Benzo[c]phenanthrene Adducts and Nogalamycin Inhibit DNA Transesterification by Vaccinia Topoisomerase
2004; Elsevier BV; Volume: 279; Issue: 22 Linguagem: Inglês
10.1074/jbc.m401203200
ISSN1083-351X
AutoresLyudmila Yakovleva, Christopher J. Handy, Jane M. Sayer, Michael C. Pirrung, Donald M. Jerina, Stewart Shuman,
Tópico(s)Bioactive Compounds and Antitumor Agents
ResumoVaccinia DNA topoisomerase forms a covalent DNA-(3′-phosphotyrosyl)-enzyme intermediate at a specific target site 5′-C+5C+4C+3T+2T+1p↓N-1 in duplex DNA. Here we study the effects of position-specific DNA intercalators on the rate and extent of single-turnover DNA transesterification. Chiral C-1 R and S trans-opened 3,4-diol 1,2-epoxide adducts of benzo[c]phenanthrene (BcPh) were introduced at single N2-deoxyguanosine and N6-deoxyadenosine positions within the 3′-G+5G+4G+3A+2A+1T-1A-2 sequence of the nonscissile DNA strand. Transesterification was unaffected by BcPh intercalation between the +6 and +5 base pairs, slowed 4-fold by intercalation between the +5 and +4 base pairs, and virtually abolished by BcPh intercalation between the +4 and +3 base pairs and the +3 and +2 base pairs. Intercalation between the +2 and +1 base pairs by the +2R BcPh dA adduct abolished transesterification, whereas the overlapping +1S BcPh dA adduct slowed the rate of transesterification by a factor of 2700, with little effect upon the extent of the reaction. Intercalation at the scissile phosphodiester (between the +1 and -1 base pairs) slowed transesterification by a factor of 450. BcPh intercalation between the -1 and -2 base pairs slowed cleavage by two orders of magnitude, but intercalation between the -2 and -3 base pairs had little effect. The anthracycline drug nogalamycin, a non-covalent intercalator with preference for 5′-TG dinucleotides, inhibited the single-turnover DNA cleavage reaction of vaccinia topoisomerase with an IC50 of 0.7 μm. Nogalamycin was most effective when the drug was pre-incubated with DNA and when the cleavage target site was 5′-CCCTT↓G instead of 5′-CCCTT↓A. These findings demarcate upstream and downstream boundaries of the functional interface of vaccinia topoisomerase with its DNA target site. Vaccinia DNA topoisomerase forms a covalent DNA-(3′-phosphotyrosyl)-enzyme intermediate at a specific target site 5′-C+5C+4C+3T+2T+1p↓N-1 in duplex DNA. Here we study the effects of position-specific DNA intercalators on the rate and extent of single-turnover DNA transesterification. Chiral C-1 R and S trans-opened 3,4-diol 1,2-epoxide adducts of benzo[c]phenanthrene (BcPh) were introduced at single N2-deoxyguanosine and N6-deoxyadenosine positions within the 3′-G+5G+4G+3A+2A+1T-1A-2 sequence of the nonscissile DNA strand. Transesterification was unaffected by BcPh intercalation between the +6 and +5 base pairs, slowed 4-fold by intercalation between the +5 and +4 base pairs, and virtually abolished by BcPh intercalation between the +4 and +3 base pairs and the +3 and +2 base pairs. Intercalation between the +2 and +1 base pairs by the +2R BcPh dA adduct abolished transesterification, whereas the overlapping +1S BcPh dA adduct slowed the rate of transesterification by a factor of 2700, with little effect upon the extent of the reaction. Intercalation at the scissile phosphodiester (between the +1 and -1 base pairs) slowed transesterification by a factor of 450. BcPh intercalation between the -1 and -2 base pairs slowed cleavage by two orders of magnitude, but intercalation between the -2 and -3 base pairs had little effect. The anthracycline drug nogalamycin, a non-covalent intercalator with preference for 5′-TG dinucleotides, inhibited the single-turnover DNA cleavage reaction of vaccinia topoisomerase with an IC50 of 0.7 μm. Nogalamycin was most effective when the drug was pre-incubated with DNA and when the cleavage target site was 5′-CCCTT↓G instead of 5′-CCCTT↓A. These findings demarcate upstream and downstream boundaries of the functional interface of vaccinia topoisomerase with its DNA target site. Poxvirus DNA topoisomerase I is important for virus replication (1Fonseca F.D. Moss B. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 11291-11296Crossref PubMed Scopus (48) Google Scholar) and a potential target for drug therapy of smallpox, in light of its unique DNA recognition specificity, compact structure, and distinctive pharmacological sensitivities compared with human topoisomerase I (2Shuman S. Golder M. Moss B. J. Biol. Chem. 1988; 263: 16401-16407Abstract Full Text PDF PubMed Google Scholar, 3Shuman S. Prescott J. J. Biol. Chem. 1990; 265: 17826-17836Abstract Full Text PDF PubMed Google Scholar, 4Morham S.G. Shuman S. J. Biol. Chem. 1992; 267: 15984-15992Abstract Full Text PDF PubMed Google Scholar, 5Sekiguchi J. Stivers J.T. Mildvan A.S. Shuman S. J. Biol. 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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 (13Shuman S. J. Biol. Chem. 1991; 266: 11372-11379Abstract Full Text PDF PubMed Google Scholar, 14Shuman S. Turner J. J. Biol. Chem. 1993; 268: 18943-18950Abstract Full Text PDF PubMed Google Scholar, 15Sekiguchi J. Shuman S. J. Biol. Chem. 1994; 269: 31731-31734Abstract Full Text PDF PubMed Google Scholar, 16Sekiguchi J. Shuman S. J. Biol. Chem. 1996; 271: 19436-19442Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar, 17Sekiguchi J. Shuman S. EMBO J. 1996; 15: 3448-3457Crossref PubMed Scopus (46) Google Scholar, 18Cheng C. Shuman S. Biochemistry. 1999; 38: 16599-16612Crossref PubMed Scopus (18) Google Scholar, 19Hwang Y. Burgin A. Bushman F. J. Biol. Chem. 1999; 274: 9160-9168Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). Recent studies have focused on delineating the features of the DNA interface that affect the kinetics of transesterification. Modifications at the scissile phosphodiester have illuminated the chemical mechanism of topoisomerase IB, the roles of the individual amino acids in either transition-state stabilization or general acid catalysis, and the parameters affecting the stability of the covalent topoisomerase-DNA intermediate (20Krogh B.O. Shuman S. Mol. Cell. 2000; 5: 1035-1041Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, 21Stivers J.T. Jagadeesh G.J. Nawrot B. Stec W.J. Shuman S. Biochemistry. 2000; 39: 5561-5572Crossref PubMed Scopus (58) Google Scholar, 22Krogh B.O. Claeboe C.D. Hecht S.M. Shuman S. J. Biol. Chem. 2001; 276: 20907-20912Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar, 23Krogh B.O. Shuman S. J. Biol. 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Another approach taken to define the interface between vaccinia topoisomerase and its cleavage site exploits position-specific covalent polycyclic aromatic hydrocarbon (PAH) 1The abbreviations used are: PAH, polycyclic aromatic hydrocarbon; BP, benzo[a]pyrene; dG, deoxyguanosine; dA, deoxyadenosine; BcPh, benzo[c]phenanthrene.1The abbreviations used are: PAH, polycyclic aromatic hydrocarbon; BP, benzo[a]pyrene; dG, deoxyguanosine; dA, deoxyadenosine; BcPh, benzo[c]phenanthrene. diol epoxide-DNA adducts (25Tian L. Sayer J.M. Kroth H. Kalena G. Jerina D.M. Shuman S. J. Biol. Chem. 2003; 278: 9905-9911Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar, 26Yakovleva L. Tian L. Sayer J.M. Kalena G.P. Kroth H. Jerina D.M. Shuman S. J. Biol. Chem. 2003; 278: 42170-42177Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). For example, to probe the DNA minor groove surface, 7,8-diol 9,10-epoxide adducts of benzo-[a]pyrene (BP) were introduced at the exocyclic N2-amino group of single deoxyguanosine (dG) positions within the non-scissile 3′-G+5G+4G+3A+2A+1N-1N-2 strand of a suicide cleavage substrate for vaccinia topoisomerase (25Tian L. Sayer J.M. Kroth H. Kalena G. Jerina D.M. Shuman S. J. Biol. Chem. 2003; 278: 9905-9911Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). The transopened BP dG adducts fit into the minor groove without perturbing helix conformation or base pairing, and the C-10 R and S diastereomers are oriented in opposite directions within the minor groove (27Cosman M. de los Santos C. Fiala R. Hingerty B.E. Singh S.B. Ibanez V. Margulis L.A. Live D. Geacintov N.E. Broyde S. Patel D.J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 1914-1918Crossref PubMed Scopus (295) Google Scholar, 28de los Santos C. Cosman M. Hingerty B.E. Ibanez V. Margulis L.A. Geacintov N.E. Broyde S. Patel D.J. Biochemistry. 1992; 31: 5245-5252Crossref PubMed Scopus (185) Google Scholar). A sharp margin of interference effects was observed, whereby +5 and -2 BP dG modifications were well tolerated, but +4, +3, and -1 BP dG adducts were severely deleterious. The stereoselective effects at the -1 nucleoside (the R diastereomer interfered, whereas the S diastereomer did not) delineated at high resolution the downstream border of the minor groove interface (25Tian L. Sayer J.M. Kroth H. Kalena G. Jerina D.M. Shuman S. J. Biol. Chem. 2003; 278: 9905-9911Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). It was inferred that BP dG inhibition of transesterification is likely caused by steric exclusion of essential constituents of the topoisomerase from the DNA minor groove. PAH diol epoxide-DNA adducts have also been exploited to probe the effects of position-specific intercalation, by introducing trans-opened 7,8-diol 9,10-epoxide adducts of BP at the exocyclic N6-amino group of deoxyadenosine (dA) positions within the nonscissile strand (26Yakovleva L. Tian L. Sayer J.M. Kalena G.P. Kroth H. Jerina D.M. Shuman S. J. Biol. Chem. 2003; 278: 42170-42177Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). The R and S BP dA adducts intercalate from the major groove on the 5′ and 3′ sides of the modified base, respectively, and perturb local base stacking (29Volk D.E. Rice J.S. Luxon B.A. Yeh H.J.C. Liang C. Xie G. Sayer J.M. Jerina D.M. Gorenstein D.G. Biochemistry. 2000; 39: 14040-14053Crossref PubMed Scopus (51) Google Scholar, 30Pradhan P. Tirumala S. Liu X. Sayer J.M. Jerina D.M. Yeh H.J.C. Biochemistry. 2001; 40: 5870-5881Crossref PubMed Scopus (46) Google Scholar, 31Zegar I.S. Charty P. Jabil R.J. Tamura P.J. Johansen T.N. Lloyd R.S. Harris C.M. Harris T.M. Stone M.P. Biochemistry. 1998; 37: 16516-16528Crossref PubMed Scopus (46) Google Scholar, 32Schurter E.J. Sayer J.M. Oh-hara T. Yeh H.J.C. Yagi H. Luxon B.A. Jerina D.M. Gorenstein D.G. Biochemistry. 1996; 34: 9009-9020Crossref Scopus (58) Google Scholar, 33Zegar I.S. Kim S.J. Johansen T.N. Horton P.J. Harris C.M. Harris T.M. Stone M.P. Biochemistry. 1996; 34: 6212-6224Crossref Scopus (80) Google Scholar). R and S BP dA modifications at +1A reduced the transesterification rate by a factor of 700 to 1000 without affecting the yield of the covalent topoisomerase-DNA complex. BP dA modifications at +2A reduced the extent of transesterification and elicited rate decrements of 200-fold and 7000-fold for the S and R diastereomers, respectively (26Yakovleva L. Tian L. Sayer J.M. Kalena G.P. Kroth H. Jerina D.M. Shuman S. J. Biol. Chem. 2003; 278: 42170-42177Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). In contrast, BP dA adducts at the -2 position had no effect on the extent of the reaction and relatively little impact on the rate of cleavage. The BP dA interference effects demarcated the -1 base pair as the “downstream” margin of the functional interface between DNA and vaccinia topoisomerase that can be affected significantly by BP intercalation. The “upstream” margin remained undefined because the effects of intercalating PAH adducts at the guanine positions of the cleavage target site were not tested. Here we extend the use of defined PAH diol epoxide-DNA adducts to probe the effects of position-specific intercalation by benzo[c]phenanthrene (BcPh) at all purines of the topoisomerase target sequence. BcPh exemplifies the sterically hindered, nonplanar “fjord-region” class of PAHs. BcPh has been studied extensively in light of the highly tumorigenic and mutagenic properties of metabolically activated BcPh diol epoxides, which react at the benzylic C1 position by trans addition of guanine N2 and adenine N6 in DNA to form the covalent trans 1S and 1R BcPh dG and BcPh dA adducts depicted in Fig. 1. Structures of duplex DNAs containing single BcPh dG adducts have established that the aromatic ring systems intercalate from the minor groove on opposite sides of the modified dG base depending on their stereochemical configuration (34Lin C.H. Huang X. Kolbanovskii A. Hingerty B.E. Amin S. Broyde S. Geacintov N.E. Patel D.J. J. Mol. Biol. 2001; 306: 1059-1080Crossref PubMed Scopus (60) Google Scholar). The S diastereomer intercalates on the 5′ side of the dG, whereas the R diastereomer intercalates on the 3′ side (Fig. 2). Structures of DNAs containing single BcPh dA adducts show that the hydrocarbon intercalates from the major groove, such that the S diastereomer is on the 3′ side of the modified dA base and the R diastereomer is on the 5′ side (Fig. 2; Refs. 35Cosman M. Fiala R. Hingerty B.E. Laryea A. Lee H. Harvey R.G. Amin S. Geacintov N.E. Broyde S. Patel D.J. Biochemistry. 1993; 32: 12488-12497Crossref PubMed Scopus (89) Google Scholar and 36Cosman M. Laryea A. Fiala R. Hingerty B.E. Amin S. Geacintov N.E. Broyde S. Patel D.J. Biochemistry. 1995; 34: 1295-1307Crossref PubMed Scopus (92) Google Scholar). The intercalated BcPh dG and BcPh dA adducts do not disrupt base pairing, but they do cause a buckling of the modified base pair and the unmodified base pair flanking the hydrocarbon (Fig. 2). An attractive feature of BcPh adducts with respect to studies of vaccinia topoisomerase is that they provide structural probes for intercalative interference effects at all of the purine bases of the nonscissile strand of the 5′-CCCTT/3′-GGGAA target site and for effects of intercalation at positions immediately 3′ of the scissile phosphodiester.Fig. 2NMR structures of R and SBcPh dG and dA adducts in duplex DNA. The structures (Ref. 34Lin C.H. Huang X. Kolbanovskii A. Hingerty B.E. Amin S. Broyde S. Geacintov N.E. Patel D.J. J. Mol. Biol. 2001; 306: 1059-1080Crossref PubMed Scopus (60) Google Scholar) of the indicated BcPh diol epoxide adducts are illustrated looking into the minor groove. The intercalated hydrocarbon moiety is colored yellow; the modified base pair is colored cyan.View Large Image Figure ViewerDownload (PPT) We report here that transesterification by vaccinia topoisomerase was suppressed by BcPh intercalation between the +4/+3, +3/+2, +2/+1, +1/-1, and -1/-2 base pairs, but intercalation between the +6/+5, +5/+4, and -2/-3 base pairs had little effect. These new findings demarcate upstream and downstream margins of the interface between DNA and vaccinia topoisomerase. In light of these results, we tested the effects of the anthracycline drug nogalamycin, which intercalates preferentially at 5′-TG dinucleotides in duplex DNA (37Smith C.K. Brannigan J.A. Moore M.H. J. Mol. Biol. 1996; 263: 237-258Crossref PubMed Scopus (26) Google Scholar, 38Williams H.E.L. Searle M.S. J. Mol. Biol. 1999; 290: 699-716Crossref PubMed Scopus (45) Google Scholar, 39Zhang X. Patel D.J. Biochemistry. 1990; 29: 9451-9466Crossref PubMed Scopus (68) Google Scholar). We find that nogalamycin inhibits the single-turnover DNA cleavage reaction of vaccinia topoisomerase and that its potency is higher when the cleavage target site is 5′-CCCTT↓G versus 5′-CCCTT↓A. We surmise that nogalamycin can inhibit the vaccinia enzyme by intercalating between the +1 and -1 base pairs. Synthesis of BcPh Diol Epoxide-adducted Oligonucleotides—Oligonucleotide 18-mers containing trans-opened S and R BcPh diol epoxide adducts (Fig. 1) were synthesized from appropriately protected 5′-O-dimethoxytrityl-3′-phosphoramidites (BcPh dA and BcPh dG phosphoramidites) as described (40Kroth H. Yagi H. Sayer J.M. Kumar S. Jerina D.M. Chem. Res. Toxicol. 2001; 14: 708-719Crossref PubMed Scopus (28) Google Scholar). Oligonucleotides containing BcPh dG were synthesized using purified S and R diastereomers of the BcPh dG phosphoramidites (41Yagi H. Ramesha A.R. Kalena G. Sayer J.M. Kumar S. Jerina D.M. J. Org. Chem. 2002; 67: 6678-6689Crossref PubMed Scopus (14) Google Scholar). BcPh dA adducts were incorporated as their mixed S/R diastereomers (42Ilankumaran P. Pannell L.K. Gebreselassie P. Pilcher A.S. Yagi H. Sayer J.M. Jerina D.M. Chem. Res. Toxicol. 2001; 14: 1330-1338Crossref PubMed Scopus (10) Google Scholar). The resulting S and R BcPh dA-adducted oligonucleotides were purified and resolved by HPLC. Absolute configurations of the BcPh dA adducts were assigned to the separated oligonucleotides after enzymatic hydrolysis to form the nucleoside adducts, which were identified by their CD spectra (43Sayer J.M. Chadha A. Agarwal S.K. Yeh H.J.C. Yagi H. Jerina D.M. J. Org. Chem. 1991; 56: 20-29Crossref Scopus (131) Google Scholar, 44Agarwal S.K. Sayer J.M. Yeh H.J.C. Pannell L.K. Hilton B.D. Pigott M.A. Dipple A. Yagi H. Jerina D.M. J. Am. Chem. Soc. 1987; 109: 2497-2504Crossref Scopus (149) Google Scholar). Details of the high performance liquid chromatography purification of the adducted oligonucleotides are provided in Supplemental Table S1. The compositions of the oligonucleotides were confirmed by matrix-assisted laser desorption ionization-mass spectrometry analysis. DNA Cleavage Substrates—The CCCTT-containing 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 modified nonscissile strand oligonucleotides at a 1:4 molar ratio of scissile:nonscissile strand. 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 and the sequences of the component strands are depicted in the figures. Vaccinia Topoisomerase—Recombinant vaccinia topoisomerase was produced in Escherichia coli BL21 by infection with bacteriophage λCE6 (2Shuman S. Golder M. Moss B. J. Biol. Chem. 1988; 263: 16401-16407Abstract Full Text PDF PubMed Google 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. Drugs—Nogalamycin was obtained from the Open Chemical Repository, Developmental Therapeutics Program, National Cancer Institute. Nogamycin was obtained from Amersham Biosciences, courtesy of Dr. Paul Aristoff. The drugs were dissolved in Me2SO at a concentration of 10 mm. The stock solutions were stored at -20 °C and diluted freshly in Me2SO for each experiment to attain the desired concentrations. Single-turnover DNA Cleavage—Reaction mixtures containing (per 20 μl) 50 mm Tris-HCl (pH 7.5), 0.3 pmol of CCCTT-containing 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. The extent of covalent complex formation was quantified by scanning the dried gel using a Fujifilm BAS2500 imager. 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 (kcl) were calculated by fitting the normalized data to the equation, 100 -%cleavage(norm) = 100e-kt. Single-turnover Religation by the Suicide Intermediate—Cleavage reaction mixtures (20 μl) containing 0.3 pmol of the 18-mer/30-mer DNA (5′ 32P-labeled on the 18-mer scissile strand and containing a G:C base pair at position -1) and 2 pmol of topoisomerase were incubated at 37 °C for 30 s to form the suicide intermediate. The reaction mixtures were adjusted to 10% Me2SO and 0, 5, 10, or 20 μm nogalamycin and incubated for 5 min at 37 °C. Religation was initiated by the simultaneous addition of NaCl to 0.5 m and a 5′ hydroxyl-terminated 18-mer acceptor strand d(GTTCCGATAGTGACTACA) to a concentration of 15 pmol/22 μl (i.e. a 50-fold molar excess over the input DNA substrate). The reaction was quenched after 15 s by adding an equal volume of buffer containing 2% SDS, 76% formamide, and 20 mm EDTA. The samples were heat-denatured and then analyzed by electrophoresis through a 17% polyacrylamide gel containing 7 m urea in TBE (90 mM Tris/64.6 mM boric acid/2.5 mM EDTA, pH 8.3). Religation of the covalently bound 12-mer strand to the 18-mer acceptor DNA will yield a5′ 32P-labeled 30-mer strand transfer product. The extent of religation is expressed as the percent of the input labeled 18-mer strand recovered as 30-mer. Position-specific BcPh Interference Effects on DNA Transesterification—Oligodeoxynucleotide 18-mers containing a single C-1 S or R BcPh diol epoxide adduct at purine positions of the nonscissile strand sequence 3′-G+5G+4G+3A+2A+1T-1A-2 were synthesized and then annealed to a 5′ 32P-labeled 34-mer scissile strand to form suicide cleavage substrates for vaccinia topoisomerase (Fig. 3). The cleavage transesterification reaction results in covalent attachment of the 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. The reaction of excess topoisomerase with the unmodified control substrate attained an end point at which 90% 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 55% of the end point value. From this datum, we calculated a single-turnover cleavage rate constant (kcl) of 0.27 s-1 (Fig. 3). We found that the S and R BcPh modifications at position +5G and the R BcPh modification at +4G slowed the transesterification rate to 0.13, 0.07, and 0.07 s-1, respectively, without significantly affecting the yield of covalently bound DNA at the reaction end point (61–65%). The kobs for cleavage of these BcPh dG-modified substrates did not increase when the concentration of topoisomerase in the reaction mixture was varied over a 4-fold range (not shown), indicating that the modestly slowed cleavage rates (2- to 4-fold compared with the unmodified control DNA) were not caused by slow noncovalent binding of topoisomerase to these substrates. In contrast, the S BcPh adduct at +4G, the R and S BcPh adducts at +3G, and the S and R adducts at +2A virtually abolished the transesterification reaction. The extents of DNA cleavage after a 24-h reaction in enzyme excess were in the range of 1–4% of the input suicide substrate (Fig. 3). Although the accumulation of the topoisomerase-DNA complex on these modified DNAs was time-dependent, the reaction clearly did not attain a useful end point for the purpose of calculating a cleavage rate constant. The end point did not increase 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 on the +4S, +3R, and +3S BcPh dG and +2S and +2R BcPh dA substrates were nonproductive with respect to transesterification, and that there was not a free equilibrium between productive and nonproductive binding modes (at least not within the 24-h period that the reactions were monitored). The S and R BcPh dA modifications at +1A reduced kcl to 0.0001 and 0.0006 s-1, respectively, without significantly affecting the end point (67–71% cleavage). Thus, +1 BcPh dA adducts elicited rate decrements of 2700-fold and 450-fold for the S and R diastereomers, respectively. Note that the interference effect was greater when the +1 BcPh dA adduct was intercalated on the 3′ side of the modified adenine base facing away from the scissile phosphodiester (Fig. 3). The BcPh dA adducts at the -2 position also displayed a strong orientation bias with respect to rate effects. The S diastereomer, which faces toward the scissile phosphodiester, reduced the cleavage rate constant to 0.003 s-1, a 90-fold decrement compared with the unmodified control DNA, whereas the R diastereomer at -2 had little impact on the rate (0.12 s-1). Neither of the -2 BcPh dA adducts affected the cleavage reaction end point. To address whether BcPh substitutions altered the site of cleavage within the 34-mer scissile strand, the products of the cleavage reactions with unmodified DNA, and the +5R, +5S, +4R, +1R, -2S, and -2R BcPh diol epoxide-modified DNAs were digested with proteinase K in the presence of SDS to remove the covalently linked topoisomerase, and the radiolabeled DNA reaction products were then analyzed by denaturing polyacrylamide gel electrophoresis. Reaction of topoisomerase with the unmodified control substrate results in the appearance of a cluster of radiolabeled species migrating faster than the input 32P-labeled 34-mer strand, which consists of the 12-mer 5′-pCGTGTCGCCCTTp linked to one or more amino acids of the topoisomerase. The same cluster was produced by proteinase K digestion of the covalent complex formed by reaction of topoisomerase with the BcPh dA and BcPh dG substrates (data not shown). Thus, the site of covalent complex formation was unchanged by the BcPh 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. Effects of Nogalamycin on DNA Transesterification by Vaccinia Topoisomerase—The exquisite sensitivity of vaccinia topoisomerase to position-specific intercalators has implications for the discovery of poxvirus-specific topoisomerase inhibitors and/or poisons as candidate antipoxviral drugs. DNA intercalating agents have been widely studied as inhibitors of other DNA topoisomerases; indeed, intercalating drugs that target topoisomerase II are mainstays of cancer chemotherapy. Existing compounds that display some activity against microbial or eukaryotic cellular topoisomerases are a reasonable starting point for screening for inhibition or poisoning of the poxvirus topoisomerase. The anthracycline drug nogalamycin intercalates in duplex DNA with sequence selectivity for 5′ pyrimidine-purine dinucleotides 5′-TG and 5′-CG (45Fox K.R. Waring M.J. Biochemistry. 1986; 25: 4349-4356Crossref PubMed Scopus (72) Google Scholar). Nogalamycin consists of a core 4-ring chromophore with a nogalose sugar and a methyl ester attached to the A ring and a bicyclic aminoglucose sugar fused to the D ring (Fig. 4). X-ray and NMR structures of drug-DNA complexes have shown that the dumbbell-shaped nogalamycin molecule intercalates with its nogalose sugar in the minor groove and its aminoglucose sugar in the major groove (37Smith C.K. Brannigan J.A. Moore M.H. J. Mol. Biol. 1996; 263: 237-258Crossref PubMed Scopus (26) Google Scholar, 38Williams H.E.L. Searle M.S. J. Mol. Biol. 1999; 290: 699-716Crossref PubMed Scopus (45) Google Scholar, 39Zhang X. Patel D.J. Biochemistry. 1990; 29: 9451-9466Crossref P
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