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• W2013320172 abstract "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 andS 7,8-diol 9,10-epoxide adducts of benzo[a]pyrene (BP) at singleN 2-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 Risomer 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. 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 andS 7,8-diol 9,10-epoxide adducts of benzo[a]pyrene (BP) at singleN 2-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 Risomer 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. 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 (1Shuman S. Biochim. Biophys. Acta. 1998; 1400: 321-337Google Scholar, 2Champoux J.J. Annu. Rev. Biochem. 2001; 70: 369-413Google Scholar, 3Zhang H. Barcelo J.M. Lee B. Kohlhagen G. Zimonjic D.B. Popescu N.C. Pommier Y. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 10608-10613Google Scholar, 4Krogh B.O. Shuman S. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 1853-1858Google Scholar). The vaccinia enzyme is distinguished from the nuclear topoisomerase I by its compact size (314 amino acids) and its site specificity in DNA transesterification (5Shuman S. Prescott J. J. Biol. Chem. 1990; 265: 17826-17836Google Scholar). 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 (6Shuman S. J. Biol. Chem. 1991; 266: 11372-11379Google Scholar). Exonuclease III footprinting suggests a two-part interaction of topoisomerase with the DNA 5′ of the site of covalent adduct formation (7Cheng C. Shuman S. Biochemistry. 1999; 38: 16599-16612Google Scholar). 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) (8Shuman S. Turner J. J. Biol. Chem. 1993; 268: 18943-18950Google Scholar). The contributions of individual phosphates to binding specificity were initially inferred from the effects of phosphate ethylation on protein binding (9Sekiguchi J. Shuman S. J. Biol. Chem. 1994; 269: 31731-31734Google Scholar). 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 (9Sekiguchi J. Shuman S. J. Biol. Chem. 1994; 269: 31731-31734Google Scholar). 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 (9Sekiguchi J. Shuman S. J. Biol. Chem. 1994; 269: 31731-31734Google Scholar). 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 (10Cheng C. Kussie P. Pavletich N. Shuman S. Cell. 1998; 92: 841-850Google Scholar, 11Redinbo M.R. Stewart L. Kuhn P. Champoux J.J. Hol W.G.J. Science. 1998; 279: 1504-1513Google Scholar). 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 (12Krogh B.O. Shuman S. Mol. Cell. 2000; 5: 1035-1041Google Scholar, 13Krogh B.O. Shuman S. J. Biol. Chem. 2002; 277: 5711-5714Google Scholar), 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) 1The abbreviations used are: BP, benzo[a]pyrene; dG, deoxyguanosine 1The abbreviations used are: BP, benzo[a]pyrene; dG, deoxyguanosine at the exocyclicN 2-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 (14Geacintov N.E. Cosman M. Hingerty B.E. Amin S. Broyde S. Patel D.J. Chem. Res. Toxicol. 1997; 10: 111-146Google Scholar, 33Cosman M. de los Santos C. Fiala R. Hingerty B.E. Ibanez V. Margulis L.A. Live D. Geacintov N.E. Broyde S. Patel D.J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 1914-1918Google Scholar, 34de los Santos C. Cosman M. Hingerty B.E. Singh S.B. Ibanez V. Margulis L.A. Geacintov N.E. Broyde S. Patel D.J. Biochemistry. 1992; 31: 5245-5252Google Scholar). Moreover, the S and Radducts have opposite orientations in the minor groove, such that theS 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 (15Pommier Y. Kohlhagen G. Pourquier P. Sayer J.M. Kroth H. Jerina D.M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 2040-2045Google Scholar, 16Pommier Y. Kohlhagen G. Laco G.S. Kroth H. Sayer J.M. Jerina D.M. J. Biol. Chem. 2002; 277: 13666-13672Google Scholar). 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 (17Arslan T. Abraham A.T. Hecht S.M. J. Biol. Chem. 1998; 273: 12383-12390Google Scholar). Vaccinia topoisomerase is more amenable to quantitatively informative interference studies because the pre-steady-state kinetic parameters for transesterification are known (18Stivers J.T. Shuman S. Mildvan A.S. Biochemistry. 1994; 33: 327-339Google Scholar, 19Wittschieben J. Shuman S. Nucleic Acids Res. 1997; 25: 3001-3008Google Scholar, 20Stivers J.T. Jagadeesh G.J. Nawrot B. Stec W.J. Shuman S. Biochemistry. 2000; 39: 5561-5572Google Scholar) and the enzyme does not relinquish its site specificity in response to a DNA lesion (21Krogh B.O. Claeboe C.D. Hecht S.M. Shuman S. J. Biol. Chem. 2001; 276: 20907-20912Google Scholar). 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 Rdiastereomer 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 ofEscherichia coli exonuclease III with the DNA minor groove. Modified oligonucleotides containing single chiral BPdG adducts (Fig. 1) were synthesized and purified on a 1.5-μmol scale as described previously (35Kroth H. Yagi H. Sayer J.M. Kumar S. Jerina D.M. Chem. Res. Toxicol. 2001; 14: 708-719Google Scholar). The modified dG was introduced by manual coupling using the diastereomeric mixture of suitably protected R andS C-10 adducted phosphoramidates (36Kroth H. Yagi H. Seidel A. Jerina D.M. J. Org. Chem. 2000; 65: 5558-5564Google Scholar). 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 (37Page J.E. Zajc B. Oh-hara T. Lakshman M.K. Sayer J.M. Jerina D.M. Dipple A. Biochemistry. 1998; 37: 9127-9137Google Scholar). Unmodified oligonucleotides were purchased from BIOSOURCEInternational. 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 [γ-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.Table IHigh performance liquid chromatography retention times and absolute configurations of 18-mer oligonucleotides containing trans opened N2-BPdG adducts at C-10OligonucleotideRetention time RdiastereomerRetention time S diastereomer5′-CGG AAT AAG GGC GAC ACG-3′ (+3)15.7,1-aOn a Hamilton PRP-1 column (10 × 250 mm, 7 mm) eluted at 3 ml/min at 60 °C with a gradient from 0–35% solvent B in solvent A over 20 min (where A is 0.1 M(NH4)2CO3, pH 7.5, and B is a 1:1 mixture of A and acetonitrile at the same pH). 16.01-bOn a Higgins analytical DNA column (4.6 × 100 mm) (Thomson Instrument Co., Clear Brook, VA) eluted at 1 ml/min at 45 °C with a gradient from 10–22 % B in A over 20 min.17.4,1-aOn a Hamilton PRP-1 column (10 × 250 mm, 7 mm) eluted at 3 ml/min at 60 °C with a gradient from 0–35% solvent B in solvent A over 20 min (where A is 0.1 M(NH4)2CO3, pH 7.5, and B is a 1:1 mixture of A and acetonitrile at the same pH). 20.31-bOn a Higgins analytical DNA column (4.6 × 100 mm) (Thomson Instrument Co., Clear Brook, VA) eluted at 1 ml/min at 45 °C with a gradient from 10–22 % B in A over 20 min.5′-CGG AAT AAG GGC GAC ACG-3′ (+4)9.3,1-cOn the Hamilton PRP-1 column eluted at 3 ml/min at 60 °C with a gradient from 11–30% B in A over 15.5 min. 11.81-dOn a Waters Xterra MS C18 analytical column (4.6 × 50 mm, 2.5 mm) eluted at 0.8 ml/min at 65 °C with a gradient from 0–15 % B in A over 7 min followed by a ramp to 22% B at 20 min.10.7,1-cOn the Hamilton PRP-1 column eluted at 3 ml/min at 60 °C with a gradient from 11–30% B in A over 15.5 min. 12.71-dOn a Waters Xterra MS C18 analytical column (4.6 × 50 mm, 2.5 mm) eluted at 0.8 ml/min at 65 °C with a gradient from 0–15 % B in A over 7 min followed by a ramp to 22% B at 20 min.5′-CGG AAT AAG GGC GAC ACG-3′ (+5)10.9,1-cOn the Hamilton PRP-1 column eluted at 3 ml/min at 60 °C with a gradient from 11–30% B in A over 15.5 min. 12.61-eOn the Xterra column eluted at 1 ml/min at 55 °C with the same gradient as d.12.1,1-cOn the Hamilton PRP-1 column eluted at 3 ml/min at 60 °C with a gradient from 11–30% B in A over 15.5 min. 14.31-eOn the Xterra column eluted at 1 ml/min at 55 °C with the same gradient as d.5′-CGG TAG AAG GGC GAC ACG-3′ (−1)10.9,1-cOn the Hamilton PRP-1 column eluted at 3 ml/min at 60 °C with a gradient from 11–30% B in A over 15.5 min. 12.31-fOn the Higgins DNA column eluted at 1 ml/min at 60 °C with a gradient from 12–22% B in A over 20 min.12.9,1-cOn the Hamilton PRP-1 column eluted at 3 ml/min at 60 °C with a gradient from 11–30% B in A over 15.5 min. 15.31-fOn the Higgins DNA column eluted at 1 ml/min at 60 °C with a gradient from 12–22% B in A over 20 min.5′-CGG AGT AAG GGC GAC ACG-3′ (−2)11.0,1-cOn the Hamilton PRP-1 column eluted at 3 ml/min at 60 °C with a gradient from 11–30% B in A over 15.5 min. 10.61-dOn a Waters Xterra MS C18 analytical column (4.6 × 50 mm, 2.5 mm) eluted at 0.8 ml/min at 65 °C with a gradient from 0–15 % B in A over 7 min followed by a ramp to 22% B at 20 min.12.0,1-cOn the Hamilton PRP-1 column eluted at 3 ml/min at 60 °C with a gradient from 11–30% B in A over 15.5 min. 11.01-dOn a Waters Xterra MS C18 analytical column (4.6 × 50 mm, 2.5 mm) eluted at 0.8 ml/min at 65 °C with a gradient from 0–15 % B in A over 7 min followed by a ramp to 22% B at 20 min.The modified base is underlined. Configurational assignments are based on the long-wavelength (320–360 nm) CD bands of the oligonucleotides, which are positive for R and negative for Sadducts.1-a On a Hamilton PRP-1 column (10 × 250 mm, 7 mm) eluted at 3 ml/min at 60 °C with a gradient from 0–35% solvent B in solvent A over 20 min (where A is 0.1 M(NH4)2CO3, pH 7.5, and B is a 1:1 mixture of A and acetonitrile at the same pH).1-b On a Higgins analytical DNA column (4.6 × 100 mm) (Thomson Instrument Co., Clear Brook, VA) eluted at 1 ml/min at 45 °C with a gradient from 10–22 % B in A over 20 min.1-c On the Hamilton PRP-1 column eluted at 3 ml/min at 60 °C with a gradient from 11–30% B in A over 15.5 min.1-d On a Waters Xterra MS C18 analytical column (4.6 × 50 mm, 2.5 mm) eluted at 0.8 ml/min at 65 °C with a gradient from 0–15 % B in A over 7 min followed by a ramp to 22% B at 20 min.1-e On the Xterra column eluted at 1 ml/min at 55 °C with the same gradient as d.1-f On the Higgins DNA column eluted at 1 ml/min at 60 °C with a gradient from 12–22% B in A over 20 min. Open table in a new tab Figure 5Effects 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 Rdiastereomers 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.View Large Image Figure ViewerDownload (PPT) The modified base is underlined. Configurational assignments are based on the long-wavelength (320–360 nm) CD bands of the oligonucleotides, which are positive for R and negative for Sadducts. Recombinant vaccinia topoisomerase was produced in E. coli (BL21) by infection with bacteriophage λCE6 (22Shuman S. Golder M. Moss B. J. Biol. Chem. 1988; 263: 16401-16407Google Scholar) 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. 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. 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. 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 mmMgCl2, 1.8 pmol of 18-mer/34-mer, and 1.0 unit ofE. 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. A pair of 18-mer strands containing a singleS 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. 2 A). 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 (18Stivers J.T. Shuman S. Mildvan A.S. Biochemistry. 1994; 33: 327-339Google Scholar, 19Wittschieben J. Shuman S. Nucleic Acids Res. 1997; 25: 3001-3008Google Scholar, 20Stivers J.T. Jagadeesh G.J. Nawrot B. Stec W.J. Shuman S. Biochemistry. 2000; 39: 5561-5572Google Scholar). 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 s−1 (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 s−1) and no effect on the reaction end point (Figs. 2 A and3.). In contrast, the Rdiastereomer at position −1 reduced the rate of transesterification by a factor of 200 (k cl = 0.002 s−1) without influencing the end point (Figs. 2 A and 3). Thek 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. 2 A). 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 input32P-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. 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. 2 B). 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 s−1 (Fig.2 B). 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 s−1 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 7and 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). 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 (8Shuman S. Turner J. J. Biol. Chem. 1993; 268: 18943-18950Google Scholar). To explore the minor groove interface with the DNA on the covalently held side of the scissile phosphodiester, we introduced Rand 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 s−1. The +5R BPdG modification had no significant effect on either the rate or extent of transesterification (k + 5R = 0.25 s−1; 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 s−1; 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 and14). 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 s−1), 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 p" @default.
• W2013320172 created "2016-06-24" @default.
• W2013320172 creator A5014112082 @default.
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