Site-specific DNA Transesterification by Vaccinia Topoisomerase
2003; Elsevier BV; Volume: 278; Issue: 43 Linguagem: Inglês
10.1074/jbc.m308079200
ISSN1083-351X
AutoresLyudmila Yakovleva, Ligeng Tian, Jane M. Sayer, Govind P. Kalena, Heiko Kroth, Donald M. Jerina, Stewart Shuman,
Tópico(s)DNA and Nucleic Acid Chemistry
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 base modifications on the rate and extent of single-turnover DNA transesterification. Chiral trans opened C-10 R and S adducts of benzo[a]pyrene (BP) 7,8-diol 9,10-epoxide were introduced at single N 6-deoxyadenosine (dA) positions within the 3′-G+5G+4G+3A+2A+1T-1A-2 sequence of the nonscissile DNA strand. The R and S BPdA adducts intercalate from the major groove on the 5′ and 3′ sides of the modified base, respectively, and perturb local base stacking. We found that R and S BPdA modifications at +1A reduced the transesterification rate by a factor of 700–1000 without affecting the yield of the covalent topoisomerase-DNA complex. BPdA modifications at +2A reduced the extent of transesterification and elicited rate decrements of 200- and 7000-fold for the S and R diastereomers, respectively. In contrast, BPdA adducts at the -2 position had no effect on the extent of the reaction and relatively little impact on the rate of cleavage. A more subtle probe of major groove contacts entailed substituting each of the purines of the nonscissile strand with its 8-oxo analog. The +3 oxoG modification slowed transesterification 35-fold, whereas other 8-oxo modifications were benign. 8-Oxo substitutions at the -1 position in the scissile strand slowed single-turnover cleavage by a factor of six but had an even greater slowing effect on religation, which resulted in an increase in the cleavage equilibrium constant. 2-Aminopurine at positions +3, +4, or +5 in the nonscissile strand had no effect on transesterification per se but had synergistic effects when combined with 8-oxoA at position -1 in the scissile strand. These findings illuminate the functional interface of vaccinia topoisomerase with the DNA major groove. 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 base modifications on the rate and extent of single-turnover DNA transesterification. Chiral trans opened C-10 R and S adducts of benzo[a]pyrene (BP) 7,8-diol 9,10-epoxide were introduced at single N 6-deoxyadenosine (dA) positions within the 3′-G+5G+4G+3A+2A+1T-1A-2 sequence of the nonscissile DNA strand. The R and S BPdA adducts intercalate from the major groove on the 5′ and 3′ sides of the modified base, respectively, and perturb local base stacking. We found that R and S BPdA modifications at +1A reduced the transesterification rate by a factor of 700–1000 without affecting the yield of the covalent topoisomerase-DNA complex. BPdA modifications at +2A reduced the extent of transesterification and elicited rate decrements of 200- and 7000-fold for the S and R diastereomers, respectively. In contrast, BPdA adducts at the -2 position had no effect on the extent of the reaction and relatively little impact on the rate of cleavage. A more subtle probe of major groove contacts entailed substituting each of the purines of the nonscissile strand with its 8-oxo analog. The +3 oxoG modification slowed transesterification 35-fold, whereas other 8-oxo modifications were benign. 8-Oxo substitutions at the -1 position in the scissile strand slowed single-turnover cleavage by a factor of six but had an even greater slowing effect on religation, which resulted in an increase in the cleavage equilibrium constant. 2-Aminopurine at positions +3, +4, or +5 in the nonscissile strand had no effect on transesterification per se but had synergistic effects when combined with 8-oxoA at position -1 in the scissile strand. These findings illuminate the functional interface of vaccinia topoisomerase with the DNA major groove. Poxvirus DNA topoisomerase is an attractive 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 (1Shuman S. Biochim. Biophys. Acta. 1998; 1400: 321-337Crossref PubMed Scopus (93) Google Scholar, 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. Chem. 1996; 271: 2313-2322Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar, 6Hwang Y. Wang B. Bushman F.D. J. Virol. 1998; 72: 3401-3406Crossref PubMed Google Scholar). Poxvirus topoisomerase, exemplified by the 314-amino acid vaccinia virus enzyme, is a prototype of the type IB DNA topoisomerase family (7Champoux J.J. Annu. Rev. Biochem. 2001; 70: 369-413Crossref PubMed Scopus (2218) Google Scholar, 8Zhang 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: 10608-10613Crossref PubMed Scopus (172) Google Scholar, 9Krogh B.O. Shuman S. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 1853-1858Crossref PubMed Scopus (71) Google Scholar). Type IB enzymes cleave and rejoin one strand of the DNA duplex through a transient DNA-(3′-phosphotyrosyl)-enzyme intermediate. Poxvirus topoisomerases bind and cleave duplex DNA at a pentapyrimidine target sequence, 5′-(T/C)CCTTp↓ (3Shuman S. Prescott J. J. Biol. Chem. 1990; 265: 17826-17836Abstract Full Text PDF PubMed Google Scholar). The T↓ nucleotide (defined as the +1 nucleotide) is linked to Tyr-274 of the vaccinia enzyme. Four conserved amino acid side chains found in all type IB topoisomerases (Arg-130, Lys-167, Arg-223, and His-265 in the vaccinia enzyme) are responsible for catalyzing the attack by tyrosine on the scissile phosphodiester to form the covalent intermediate (10Wittschieben J. Shuman S. Nucleic Acids Res. 1997; 25: 3001-3008Crossref PubMed Scopus (73) Google Scholar, 11Petersen B.Ø. Shuman S. J. Biol. Chem. 1997; 272: 3891-3896Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 12Cheng C. Wang L.K. Sekiguchi J. Shuman S. J. Biol. Chem. 1997; 272: 8263-8269Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). The topoisomerase binds to DNA as a C-shaped protein clamp formed by an 80-amino acid N-terminal domain that contacts the DNA major groove and a 234-amino acid C-terminal catalytic domain that interacts with the scissile phosphate on the minor groove face of the double helix (13Sharma A. Hanai R. Mondragon A. Structure (Lond.). 1994; 2: 767-777Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 14Sekiguchi J. Shuman S. J. Biol. Chem. 1994; 269: 31731-31734Abstract Full Text PDF PubMed Google Scholar, 15Sekiguchi J. Shuman S. EMBO J. 1996; 15: 3448-3457Crossref PubMed Scopus (46) Google Scholar, 16Cheng C. Kussie P. Pavletich N. Shuman S. Cell. 1998; 92: 841-850Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar, 17Cheng C. Shuman S. J. Biol. Chem. 1998; 273: 11589-11595Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar, 18Hwang Y. Park M. Fischer W.H. Burgin A. Bushman F. Virology. 1999; 262: 479-491Crossref PubMed Scopus (11) Google Scholar). The crystal structure of vaccinia topoisomerase in the free state (16Cheng C. Kussie P. Pavletich N. Shuman S. Cell. 1998; 92: 841-850Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar) showed that several of the catalytic residues are either disordered (Arg-130) or out of position to perform transesterification chemistry (Lys-167, Tyr-274), implying that the active site is not pre-assembled. It was proposed that formation of a competent active site is triggered by recognition of the DNA target site. The DNA features that contribute to target site recognition and catalysis have been examined by nuclease footprinting, modification interference, modification protection, analog substitution, and UV cross-linking techniques (14Sekiguchi J. Shuman S. J. Biol. Chem. 1994; 269: 31731-31734Abstract Full Text PDF PubMed Google Scholar, 15Sekiguchi J. Shuman S. EMBO J. 1996; 15: 3448-3457Crossref PubMed Scopus (46) Google Scholar, 19Shuman S. J. Biol. Chem. 1991; 266: 11372-11379Abstract Full Text PDF PubMed Google Scholar, 20Shuman S. Turner J. J. Biol. Chem. 1993; 268: 18943-18950Abstract Full Text PDF PubMed Google Scholar, 21Sekiguchi J. Shuman S. J. Biol. Chem. 1996; 271: 19436-19442Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar, 22Cheng C. Shuman S. Biochemistry. 1999; 38: 16599-16612Crossref PubMed Scopus (18) Google Scholar, 23Hwang Y. Burgin A. Bushman F. J. Biol. Chem. 1999; 274: 9160-9168Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). The results show that vaccinia topoisomerase makes contact with several nucleotide bases and the sugar-phosphate backbone of DNA within and immediately flanking the CCCTT element. Recent studies have focused on delineating the features of the DNA interface that affect the kinetics of transesterification. In particular the effects of modifications at the scissile phosphodiester have illuminated the chemical mechanism of topoisomerase IB and the roles of the individual amino acids in either transition-state stabilization or general acid catalysis (24Krogh B.O. Shuman S. Mol. Cell. 2000; 5: 1035-1041Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, 25Stivers J.T. Jagadeesh G.J. Nawrot B. Stec W.J. Shuman S. Biochemistry. 2000; 39: 5561-5572Crossref PubMed Scopus (59) Google Scholar, 26Krogh 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, 27Krogh B.O. Shuman S. J. Biol. Chem. 2002; 277: 5711-5714Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). Our expectation is that fuller knowledge of the other functionally relevant DNA contacts (i.e. other than to the scissile phosphate) will facilitate the design of small molecule inhibitors that either block transesterification or trap the poxvirus topoisomerase in a catalytically inactive conformation. Interference by polycyclic aromatic hydrocarbon DNA adducts allowed us to map the minor groove interface between vaccinia topoisomerase and its cleavage site (28Tian 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). This approach entailed the introduction of 7,8-diol 9,10-epoxide adducts of benzo[a]pyrene (BP) 1The abbreviations used are: BP, benzo[a]pyrene; HPLC, high performance liquid chromatography; AP, 2-aminopurine deoxyriboside. at the exocyclic N 2-amino group of single deoxyguanosine (dG) positions within the nonscissile 3′-G+5G+4G+3A+2A+1N-1N-2 strand of a suicide cleavage substrate for vaccinia topoisomerase. The trans opened BPdG 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 (29Cosman 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, 30de 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). 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. 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. It was inferred that BPdG inhibition of transesterification is likely caused by steric exclusion of essential constituents of the topoisomerase from the DNA minor groove. These results highlight the merits of testing minor groove sequence-reading polyamides (31White S. Szewcyzk J.W. Turner J.M. Baird E.E. Dervan P.B. Nature. 1998; 391: 468-471Crossref PubMed Scopus (1347) Google Scholar) as antipoxviral agents. Here we extend the use of defined polycyclic aromatic hydrocarbon DNA adducts to probe the effects of position-specific intercalation via the major groove. We introduce 7,8-diol 9,10-epoxide adducts of BP at the exocyclic N 6-amino group of single deoxyadenosine (dA) positions within the nonscissile strand. These BPdA adducts, shown in Fig. 1A, are derived from trans opening with inversion at C-10 of the (-)-(7S,8R,9R,10S) and (+)-(7R,8S,9S,10R) enantiomers of the 7,8-diol 9,10-epoxides in which the benzylic 7-hydroxyl group and the epoxide oxygen are trans. Two-dimensional NMR structures of duplex DNAs containing single BPdA adducts have established that the aromatic ring systems intercalate via the major groove on opposite sides of the modified dA* base depending on their configuration at C-10 (32Volk 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 (52) Google Scholar, 33Pradhan 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, 34Zegar I.S. Chary 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, 35Schurter 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, 36Zegar 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). The pyrenyl moiety of the C-10 R diastereomer intercalates on the 5′ side of the dA*, whereas the S diastereomer intercalates on the 3′ side (e.g. as depicted schematically in Fig. 1B). BPdA-modified DNAs retain B-form helix conformation along the duplex except at the lesion site, where base stacking is perturbed by the intercalated aromatic ring system. The intercalated hydrocarbon wedges apart the two flanking bases of the complementary strand and causes a buckling of the modified base pair and the base pair flanking the hydrocarbon. Fortuitously, an NMR structure of the R diastereomer of BPdA-modified duplex DNA has been determined (32Volk 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 (52) Google Scholar) in the sequence context 5′-d(GCCCTT)-3′/3′-d(CGGGA* A)-5, which corresponds to the target sequence for vaccinia topoisomerase. This structure, shown in Fig. 2, depicts what would be expected for a suicide cleavage substrate containing an R BPdA diastereomer at the +2A position of the nonscissile strand.Fig. 2Structure of a DNA duplex with a trans R BPdA adduct at the +2 position of a target site for topoisomerase cleavage. The NMR structure of a 12-bp duplex DNA containing a single BPdA modification (A*) within the sequence 5′-d(GCCCTT)-3′/3′-d(CGGGA* A)-5′ is from Protein Data Bank entry 1FYY (32Volk 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 (52) Google Scholar). The +3 C:G, +2 T:A*, and +1 T:A base pairs are indicated. The topoisomerase cleavage site is denoted by the arrow. The intercalated hydrocarbon moiety of the BPdA nucleoside is colored yellow and is indicated by the red asterisk (*). The dA nucleoside of BPdA is colored pink.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Vaccinia topoisomerase is amenable to quantitatively informative interference studies, because the pre-steady-state kinetic parameters for transesterification are known (10Wittschieben J. Shuman S. Nucleic Acids Res. 1997; 25: 3001-3008Crossref PubMed Scopus (73) Google Scholar, 25Stivers J.T. Jagadeesh G.J. Nawrot B. Stec W.J. Shuman S. Biochemistry. 2000; 39: 5561-5572Crossref PubMed Scopus (59) Google Scholar), and the enzyme does not relinquish its site specificity in response to a DNA lesion (26Krogh 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, 28Tian 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). We report here that R and S BPdA modifications at the +1A and +2A positions drastically reduced the transesterification rate (by factors of 200–7000), whereas BPdA adducts at the -2 position had little impact on the rate of cleavage. We also use more subtle base modifications (8-oxoguanine, 8-oxoadenine, and 2-aminopurine) to delineate the functional DNA interface of vaccinia topoisomerase with the major groove. Notable findings are that the +3 oxoguanine modification of the nonscissile strand significantly slows the cleavage rate, with no effect on religation, implying that contacts with or near +3G in the major groove are important for triggering assembly of the active site. 8-Oxo substitutions immediately 3′ of the cleavage site on the scissile strand act as mild topoisomerase poisons, i.e. they slow the rate of cleavage modestly but have a greater retarding effect on the rate of religation, which increases the cleavage equilibrium constant. Modified DNA Oligonucleotides—Oligonucleotide 18-mers containing trans opened C-10 S and R BPdA adducts (Fig. 1) were synthesized and purified as described (37Pommier Y. Laco S. Kohlhagen G. Sayer J.M. Kroth H. Jerina D.M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10739-10744Crossref PubMed Scopus (50) Google Scholar, 38Lakshman M.K. Sayer J.M. Jerina D.M. J. Am. Chem. Soc. 1991; 113: 6589-6594Crossref Scopus (58) Google Scholar, 39Lakshman M.K. Sayer J.M. Yagi H. Jerina D.M. J. Org. Chem. 1992; 57: 4585-4590Crossref Scopus (57) Google Scholar) using the appropriately protected 5′-O-dimethoxytrityl-3′-phosphoramidites (BPdA phosphoramidites) as their mixed S/R diastereomers at C-10 (for synthesis of oligonucleotides +2S/+2R BPdA and +1S/+1R BPdA) or from the separated C-10 R and C-10 S BPdA phosphoramidites (for oligonucleotides -2S BPdA and -2R BPdA). The S and R BPdA-adducted oligonucleotides were purified by HPLC. For chromatographic conditions and retention times see Supplemental Table S1. For oligonucleotides that were synthesized as the S/R diastereomeric pairs, absolute configurations were assigned to the separated oligonucleotides on the basis of their long wavelength (330–360 nm) circular dichroism spectra, which exhibited negative bands for the C-10 S diastereomers and positive bands for the C-10 R diastereomers (52Christner D.F. Lakshman M.K. Sayer J.M. Jerina D.M. Dipple A. Biochemistry. 1994; 33: 14297-14305Crossref PubMed Scopus (51) Google Scholar), analogous to those observed for the adducts as the free nucleosides (53Sayer 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). Oligonucleotides containing 7,8-dihydro-8-oxodeoxyguanosine (oxoG) were prepared by automated synthesis using oxoG phosphoramidite purchased from Glen Research (Sterling, VA). Following the supplier's instructions, 0.25 m 2-mecaptoethanol was added to the ammonia solution used to cleave the oligonucleotide from the support and remove base-labile protecting groups. Mercaptoethanol was removed by gel filtration through a NAP-25 column (Amersham Biosciences) equilibrated with 0.1 m ammonium acetate. The 5′-DMT-protected oxoG oligonucleotides were purified by HPLC followed by acid cleavage of the DMT-protecting groups. The deprotected oxoG oligonucleotides were then repurified by HPLC (see Table S2 for chromatographic conditions and retention times). Selected oxoG oligonucleotides were analyzed by electrospray mass spectrometry; the observed masses were within 1 Da of the calculated values. Oligonucleotides containing 7,8-dihydro-8-oxodeoxyadenosine (oxoA) or 2-aminopurine deoxyriboside (AP), synthesized on a 0.2 μmol scale and HPLC-purified, were purchased from Oligos Etc., Inc. (Wilsonville, OR). DNA 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 to 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 (2) 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. Single-turnover DNA Cleavage—Reaction mixtures containing (per 20 μl) 50 mm Tris-HCl (pH 7.5), 0.3 pmol of 5′ 32P-labeled DNA substrate, and 75 or 150 ng (2 or 4 pmol) of vaccinia topoisomerase were incubated at 37 °C. The reactions were initiated by the addition of topoisomerase to prewarmed reaction mixtures. 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 protein. The extent of covalent complex formation was quantified by scanning the dried gel with a Fujifilm BAS-2500 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 (k cl) were calculated by fitting the normalized data to the equation 100 - % cleavage(norm) = 100e -kt. Cleavage Site Specificity—Reaction mixtures (20 μl) containing 50 mm Tris-HCl (pH 7.5), 0.3 pmol of unmodified or BPdA-modified 34-mer/18-mer DNA, and 75 ng of vaccinia topoisomerase were incubated at 37 °C for either 5 min (unmodified, -2S and -2R BPdA), 4 h (+1R and +1S BPdA), 7 h (+2S BPdA), or 10 h (+2R BPdA). 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 electrophoresis through a 17% 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. Single-turnover Religation by the Suicide Intermediate—Cleavage reaction mixtures containing (per 20 μl) 0.3 pmol of the 18-mer/30-mer DNA (containing either an A:T or G:C base pair at position -1) and 75 ng of topoisomerase were incubated at 37 °C to form the suicide intermediate. Religation was initiated by the simultaneous addition of NaCl to 0.5 m concentration and a 5′ hydroxyl-terminated 18-mer acceptor strand, either d(ATTCCGATAGTGACTACA), d(GTTCCGATAGTGACTACA), or d(oxoGTTCCGATAGTGACTACA) to a concentration of 15 pmol/22 μl (i.e. a 50-fold molar excess over the input DNA substrate). Aliquots (22 μl) were withdrawn at 5, 10, 20, 30, 60, 120, and 300 s and quenched immediately in an equal volume of buffer containing 2% SDS, 76% formamide, and 20 mm EDTA. A time 0 sample was withdrawn before the addition of the acceptor strand. The samples were heat-denatured and then analyzed by electrophoresis through a 17% polyacrylamide gel containing 7 m urea in TBE. Religation of the covalently bound 12-mer strand to the 18-mer acceptor DNA will yield a 5′ 32P-labeled 30-mer strand transfer product. The extent of religation (expressed as the percent of the input labeled 18-mer strand recovered as 30-mer) was plotted as a function of reaction time. The data were normalized to the end point values, and k rel was determined by fitting the data to the equation (100 - %Relnorm) = 100e -kt. Equilibrium Cleavage—Three 30-mer CCCTT-containing DNA oligonucleotides, 5′-d(CGTGTCGCCCTT↓ATTACGATAGTGACTACA), 5′-d(CGTGTCGCCCTT↓GTTCCGATAGTGACTACA), and 5′-d(CGTGTCGCCCTT↓oxoGTTCCGATAGTGACTACA), were 5′ 32P-labeled, then gel-purified and annealed to unlabeled complementary 30-mer strands to form duplexes containing 12 bp of DNA 5′ of the topoisomerase cleavage site (↓) and 18 bp 3′ of the cleavage site. Cleavage reaction mixtures containing (per 20 μl) 50 mm Tris-HCl (pH 7.5), 0.3 pmol of 30-mer/30-mer DNA, and 9, 18, 37, 75, 150, or 300 ng of topoisomerase were incubated at 37 °C for 5 min. The reaction was quenched by adding SDS to 0.5%. The samples were then digested for 60 min at 45 °C with 10 μg of proteinase K. The volume was adjusted to 100 μl, and the digests were then extracted with an equal volume of phenol/chloroform. DNA was recovered from the aqueous phase by ethanol precipitation. The pelleted material was resuspended in formamide and analyzed by electrophoreses through a 17% polyacrylamide gel containing 7 m urea in TBE. The cleavage product, a 32P-labeled 12-mer bound to a short peptide, was well resolved from the input 30-mer substrate. The extent of strand cleavage was quantitated by scanning the gel. Position-specific BPdA Interference Effects on DNA Transesterification—Oligodeoxynucleotide 18-mers containing a single C-10 S or R BPdA adduct at positions +2, +1, or -2 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. 1B). 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 85% of the end point value. From this datum, we calculated a single-turnover cleavage rate constant (k cl) of 0.42 s-1 (Fig. 1B). We found that S and R BPdA modifications at position +1A slowed the transesterification rate to 0.0004 and 0.0006 s-1, respectively, without significantly affecting the yield of covalently bound DNA at the reaction end point (81–87%). The k obs for cleavage of the BPdA-modified substrates did not increase when the concentration of topoisomerase in the reaction mixture was increased 2-fold (not shown), suggesting that the slowed cleavage rates were not caused by a defect in initial noncovalent binding of topoisomerase to the substrate. Thus, the +1 BPdA adducts elicited 700–1000-fold rate decrements; there was no significant stereoisomer effect. The S and R BPdA modifications at +2A reduced k cl to 0.0018 and 0.00006 s-1, respectively. The extents of transesterification were also reduced, so that only 18–22% of the input DNA became covalently bound to the enzyme. 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 on the +2 BPdA 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 time frame that the reactions were monitored). Thus, +2 BPdA adducts elicited rate decrements of 200- and 7000-fold for the S and R diastereomers, respectively, with a significant stereospecific interference effect (S/R = 30). Note that the interference effect was greater when the +2 BPdA adduct was intercalated on the 5′ side facing toward the scissile phosphodiester (Fig. 1B). BPdA adducts at the -2 position had no effect on the extent of the reaction and relatively little impact on the rate of cleavage compared with the BPdA adducts within the 3-GGGAA recognition motif. The cleavage rate constants were 0.05 and 0.23 s-1 for the -2 S and R BPdA diastereomers, respectively. The modest stereoselective effect at the -2A position (R/S = 4) is such that interference occurred only when the BPdA adduct was intercalated on the 3′ side facing toward the scissile phosphodiester. Taken together, the BPdA interference effects define the -1 base pair as the “downstream” margin of the functional interface between DNA and vaccinia topoisomerase that can be affected significantly by polycyclic aromatic hydrocarbon intercalation (Fig. 1B). BPdA Adducts Do Not Alter the Cleavage Site of Vaccinia Topoisomerase—To address whether the +2, +1, or -2 BPdA substitu
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