodified control substrate resulted in the appearance of a cluster of radiolabeled species migrating faster than the input ³²P-labeled 34-mer strand (Fig. 4, lane 2). The cluster consists of the 12-mer 5'-pCGTGTCGCCCTTp linked to one or more amino acids of the topoisomerase. Detection of the covalent oligonucleotide-peptide complex was completely dependent on prior digestion of the sample with proteinase K (not shown). This is because the labeled DNA does not migrate into the polyacrylamide gel when it is bound covalently to the topoisomerase polypeptide. The instructive finding was that the same cluster was produced by proteinase K digestion of the covalent complex formed by reaction of topoisomerase with the substrates containing 1R or 1S BPdG adducts on the nonscissile strand (Fig. 4, lanes 3 and 4). Thus, the site of covalent complex formation was unchanged by the BPdG modifications. Any shift in the cleavage site, and hence the size of the covalently bound oligonucleotide, would have been readily detected by an altered mobility of the array of labeled oligonucleotide-peptide complexes.

View larger version (65K): [in this window] [in a new window]   Fig. 4.   BPdG adducts do not affect the cleavage site specificity of vaccinia topoisomerase. Cleavage reactions and subsequent treatments of the reaction products were performed as described under "Experimental Procedures." An autoradiogram of the polyacrylamide gel is shown. The control substrates contained no modifications (). Other substrates contained BPdG adducts in the nonscissile strands at the positions specified. The positions of the 34-mer scissile strand and the cluster of covalent peptide-DNA complexes are indicated on the right.

BPdG Adducts at the 2 Nucleoside Do Not Interfere with Transesterification-- We altered the sequence of the DNA strand 3' of the CCCTT cleavage site so as to place a C:G base pair at the 2 position (Fig. 2B). Synthetic 18-mer strands containing a single R or S BPdG adduct at the 2 nucleoside of the nonscissile strand were annealed to the complementary 5' ³²P-labeled 34-mer scissile strand to form a 34-mer/18-mer suicide substrate. The reaction of topoisomerase with the unmodified control substrate attained an end point of 89% covalent enzyme-DNA formation with a cleavage rate constant of 0.25 s¹ (Fig. 2B). The 2S and 2R BPdG adducts had no interfering effects on the rate or extent of transesterification. Indeed, the cleavage of both 2 adducts was about twice as fast as the unmodified DNA. (A rate constant of 0.5 s¹ is at the upper limit of what we can measure by manual assay.) Analysis of the cleavage products by PAGE after proteinase K digestion showed that the 2R and 2S BPdG modifications did not alter the site of topoisomerase transesterification to the scissile strand (Fig. 4, lanes 7 and 8). Taken together, the 1 and 2 BPdG interference experiments define the "downstream" margin of the minor groove interface between vaccinia topoisomerase and its target site, said margin being between the +1 and 1 base pairs (Fig. 2).

Effects of BPdG Adducts within the 3'-GGGAA Sequence of the Nonscissile Strand-- Previous studies suggested that N7 methylation of the +5, +4, and +3 guanines in the major groove interfered with noncovalent binding of vaccinia topoisomerase to its target site (8). To explore the minor groove interface with the DNA on the covalently held side of the scissile phosphodiester, we introduced R and S BPdG adducts at the +5, +4, and +3 nucleosides of the 3'-GGGAA sequence and measured the rate and extent of single turnover transesterification by vaccinia topoisomerase on the BPdG-modified substrates (Fig. 5). The unmodified substrate was cleaved to an extent of 93% of the input-labeled DNA with an apparent rate constant of 0.37 s¹. The +5R BPdG modification had no significant effect on either the rate or extent of transesterification (k_+ 5R = 0.25 s¹; 87% end point cleavage). The +5S isomer had only a modest (3-fold) slowing effect on the cleavage rate and little impact on the end point (k_+ 5S = 0.12 s¹; 80% yield) (Fig. 5). The electrophoretic mobility of the cluster of proteinase K-digested reaction products formed with the +5S and +5R BPdG substrates was unaltered compared with the cluster produced by cleavage of unmodified DNA (Fig. 4, lanes 13 and 14).

View larger version (25K): [in this window] [in a new window]   Fig. 5.   Effects of BPdG adducts at positions +3, +4, and +5 of the nonscissile strand on the rate and extent of DNA transesterification by vaccinia topoisomerase. The full structure of the 34-mer/18-mer CCCTT-containing substrate is shown at the bottom of the figure. Detailed views of the CCCTT target site in the unmodified control DNA and the BPdG-modified substrates are illustrated with the numerical coordinates of the nucleotides indicated above the unmodified scissile strand sequence. The S and R diastereomers of the BPdG adducts are depicted as horizontal bars in their respective orientations from the site of covalent attachment to guanine on the nonscissile strand. The topoisomerase cleavage rate constants and cleavage endpoints are indicated to the right of each structure.

Phasing the BPdG modifications 1 or 2 nucleotides closer to the scissile phosphodiester dramatically reduced the rate and the extent of the DNA cleavage reaction (Fig. 5). The reactions with the +4R, +4S, +3R, and +3S BPdG-modified substrates occurred with similar rates of approach to the reaction endpoints (k_cl = 0.0002 s¹), which were attained when 18%, 6.3%, 7.4%, and 5.8% of the input DNA was transferred to the topoisomerase polypeptide. Neither the rate nor the end point increased when the concentration of topoisomerase was doubled, implying that the reaction was not limited by the noncovalent binding step. Rather, we surmise that the majority of the topoisomerase binding events are nonproductive with respect to transesterification and that there is not a free equilibrium between productive and nonproductive binding modes (at least not within the 24-h time-frame in which the reactions were monitored). Although the yields were low, the mobility of the proteinase K-digested products formed on the +4R- and +4S-modified substrates was typical of the pattern seen with the unmodified DNA (Fig. 4, lanes 11 and 12). We suspect that the presence of one additional labeled species migrating above the main cluster of DNA-peptide complexes reflects incomplete proteolysis. (Were there a shift in the cleavage site, we would expect to see phasing of the entire cluster rather than only one additional species.)

The >1000-fold rate decrement elicited by the +4 and +3 BPdG adducts illustrates the strong requirement for access to the DNA minor groove within the segment spanning the +4C:G, +3C:G, and +2T:A base pairs. Comparison to the benign effects of the +5 BPdG modifications delineates an upstream margin for the minor groove interface between the +5C:G and +4C:G base pairs (Fig. 5 and "Discussion" below).

Effects of BPdG Adducts on Exonuclease III-- E. coli exonuclease III catalyzes unidirectional digestion of duplex DNA from the 3' end to liberate 5' dNMP products. The phosphodiesterase activity of exonuclease III is impeded by various chemical modifications of the phosphate backbone (21, 23, 24). Here we explored the effects of BPdG adducts in the minor groove on exonuclease III. 5' ³²P-labeled 18-mers containing the unmodified or BPdG-modified complement of the topoisomerase cleavage site were annealed to an unlabeled complementary 34-mer strand, and the duplexes were incubated with exonuclease III. The 5'-labeled digestion products were resolved by denaturing gel electrophoresis (Fig. 6). All of the unmodified control 18-mer was converted after 10-25 min to 5'-labeled species 6-7 nucleotides in length. A ladder of partially digested strands was evident at 2 min. Strands containing 3' dCMP termini were underrepresented in the ladder in keeping with the intrinsically faster rate of hydrolysis of dCMP by exonuclease III (25). Initial digestion of the +5, +4, and +3 BPdG-modified DNAs by exonuclease III was unaffected as gauged by the rate of decay of the full-length 18-mer strand (Fig. 6). Note that the electrophoretic mobility of the BPdG strands is retarded compared with the unmodified control strand and that the R diastereomer migrates more slowly than the S diastereomer in each case. Electrophoretic mobility differences for DNAs containing R and S BPdG diastereomers have been reported previously (38). These differences notwithstanding, we were able to assign the 3' ends of each of the digestion products using the partial digest ladder of each substrate seen at the 2-min time point.

View larger version (58K): [in this window] [in a new window]   Fig. 6.   Exonuclease III digestion of BPdG-modified DNAs. 5' 32P-labeled control and BPdG-containing 18-mer oligonucleotides were annealed to a 34-mer to form the tailed duplex molecule shown below the autoradiogram. The DNAs were digested with exonuclease III for 0, 2, 10, or 25 min, and the products were analyzed by PAGE. The nucleotide sequences of the 18-mers are displayed next to the cleavage ladders with each letter specifying the 3' nucleotide of the indicated radiolabeled species. Black diamonds mark the sites of BPdG modification.

Placement of R and S BPdG adducts at position +3 resulted in a strong kinetic roadblock to exonuclease III digestion located 2-4 nucleotides 3' of the site of the modification that was apparent at 2 min (the adduct sites are indicated by  in Fig. 6). The enzyme progressed to within 2-3 nucleotides of the adduct site by 25 min and no further. A similar kinetic pattern of arrest was seen with the +4 and +5 BPdG adducts, except that the arrest sites were phased by 1 nucleotide (for +4 BPdG) and 2 nucleotides (for +5 BPdG) in the 3' direction. In each case, exonuclease III was blocked within 2 min at positions 2-4 nucleotides 3' of the modified base and hydrolysis could not proceed beyond the position 2 nucleotides 3' of the adduct (Fig. 6). We discerned no significant stereoisomer effects on the digestion ladder at positions +3 or +4. In the case of the +5R BPdG-modified substrate, exonuclease III was sharply arrested 3 nucleotides prior to the lesion, whereas about half of the +5S isomer was digested to within 2 nucleotides of the lesion (Fig. 6). These results underscore the notion that exonuclease III surveys the minor groove ahead of the active site of catalysis and that space-filling lesions in the minor groove strongly impede its progress.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The Topoisomerase Minor Groove Interface-- By introducing chiral BPdG adducts at five positions of the nonscissile strand, we have defined the dimensions of the functional interface of vaccinia topoisomerase with the DNA minor groove. We observe discrete margins for interference effects on upstream (+) and downstream () of the scissile phosphodiester, whereby +5 and 2 BPdG modifications have little effect on the rate or extent of transesterification, while +4, +3, and 1 BPdG adducts interfere strongly. The simple interpretation of the data is that BPdG inhibition of transesterification is a consequence of steric exclusion of constituents of the enzyme from the DNA minor groove. This view is consistent with NMR data showing that single R and S BPdG adducts do not perturb base pairing or B-form helical conformation in a model synthetic oligonucleotide (14, 33, 34). Alternatively, the BPdG adducts in the minor groove may interfere with a topoisomerase-induced conformational change of the DNA (entailing unpairing of the +1T base) that precedes the transesterification reaction (26, 27).

The stereoselective effects at the 1 nucleoside (the R diastereomer interferes, whereas the S diastereomer does not) delineate at reasonably high resolution the downstream border of the minor groove interface. Fig. 7 uses the NMR structures (14, 33, 34) to model the 1S and 1R BPdG-modified duplexes; the position of the scissile phosphodiester (+1) on the CCCTT strand is indicated by the arrow. Note that the non-interfering S diastereomer has the aromatic pyrenyl moiety pointing away from the scissile phosphate, whereas the strongly interfering R diastereomer has the pyrenyl component pointing toward the +1 phosphate. It is evident that the 50-fold greater interference by the R diastereomer correlates with occlusion of the minor groove over the +1T:A base pair and part of the +2T:A base pair, said surface being exposed in the model of the S diastereomer. The critical area is highlighted by the blue ellipse in Fig. 7. One can similarly model the 2BPdG substrates on the NMR structure by shifting the scissile phosphate by one position (downward in the view shown in Fig. 7). This maneuver indicates that neither the 2R or 2S BPdG adducts overlap the critical interfering surface, thereby explaining why the 2BPdG adducts do not inhibit the transesterification reaction.

View larger version (70K): [in this window] [in a new window]   Fig. 7.   Downstream margin of the topoisomerase minor groove interface. Models of B-DNA containing 1R and 1S BPdG adducts on the nonscissile strand are shown with the site of transesterification of topoisomerase to the scissile strand (+1) indicated by the yellow arrows. The observed rate constants for topoisomerase cleavage of the respective diastereomers are shown below the structures.

BPdG adducts at the +4 and +3 guanines of the 3'-GGGAA recognition sites decreased the rate and the extent of transesterification by vaccinia topoisomerase. The decreased yield of product was not corrected by increasing enzyme concentration, implying that initial binding was not rate-limiting but was predominantly nonproductive. The sequence specificity of vaccinia topoisomerase is dictated principally at the level of transesterification chemistry rather than noncovalent binding. The enzyme cleaves exclusively at (T/C)CCTT or related pentapyrimidine sites, but the noncovalent binding affinity for CCCTT-containing DNA is only 8-fold higher than its affinity for non-CCCTT DNA under the standard reaction conditions (28). The transition from nonproductive to productive binding entails the assembly of a catalytically competent active site (which is not preformed in the free enzyme) in which the essential functional groups (Arg-130, Lys-167, Arg-223, His-265, and Tyr-274) are oriented properly at the scissile phosphodiester (10, 12, 13). The decreased cleavage seen with the +4 and +3 BPdG adducts may reflect their inhibition of this conformational transition, which could entail penetration of the enzyme into the minor groove within the CCCTT element.

A notable finding was that the +5S BPdG adduct has only a 3-fold effect on the cleavage rate constant and little impact on the end point, whereas the +4R BPdG modification strongly suppressed the rate and extent of the reaction (Fig. 5). This disparity is surprising at first given that the +5S adduct (which points toward the scissile phosphate) and the +4R adduct (which points away from the scissile phosphate) are predicted to overlap substantially in the minor groove. We speculate that the functional differential between +4R and +5S BPdG modifications arises because the three nonplanar hydroxyl substituents of the cyclohexene ring of the +4R BPdG adduct occlude the topoisomerase interface, whereas the edge of the planar pyrenyl ring of +5S BPdG does not penetrate into the enzyme's interaction zone in the minor groove. Our results fix a sharp (and structurally subtle) upstream margin of the minor groove interface of vaccinia topoisomerase near the +4C:G base pair.

Minor Groove Interactions of Exonuclease III-- We also applied the BPdG interference method to probe the interactions of exonuclease III with the DNA minor groove. Exonuclease III employs a one-step in-line mechanism in which an activated water attacks the scissile phosphodiester (which is coordinated to an essential divalent cation) and expels the 3'-O of the upstream nucleotide of the DNA strand (29). Exonuclease III is clearly impeded from hydrolyzing the two phosphodiester linkages immediately 3' of a BPdG adduct; indeed it displays feeble activity toward the phosphodiesters located 3 or 4 nucleotides 3' of the BPdG nucleoside. We presume that the occupancy of the minor groove precludes the approach of the active site to these protected phosphodiesters because the enzyme itself penetrates the minor groove when it binds to the 3' terminal dNMP of the substrate.

Exonuclease III is structurally related to DNase I and to the abasic nuclease HAP1 (29, 30). DNase I binds to DNA in the minor groove (31), and models of exonuclease III and HAP1 DNA interactions also invoke minor groove contacts, including contacts of a conserved loop with nucleotides 5' of the scissile phosphodiester (30). We showed previously that a 2'-5' phosphodiester modification arrested exonuclease III at positions 1 and 2 nucleotides prior to the encounter of its active site with the 2'-5' linkage, which was itself poorly hydrolyzed (21). Taken together, the 2'-5'-phosphate and BPdG interference experiments indicate that exonuclease III surveys the phosphate backbone and the minor groove advance of the active site.

Although the S and R diastereomers of trans BPdG are oppositely oriented in the minor groove of duplex DNA, we did not observe consistent differences in the sites or duration of the blocks to exonuclease III digestion by the S versus R adducts. This contrasts with the stereoselective impediments to exonucleolytic digestion of a BPdG-modified single-stranded DNA oligonucleotide by snake venom and spleen phosphodiesterases (32). Snake venom phosphodiesterase, which has the same 3' to 5' directionality as exonuclease III, is arrested either at the adducted guanine nucleotide (R diastereomer) or else 1 nucleotide 3' of the adduct (S diastereomer). Thus, exonuclease III senses the BPdG adduct at a greater distance than does snake venom phosphodiesterase.

	ACKNOWLEDGEMENT

We are grateful to Dr. Yves Pommier (National Institutes of Health) who played a key role in the initial conception of this project and in catalyzing the collaboration between the S. S. and D. M. J. laboratories.

	FOOTNOTES

^* This work was supported by National Institutes of Health Grant GM46330.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed. Tel.: 212-639-7145; Fax: 212-717-3623; E-mail: s-shuman@ski.mskcc.org.

Published, JBC Papers in Press, January 10, 2003, DOI 10.1074/jbc.M212468200

	ABBREVIATIONS

The abbreviations used are: BP, benzo[a]pyrene; dG, deoxyguanosine.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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