Title: Different Effects on Human Topoisomerase I by Minor Groove and Intercalated Deoxyguanosine Adducts Derived from Two Polycyclic Aromatic Hydrocarbon Diol Epoxides at or Near a Normal Cleavage Site
Abstract: Topoisomerase I (top1) relieves supercoiling in DNA by forming transient covalent cleavage complexes. These cleavage complexes can accumulate in the presence of damaged DNA or anticancer drugs that either intercalate or lie in the minor groove. Recently we reported that covalent diol epoxide (DE) adducts of benzo[a]pyrene (BaP) at the exocyclic amino group of G(+1) block cleavage at a preferred cleavage site (∼CTT—G(+1)G(+2)A∼) and cause accumulation of cleavage products at remote sites. In the present study, we have found that the 10S G(+2) adduct of BaP DE, which lies toward the scissile bond in the minor groove, blocks normal cleavage, whereas the 10R isomer, which orients away from this bond, allows normal cleavage but blocks religation. In contrast to BaP, the pair of benzo[c] phenanthrene (BcPh) DE adducts at G(+2), which intercalate from the minor groove either between G(+1)/G(+2) or between G(+2)/A, allow normal cleavage but block religation. Both intercalated BcPh DE adducts at G(+1) suppress normal cleavage, as do both groove bound BaP DE adducts at this position. These studies demonstrate that these DE adducts provide a novel set of tools to study DNA topoisomerases and emphasize the importance of contacts between the minor groove and top1′s catalytic site. Topoisomerase I (top1) relieves supercoiling in DNA by forming transient covalent cleavage complexes. These cleavage complexes can accumulate in the presence of damaged DNA or anticancer drugs that either intercalate or lie in the minor groove. Recently we reported that covalent diol epoxide (DE) adducts of benzo[a]pyrene (BaP) at the exocyclic amino group of G(+1) block cleavage at a preferred cleavage site (∼CTT—G(+1)G(+2)A∼) and cause accumulation of cleavage products at remote sites. In the present study, we have found that the 10S G(+2) adduct of BaP DE, which lies toward the scissile bond in the minor groove, blocks normal cleavage, whereas the 10R isomer, which orients away from this bond, allows normal cleavage but blocks religation. In contrast to BaP, the pair of benzo[c] phenanthrene (BcPh) DE adducts at G(+2), which intercalate from the minor groove either between G(+1)/G(+2) or between G(+2)/A, allow normal cleavage but block religation. Both intercalated BcPh DE adducts at G(+1) suppress normal cleavage, as do both groove bound BaP DE adducts at this position. These studies demonstrate that these DE adducts provide a novel set of tools to study DNA topoisomerases and emphasize the importance of contacts between the minor groove and top1′s catalytic site. Topoisomerase I (top1) 1The abbreviations used are: top1human DNA topoisomerase IBaPbenzo[a]pyreneBcPhbenzo[c]phenanthrene, DE, diol epoxideDE-2diol epoxide isomer in which the benzylic 7-hydroxyl group and the epoxide oxygen are transis a nuclear enzyme that is essential for the regulation of DNA topology, transcription, replication, and probably DNA recombinations. The enzyme's catalytic mechanism is well characterized. Nucleophilic attack on the DNA backbone by a catalytically essential tyrosine (Tyr723) of the enzyme breaks one of the DNA strands by forming a covalent phosphotyrosyl bond with the 3′-end of the DNA. This break provides a swivel point for DNA relaxation. Once supercoiling has been relieved, the 5′-hydroxyl of the cleaved DNA acts as a nucleophile toward the phosphotyrosyl bond and restores the DNA backbone, thus releasing the enzyme for further catalytic cycles (reviewed in Refs. 1.Wang J.C. Annu. Rev. Biochem. 1996; 65: 635-692Crossref PubMed Scopus (2086) Google Scholar and 2.Champoux J.J. Annu. Rev. Biochem. 2001; 70: 369-413Crossref PubMed Scopus (2218) Google Scholar). human DNA topoisomerase I benzo[a]pyrene benzo[c]phenanthrene, DE, diol epoxide diol epoxide isomer in which the benzylic 7-hydroxyl group and the epoxide oxygen are trans Top1 can be trapped in a covalent complex with DNA by a variety of lesions including abasic sites, mismatches, oxidative lesions, base methylation (O6-methyl guanine, 5-methylcytosine), base alkylations (vinyl adducts), and DNA strand breaks (reviewed in Ref. 3.Pourquier P. Pommier Y. Adv. Cancer Res. 2001; 80: 189-216Crossref PubMed Google Scholar). Top1 is an important target of several anticancer drugs. Camptothecin and its derivatives are believed to kill cancer cells by specifically trapping top1 and preventing the religation of the covalent top1 cleavage complexes. Several other DNA intercalating and minor groove-binding drugs have also been reported to trap these top1 cleavage complexes (reviewed in Ref. 4.Pommier Y. Pourquier P. Fan Y. Strumberg D. Biochim. Biophys. Acta. 1998; 1400: 83-105Crossref PubMed Scopus (549) Google Scholar). However, little is known about how these noncovalently bound drugs block religation and so prevent release of functional top1. Such structural information should prove highly valuable in designing new top1 inhibitors. We have recently utilized covalent DNA adducts (Fig. 1A) derived from trans ring opening of benzo[a]pyrene 7,8-diol-9,10-epoxides (BaP DE, two enantiomers of the diastereomer in which the benzylic 7-hydroxyl group and the epoxide oxygen are trans) by the exocyclic amino groups of the purine bases as probes of the catalytic activity of top1 (5.Pommier Y. Kohlhagen G. Pourquier P. Sayer J.M. Kroth H. Jerina D.M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 2040-2045Crossref PubMed Scopus (65) Google Scholar, 6.Pommier Y. Kohlhagen G. Laco G.S. Sayer J.M. Kroth H. Jerina D.M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10739-10744Crossref PubMed Scopus (50) Google Scholar). These adducts were introduced into a 22-mer DNA sequence, which contains a single high affinity (7.Bonven B.J. Gocke E. Westergaard O. Cell. 1985; 41: 541-551Abstract Full Text PDF PubMed Scopus (216) Google Scholar) top1 cleavage site (between T andX(+1) in ∼CTT-X(+1)G(+2)A∼), where X is either G or A, Fig. 1B) and is derived from the oligonucleotide used for determination of the crystal structure of human top1 bound to this DNA substrate (8.Redinbo M.R. Champoux J.J. Hol W.G. Biochemistry. 2000; 39: 6832-6840Crossref PubMed Scopus (133) Google Scholar). These adducts are extremely powerful probes of enzyme-DNA contacts, since their solution conformations are known from two-dimensional NMR studies. For the BaP DE adducts at the exocyclic N2-amino group of dG (X = G, Fig. 1B), the trans 10Sadduct (S-absolute configuration at the point of attachment of the amine to the hydrocarbon) lies in the minor groove with the aromatic portion pointing toward the 5′-end of the adducted strand, as shown in Fig. 2A (9.Cosman 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). The 10R adduct lies toward the 3′-end (Fig. 2, A andB) (10.de 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 previously found (5.Pommier Y. Kohlhagen G. Pourquier P. Sayer J.M. Kroth H. Jerina D.M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 2040-2045Crossref PubMed Scopus (65) Google Scholar) that both of these diastereomeric BaP DE trans adducts at G(+1) suppress normal top1 cleavage and induce new cleavages at positions 3–6 bases away from the adducted G. In marked contrast, when X = A the trans-opened BaP DE adducts at N6 are intercalated (10S toward the 3′-end) from the major groove (Fig. 2C) (11.Yeh H.J. Sayer J.M. Liu X. Altieri A.S. Byrd R.A. Lakshman M.K. Yagi H. Schurter E.J. Gorenstein D.G. Jerina D.M. Biochemistry. 1995; 34: 13570-13581Crossref PubMed Scopus (79) Google Scholar, 12.Volk D.E. Rice J.S. Luxon B.A. Yeh H.J. Liang C. Xie G. Sayer J.M. Jerina D.M. Gorenstein D.G. Biochemistry. 2000; 39: 14040-14053Crossref PubMed Scopus (52) Google Scholar, 13.Zegar 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). Quite interestingly, these adducts initially appear invisible to the enzyme in that normal cleavage occurs but religation is blocked (6.Pommier Y. Kohlhagen G. Laco G.S. Sayer J.M. Kroth H. Jerina D.M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10739-10744Crossref PubMed Scopus (50) Google Scholar).Figure 2Schematic representation of the effects of BaP and BcPh adducts on top1-mediated DNA cleavage. The DNA is shown in gray with the base pairs shown vertically and numbered as in Fig. 1. Adducts lying in the minor groove are shown ashorizontal rectangles and intercalated adducts asvertical rectangles. Covalent attachment sites are represented by the closed circles. A shows the adducts that prevent top1-mediated DNA cleavage at the normal site and induce cleavage at a 15-mer site between positions −2 and −3 (not shown). B and C show the adducts that permit normal cleavage and trap top1 at its high affinity cleavage site between positions −1 and +1. The horizontal line separates the dG adducts lying in or intercalated from the minor groove (A and B) from the dA adducts intercalated from the major groove (C).View Large Image Figure ViewerDownload Hi-res image Download (PPT) The present study was designed to investigate the effects of additional unique structural motifs on the activity of top1. Here we examine the consequences of placing the minor groove-bound trans 10R and 10S BaP dG adducts at the +2 position downstream from the scissile bond. We also examine the effects of DE dG adducts (Fig. 1A) derived from trans opening of benzo[c]phenanthrene 3,4-diol 1,2-epoxide (BcPh DE) at positions +1 and +2. These BcPh adducts differ dramatically from the BaP adducts in that the aromatic portion of the hydrocarbon is intercalated from the minor groove into the DNA helix (14.Lin 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), as shown in Fig. 2 (trans-S adduct intercalated toward the 5′-end of the adducted strand, panel C) (14.Lin 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), and were consequently expected to have a very different “footprint” from the groove-bound BaP dG adducts in terms of their interactions with the enzyme at the cleavage site. Human recombinant top1 was purified from Baculovirus-infected cells as described previously (15.Pourquier P. Ueng L.-M. Fertala J. Wang D. Park H.-J. Essigman J.M. Bjornsti M.-A. Pommier Y. J. Biol. Chem. 1999; 274: 8516-8523Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). Terminal deoxynucleotidyl transferase was purchased from Invitrogen. [α-32P]Cordycepin 5′-triphosphate was purchased from PerkinElmer Life Sciences; Polyacrylamide from Bio-Rad. Camptothecin was provided by Drs. M. C. Wani and M. E. Wall (Research Triangle Institute, Research Triangle Park, NC). Ten-mm aliquots of camptothecin in dimethyl sulfoxide (Me2SO) were stored at −20 °C and then thawed and diluted in reaction buffer just before use. The adducted oligonucleotides described in this and a previous study (5.Pommier Y. Kohlhagen G. Pourquier P. Sayer J.M. Kroth H. Jerina D.M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 2040-2045Crossref PubMed Scopus (65) Google Scholar) are 22-mers, 5′-[AAA AAG ACT TG(+1)G(+2) AAA AAT TTT T]-3′ with modified deoxyguanosine residues at G(+1) or G(+2) corresponding to the adducts formed upon trans opening of (+)- or (−)-BaP DE at C10 and of (+)- or (−)-BcPh DE at C1 (Fig. 1A). The oligonucleotides containing BaP adducts at G(+1) and their effects on top1 were described by us in previous work (5.Pommier Y. Kohlhagen G. Pourquier P. Sayer J.M. Kroth H. Jerina D.M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 2040-2045Crossref PubMed Scopus (65) Google Scholar). The 22-mers modified at G(+2) with the trans-opened BaP and BcPh DE adducts, as well as shorter marker oligonucleotides corresponding to their potential cleavage products generated by top1, were synthesized and characterized in a separate study (16.Kroth H. Yagi H. Sayer J.M. Kumar S. Jerina D.M. Chem. Res. Toxicol. 2001; 14: 708-719Crossref PubMed Scopus (28) Google Scholar). The two diastereomeric 22-mers containing trans-opened BcPh DE-2 adducts at G(+1) were prepared by the methods described therein. High performance liquid chromatography retention times, configurational assignments, and major CD bands for these two new oligonucleotides are given in Table I. The CD spectra of these 22-mers containing BcPh DE-2 adducts at G(+1) are almost identical to the spectra for the corresponding oligonucleotides with these adducts at G(+2) (16.Kroth H. Yagi H. Sayer J.M. Kumar S. Jerina D.M. Chem. Res. Toxicol. 2001; 14: 708-719Crossref PubMed Scopus (28) Google Scholar). Each of the new BcPh DE-2 adducted 22-mers (Table I) gave a mass of 7070 daltons (calculated for C237H286N87O129P217068 daltons).Table IHPLC retention times and absolute configurations of oligonucleotides, 5′-AAA AAG ACT TG*G AAA AAT TTT T-3′ (where the modified base (G(+1)) is indicated as G*), containing N2-deoxyguanosine adducts corresponding to trans opening of BcPh DE-2Parent diol epoxideAbsolute configuration at C11-aAssignments are based on CD spectra of the individual N2-dG adducts (36, 37) after enzymatic digestion (38), as well as on comparison of the CD spectra for the present oligonucleotides with those of the analogous oligonucleotides with the trans opened R and Sadducts of BcPh DE-2 at G(+2) (16).Retention time1-bOn a Higgins DNA column (4.6 × 100 mm, 5 μm) at 40 °C eluted at 1.5 ml/min with a gradient of acetonitrile in 0.1 m (NH4)2CO3, pH 7.5, that increased the acetonitrile composition from 5 to 11% over 20 min.CD spectrum (intensity)1-cIn MeOH, normalized to 1.0 absorbance unit at 260 nm.minnm(+)-(1S,2R,3R,4S)-DE-2R17.9254 (−12.8); 279 (+12.3)(−)-(1R,2S,3S,4R)-DE-2S18.9246 (−10.3); 262 (+15.9)1-a Assignments are based on CD spectra of the individual N2-dG adducts (36.Cheng S.C. Hilton B.D. Roman J.M. Dipple A. Chem. Res. Toxicol. 1989; 2: 334-340Crossref PubMed Scopus (374) Google Scholar, 37.Moore P.D. Koreeda M. Wislocki P.G. Levin W. Conney A.H. Yagi H. Jerina D.M. Jerina D.M. Drug Metabolism Concepts. American Chemical Society, Washington, D. C.1977: 127-154Google Scholar) after enzymatic digestion (38.Sayer 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), as well as on comparison of the CD spectra for the present oligonucleotides with those of the analogous oligonucleotides with the trans opened R and Sadducts of BcPh DE-2 at G(+2) (16.Kroth H. Yagi H. Sayer J.M. Kumar S. Jerina D.M. Chem. Res. Toxicol. 2001; 14: 708-719Crossref PubMed Scopus (28) Google Scholar).1-b On a Higgins DNA column (4.6 × 100 mm, 5 μm) at 40 °C eluted at 1.5 ml/min with a gradient of acetonitrile in 0.1 m (NH4)2CO3, pH 7.5, that increased the acetonitrile composition from 5 to 11% over 20 min.1-c In MeOH, normalized to 1.0 absorbance unit at 260 nm. Open table in a new tab Single-stranded oligonucleotides were 3′-end-labeled with α-32P-labeled cordycepin, as described previously (15.Pourquier P. Ueng L.-M. Fertala J. Wang D. Park H.-J. Essigman J.M. Bjornsti M.-A. Pommier Y. J. Biol. Chem. 1999; 274: 8516-8523Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar, 17.Pommier Y. Jenkins J. Kohlhagen G. Leteurtre F. Mutat. Res. 1995; 337: 135-145Crossref PubMed Scopus (53) Google Scholar). Annealing to the complementary strand was performed in 1× annealing buffer (10 mmTris-HCl, pH 7.8, 100 mm NaCl, 1 mm EDTA) by heating the reaction mixture to 95 °C and overnight cooling to room temperature. DNA substrates (∼50 fmol/reaction) were incubated with 5 ng of top1 with or without camptothecin (1 μm) for the indicated times at 25 °C in 10 μl of reaction buffer (10 mm Tris-HCl, pH 7.5, 50 mm KCl, 5 mm MgCl2, 0.1 mm EDTA, 15 μg/ml bovine serum albumin, final concentrations). Reactions were stopped by adding sodium dodecyl sulfate (SDS) (final concentration 0.5%). For reversal experiments, the SDS stop was preceded by the addition of NaCl to a final concentration of 0.35 m followed by incubation for 30 min at 25 °C. For the heat reversal experiments, the SDS stop was preceded by heating the samples to 65 °C. Sequencing of oligonucleotides was performed by using the Maxam-Gilbert purine sequencing protocol (18.Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Before loading of the electrophoresis, 3.3 volumes of Maxam-Gilbert loading buffer (98% formamide, 0.01m EDTA, 10 mm NaOH, 1 mg/ml xylene cyanol, and 1 mg/ml bromphenol blue) were added to reaction mixtures. Sixteen percent denaturing polyacrylamide gels (7 m urea) were run at 40 V/cm at 50 °C for 2–3 h and dried on Whatman No. 3MM paper sheets. Imaging and quantitations were performed using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA). Fig. 3A shows our previously reported (5.Pommier Y. Kohlhagen G. Pourquier P. Sayer J.M. Kroth H. Jerina D.M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 2040-2045Crossref PubMed Scopus (65) Google Scholar) results (repeated for comparison purposes) on top1-mediated DNA cleavage of the upper strand of the oligonucleotide containing BaP dG adducts at position G(+1), in comparison with a new experiment utilizing the same adducts at G(+2). In the absence of adduct, top1 did not produce detectable cleavage (lane 2, control), and camptothecin was required for observing the expected 13-mer cleavage product (lane 3, control). When BaP adducts were present at the G(+1) position, the formation of the 13-mer cleavage product was suppressed, and top1 cleavage was shifted in such a way that a 15-mer product was observed independently of camptothecin, and an 18-mer product was observed in the presence of camptothecin. The trans-R adduct had a stronger effect than the trans-S adduct (see Fig. 3B for quantitation). Different effects were observed when the BaP adduct was at the G(+2) position (right side of Fig. 3A). The trans-R adduct induced the accumulation of a large amount of cleavage product, which, in contrast to the control, did not require the presence of camptothecin. In the presence of camptothecin, cleavage at this site was almost irreversible in the presence of 0.35m NaCl. The cleavage band migrated above the 13-mer cleavage product observed for the control oligonucleotide and below the 15-mer cleavage product observed for the oligonucleotides containing BaP G(+1) adducts. Because the migration of the oligonucleotides containing BaP adducts was dependent on the presence and type of BaP adducts (see 23-mer bands), short BaP dG adduct-containing oligonucleotides of known length (14-, 15-, and 16-mers, including the labeled cordycepin residue) were used as markers to establish the length of this cleavage product. The cleavage product derived from the BaP trans-R G(+2)-adducted oligonucleotide migrated in a position consistent with its being a 13-mer. Thus, the presence of a BaP trans-R dG adduct at the +2 position increased cleavage at the normal top1 high affinity site. In marked contrast to the trans-R G(+2) adduct, a BaP trans-S G(+2) adduct suppressed top1-mediated DNA cleavage at the 13-mer site and induced cleavage upstream, resulting in a 15-mer cleavage product (see sequence at the bottom of Fig. 3A) that was independent of camptothecin. Further reversal experiments were carried out with the oligonucleotide containing the BaP trans-R dG(+2) adduct. Heat reversal is commonly used to determine the stability of top1 cleavage complexes (19.Hsiang Y.H. Liu L.F. Cancer Res. 1988; 48: 1722-1726PubMed Google Scholar). Fig. 4 shows that a significant fraction of the top1 cleavage product (13-mer) from the BaP trans-R G(+2) adduct resisted heat treatment. This result is consistent with the stabilization of this cleavage product to salt reversal (see Fig. 3A and results above) (5.Pommier Y. Kohlhagen G. Pourquier P. Sayer J.M. Kroth H. Jerina D.M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 2040-2045Crossref PubMed Scopus (65) Google Scholar). Together, these observations suggest that the enhancement of top1 cleavage by the BaP trans-R G(+2) adduct is due at least in part to an inhibition of top1-mediated DNA religation. We previously reported (6.Pommier Y. Kohlhagen G. Laco G.S. Sayer J.M. Kroth H. Jerina D.M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10739-10744Crossref PubMed Scopus (50) Google Scholar) that a BaP dG adduct at the +1 position of the oligonucleotide induces the formation of top1 cleavage sites on the lower strand, complementary to the adducted strand. We compared these results with the effects of the BaP dG(+2) adducts. In the unmodified oligonucleotide, cleavage of the lower strand by top1 is minimal (Fig. 5, lane 3,Control). The presence of BaP dG adducts (both Sand R) at positions +1 or +2 induced top1 cleavage of the lower strand upstream from the normal cleavage site, resulting in 8-, 16-, and 17-mer cleavage products (Fig. 5). The 16-mer was observed only in the presence of camptothecin, whereas the 8-mer was observed in the absence of camptothecin. The 8-mer presumably represent “suicide” products resulting from diffusion away of the short, T-rich 8-mer fragment so that the cleavage complex cannot religate. Thus, the presence of BaP dG adducts either at the +1 or +2 position results in the formation of top1 cleavage products at positions flanking the adducts on both strands of the modified oligonucleotide. The adducts at G(+2) appeared to be somewhat more effective in promoting 16-mer formation than were the adducts at G(+1). We then tested the effects of the intercalated BcPh dG adducts at position +1 of the same oligonucleotide (Fig. 6). As in the case of the BaP G(+1) adducts, top1-mediated DNA cleavage was: 1) completely suppressed at the high affinity 13-mer site, 2) induced upstream in the absence of camptothecin (15-mer cleavage product), and 3) induced at the 18-mer site in the presence of camptothecin. Cleavage at both of these sites was reversible (Fig. 6). Next we looked at the effects of BcPh dG adducts at G(+2) (Fig. 7). Both the trans-S- and the trans-R-adducted oligonucleotides trapped top1 at the high affinity (13-mer) site independently of camptothecin. However, the oligonucleotide containing the trans-S adduct showed little cleavage at any site, even in the presence of camptothecin. Cleavage of the trans-R adducted oligonucleotide was markedly more efficient. Reversal experiments in the presence of 0.35 mNaCl showed that the cleavage observed with the BcPh trans-RdG(+2) adduct reversed more slowly than cleavage of the corresponding unmodified oligonucleotide observed in the presence of camptothecin (Fig. 8). Similarly, the cleavage product from the BcPh trans-R-adducted oligonucleotide failed to undergo religation upon treatment at 65 °C. These results demonstrate that, as in the case of the BaP adduct which lies in the minor groove, a BcPh adduct intercalated in the DNA from the minor groove one base downstream from the high affinity top1 cleavage site traps the cleavage complex by inhibiting its religation and does so more effectively than does camptothecin.Figure 7Top1 cleavage complexes observed in the presence of BcPh adducts at the G(+2) position. The oligodeoxynucleotide was labeled on the upper strand with α-32P-labeled cordycepin (A).control, unmodified DNA, unmodified oligodeoxynucleotide;BcP(S), trans-S-BcPh adduct at G(+2);BcP(R), trans-R-BcPh adduct at G(+2). Lanes A, DNA alone; lanes B, + top1; lanes C, + top1 + camptothecin. Reactions were for 15 min at 21 °C.13, 14, and 15 correspond to 13-, 14-, and 15-mer oligodeoxynucleotides labeled at their 3′-end with α-32P-labeled cordycepin and containing a trans-S- or trans-R-BcPh adduct at their G(+2) position.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 8Trapping of top1 cleavage complexes observed in the presence of the trans-R-BcPh G(+2) adduct.control, unmodified DNA. Lane 1, DNA, no treatment; lanes 2 and 9–13, DNA + top1 in the absence of camptothecin; lanes 3–7, DNA + top1 + camptothecin. Reactions were for 15 min at 21 °C in lanes 2, 3, and 9. Reversibility of the top1 cleavage complexes was studied after addition of 0.35 mNaCl (lanes 4–6 and 10–13) for 3, 10, and 30 min as indicated above the lanes or after heating the samples at 65 °C for 30 min.View Large Image Figure ViewerDownload Hi-res image Download (PPT) When incorporated near the scissile bond of a DNA substrate, covalent adducts derived from trans ring opening of BaP and BcPh DE-2 by the exocyclic amino groups of purines have remarkable effects on the nicking-closing activity of human top1 (summarized in Fig. 2). Depending on their position in the DNA sequence relative to the top1 cleavage site and their orientation in the DNA, these adducts either: 1) prevent top1 from cleaving its DNA substrate at the normal position while enhancing cleavage at other, remote sites (Fig. 2A) or 2) permit cleavage at the normal site but inhibit religation, resulting in accumulation (trapping) of the top1-DNA cleavage complex, even in the absence of camptothecin (Fig. 2, B and C). Structures for all these adducts are known from solution NMR studies (9.Cosman 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, 10.de 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, 11.Yeh H.J. Sayer J.M. Liu X. Altieri A.S. Byrd R.A. Lakshman M.K. Yagi H. Schurter E.J. Gorenstein D.G. Jerina D.M. Biochemistry. 1995; 34: 13570-13581Crossref PubMed Scopus (79) Google Scholar, 12.Volk D.E. Rice J.S. Luxon B.A. Yeh H.J. Liang C. Xie G. Sayer J.M. Jerina D.M. Gorenstein D.G. Biochemistry. 2000; 39: 14040-14053Crossref PubMed Scopus (52) Google Scholar, 13.Zegar 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, 14.Lin 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, 20.Cosman 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, 21.Cosman M. Fiala R. Hingerty B.E. Laryea A. Lee H. Harvey R.G. Amin S. Geacintov N.E. Broyde S. Patel D. Biochemistry. 1993; 32: 12488-12497Crossref PubMed Scopus (89) Google Scholar). As shown in Fig. 2, the trans-opened adducts from BaP and BcPh DE-2 may either lie in the minor groove (BaP DE dG adducts), intercalate from the minor groove (BcPh DE dG adducts), or intercalate from the major groove (dA adducts from both hydrocarbons). Their orientation relative to the DNA strand depends on the configuration at the site of attachment of the hydrocarbon to the base as shown in Fig. 2. In the case of trans-opened dG adducts, both minor groove-bound BaP S-adducts and intercalated BcPhS-adducts (9.Cosman 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, 14.Lin 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) orient toward the 5′-end of the adducted strand, whereas the corresponding R-adducts (10.de 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, 14.Lin 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) orient toward the 3′-end. For trans-opened dA adducts from both hydrocarbons, the orientations relative to the adducted strand are opposite from those of the corresponding dG adducts, such that S-dA adducts intercalate toward the 3′-end (11.Yeh H.J. Sayer J.M. Liu X. Altieri A.S. Byrd R.A. Lakshman M.K. Yagi H. Schurter E.J. Gorenstein D.G. Jerina D.M. Biochemistry. 1995; 34: 13570-13581Crossref PubMed Scopus (79) Google Scholar, 20.Cosman 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, 22.Pradhan P. Tirumala S. Liu X. Sayer J.M. Jerina D.M. Yeh H.J. Biochemistry. 2001; 40: 5870-5881Crossref PubMed Scopus (46) Google Scholar) and R-adducts toward the 5′-end (12.Volk D.E. Rice J.S. Luxon B.A. Yeh H.J. Liang C. Xie G. Sayer J.M. Jerina D.M. Gorenstein D.G. Biochemistry. 200