Nucleic Acids Research, Vol. 18, No. 8 1983

Nucleic Acids Research, Vol. 18, No. 8

Distamycin inhibition of topoisomerase I-DNA interaction: mechanistic analysis

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Uffe H.Mortensen, Tinna Stevnsner+, Susanne Krogh, Kjeld Olesen, Ole Westergaard and Bjarne J.Bonven* Department of Molecular Biology and Plant Physiology, University of Aarhus, DK-8000 Arhus C, Denmark Received January 24, 1990; Accepted March 16, 1990

ABSTRACT Inhibition of eukaryotic DNA topoisomerase I by the minor groove binding ligand, distamycin A, was investigated. Low concentrations of the ligand selectively prevented catalytic action at a high affinity topoisomerase I binding sequence. A restriction enzyme protection assay indicated that the catalytic cycle was blocked at the binding step. Distamycin binding sites on DNA were localized by hydroxyl radical footprinting. A strongly preferred site mapped to a homopolymeric (dA) * (dT)-tract partially included in the essential topoisomerase I binding region. Mutational elimination of the stable helix curvature associated with this ligand binding site demonstrated that (i) the intrinsic bend was inessential for efficient binding of topoisomerase 1, and (ii) distamycin inhibition did not occur by deformation of a stable bend. Alternative modes of inhibition are discussed. INTRODUCTION The pyrrole amide antibiotics of Streptomyces are A-T directed, non-intercalative DNA binders (reviewed in 1). Their mode of DNA binding has been studied by various footprinting (2,3) and NMR (4,5) techniques, and two compounds, netropsin and distamycin, have been cocrystallized with defined DNA oligonucleotides (6,7). These ligands bind within the minor groove of B-DNA, replacing the spine of hydration. Amide groups in the oligopeptide backbone of the antibiotics form bifurcated hydrogen bonds to N3 of adenine or o2 of thymine in adjacent bases on opposite strands (6,7). Further stabilization is provided by van der Waal's interactions between the pyrrole rings and the walls of the minor groove as well as electrostatic interactions between the terminal cationic group(s) and phosphates of the DNA backbone. The A-T preference is believed to arise from (i) steric hindrance in the minor groove by the exocyclic C2 amino group of guanine, and (ii) optimization of van der Waal's interactions in the distinctively narrow minor groove associated with A * T pairs. Distamycin induces local structural distortions of DNA, such as elimination of stable helix curvature

(8). This ligand is known to inhibit a wide range of biological (reviewed in 1) and interfere with various protein-DNA interactions in vitro (1, 9, 10 and 11), including topoisomerase I and II catalysis (12, 13). Eukaryotic topoisomerase I interacts with DNA in a non specific, but sequence dependent manner. The minimal binding site requirements defined by the 4-5 base pair degenerate consensus sequence (14-18) give no indication of a specific sequence code for recognition. We have recently observed a conserved structural motif associated with the core (consensus) part of topoisomerase I sites, indicating that local aspects of helix geometry set the minimal requirements for site selection (19). The binding efficiency at sites fulfilling the minimal requirements is modulated by flanking sequences. To shed light on these context effects, we have studied a high affinity site, in which the core part is embedded between two A-tracts (oligo d(A) * d(T)-tracts) (20-24, 26). Footprinting data showed that both A-tracts are held in close proximity to the enzyme, suggesting that both may be involved in determination of binding affinity. However, the two regions are functionally distinct. Thus, the A-tract located 5' to the core is partially included in the region essential for topoisomerase I action, whereas no essential contacts occur in the A-tract located to the 3'-side (24). In this report, we extend our studies on these binding site sub-domains using distamycin as a molecular probe. processes

EXPERIMENTAL PROCEDURES Enzyme Purification and Topoisomerase I Reactions Topoisomerase I was purified from exponentially grown cultures of Tetrahymena thermophila, strain B1868-VH, using a modification of the method described by Ishii et al. (25,26). The electrophoretically pure preparation had a specific activity of 5 x 106 units (as defined in ref. 26) per mg protein. Topoisomerase I mediated DNA relaxation of supercoiled DNA substrates was performed in 10 mM Tris-HCl, pH = 7.5, 150 mM NaCl, 3 mM CaCl2, 1 mM MgCl2, 0.1 mM EDTA, 20 tig/ml bovine serum albumin at 30°C. Reaction products were analyzed by electrophoresis in 1 % (w/v) agarose gels and

To whom correspondence should be addressed + Present address: National Cancer Institute, National Institute of Health, Bethesda, MD 20892, USA

*

1984 Nucleic Acids Research, Vol. 18, No. 8

visualized by ethidium bromide staining and UV transillumination. Sodium dodecyl sulfate (SDS)-mediated topoisomerase cleavage of end-labeled DNA fragments followed the procedure detailed in (26). In distamycin A containing reactions, DNA was incubated with the ligand for 15 min at 30°C in topoisomerase I reaction buffer prior to addition of enzyme.

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Plasmids The plasmids pNC1, PNC5A, pUCNC 14, and pUCNC15 or fragments thereof were used as substrates in topoisomerase I reactions. pNC 1 was constructed by inserting a synthetic topoisomerase I recognition sequence between the EcoRV- and SphI-sites of pBR322 as detailed in (26). The plasmid pNC5 is identical to pNC 1 except for a point mutation in the recognition sequence rendering it non-functional (23,26). pNC5A was derived from pNC5 by deletion of vector sequences located between the NlaI sites corresponding to coordinates 769 and 1283 on the original pBR322 map. In pUCNC 14, the oligonucleotide: 5'-AAAAAAGACTTAGAAAAATTTTTAAAG-3' 3'-GTACTTTTTTCTGAATCTTTTTAAAAATTTC-5' was inserted between the SphI- and HincII-sites of pUC 19. pUCNC 15 contains the oligonucleotide: 5'-TATATAGACTTAGAAAAATTTTTAAAG-3' 3'-GTACATATATCTGAATCTTTTTAAAAATTTC-5'

inserted between the same sites.

Hydroxyl Radical Footprinting Generation of hydroxyl radical by reacting Fe(11) * EDTA with H202 followed the principles in (3,27). For analysis of the scissile strand, the approximately 250 bp EcoRI-PvuII fragment of pUCNC14 or pUCNC15 was 3'-end labeled at the EcoRIsite using [t-32P] dATP and the Klenow fragment of E.coli DNA polymerase I. For analysis of the complementary strand, the EcoRI-PvuII fragment was 5'-end labeled at the EcoRI-site using polynucleotide kinase and [y-32P] ATP . Approximately 0,1 pmol end-labeled fragment was incubated with distamycin A in 70 1l Tris-HCl, pH = 7.0 for 15 min at 30°C. A freshly prepared mixture containing 8 4d 0.3 mM Fe(NH4)2 * (SO4)2, 8 A1 0.6 mM EDTA pH = 7.4, 6 ,u H20, 8 ,tl 30 mM sodium ascorbate, and 8 10.9% H202 was added. After 2 min at 30°C the formation of hydroxyl radical was terminated by sequential addition of 10 i1 0.3 M thio urea, 32 d41 0.2 M EDTA, 200 ,ul 0.3 M NaCl, and 750 Al ethyl alcohol. Precipitated DNA was reprecipitated twice and processed for sequencing gel electrophoresis (28).

Autoradiography and Densitometry Autoradiography of sequencing gels was according to (28). Densitometer scans were obtained from autoradiograms optimized for linear film response as described (26). Agarose gels containing ethidium bromide stained DNA bands were photographed and relative DNA concentrations determined by densitometry of photographic negatives. In DNA relaxation time-course experiments, the relative amount of supercoiled substrate converted was determined as 1-

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Fig. 1. Distamycin Inhibits Topoisomerase I Cleavage. The 3'-end labeled 1.6 Kbp HindlII-PvuII fragment of pNC1 was used as substrate. Each reaction contained 5 fmol fragment, 30 units topoisomerase I and distamycin as indicated. DNA and distamycin were preincubated for 15 min at 30°C prior to addition of topoisomerase I. The incubations were continued for 15 min and termninated with 1% SDS. The resulting DNA was denatured and displayed on a 6% polyacrylamide/urea sequencing gel. The distamycin per basepair ratios were: 0 (lane 1), .01 (lane 2), .1 (lane 3), .3 (lane 4), .6 (lane 5), 1.5 (lane 6). The arrow marks the band generated by topoisomerase I cleavage.

where SCO was the area under the peak of supercoiled DNA at time zero, and SCt was the area of this peak at the time t.

RESULTS Sequence-Dependent Inhibition of Topoisomerase I-DNA Interaction by Distamycin The plasmid pNC 1 contains a topoisomerase I recognition sequence supporting a higher catalytic rate than any other known site (23). When present on linear fragments, this site binds topoisomerase I with a KD of approximately 10-10 M. The nicking-closing equilibrium of the system is such that one third

Nucleic Acids Research, Vol. 18, No. 8 1985

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B. Fig. 2. The Influence of Distamycin on Relaxation. Equimolar mixtures of the supercoiled forms of the plasmid pair pNC 1 and pNC5A were relaxed in the presence of topoisomerase I as previously described (23). Panel A, no distamycin, Panel B, distamycin present (.005 distamycin per basepair). Samples were withdrawn from the two parallel reactions at the following time points (min): 0 (lane 1), 1 (lane 2), 2 (lane 3), 3 (lane 4), 4 (lane 5), 5 (lane 6), 6 (lane 7), 8 (lane 8), 10 (lane 10), and 15 (lane 11), and analyzed by agarose gel electrophoresis. Panel C, graphic representation of relaxation time-courses. The degree of substrate conversion was determined densitometrically from the gel (for details, see 'Experimental Procedures') and plotted as a function of time. Closed circles, pNC 1 without distamycin; closed triangles, pNC5A without distamycin; open circles, pNCl with distamycin; open triangles, pNC5A with distamycin.

of the complexed substrate exists in the nicked state (24). In Fig. 1, an appropriate fragment of pNC1 was single-end labeled in the scissile strand and incubated with a saturating amount of topoisomerase I. Complexes in the nicked state of the catalytic cycle were arrested by rapid denaturation with SDS. Subsequent sequencing gel analysis showed that the anticipated approximately 30% of the input DNA was sequence-specifically nicked at the recognition sequence (lane 1). Preincubation of the DNA substrate with distamycin reduced the amount of cleavage in a dosedependent manner (lanes 2-6). Fifty per cent inhibition occurred at a distamycin-DNA ratio of one ligand molecule per ten basepairs, and cleavage was completely quenched after a three to five fold increase of the ligand concentration. To assess the sequence-specificity of distamycin inhibition, we exploited the observation (23) that plasmids containing the recognition sequence are relaxed at a higher rate than control plasmids in the presence of topoisomerase I. Fig. 2A shows a time-course of topoisomerase I mediated relaxation of an equimolar mixture of pNC 1 and pNC5A (the control plasmid, pNC5A, is identical to pNC 1, except for a point mutation inactivating the recognition sequence and a moderate deletion in the vector part). The slowly migrating form I band (supercoiled pNC 1) disappeared more readily than the fast form I band (supercoiled pNC5A). To determine the rates, the degree of substrate conversion was determined densitometrically and plotted as a function of time. The resulting curves (panel C) provides a quantitative estimate of the difference between the initial relaxation rate of pNC 1 (closed circles) and pNC5A (closed triangles). Panel B shows that the rate difference was eliminated in the presence of distamycin. The ligand concentration sufficient to equalize the rates (one distamycin per 200 base pairs) only

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Fig. 3. Topoisomerase I Binding Assayed by Restriction Enzyme protection. The DNA substrate was as in Fig. 1. Each reaction contained 5 fmol DNA, preincubated with distamycin as indicated, and 30 units topoisomerase I. Following addition of 0.01 units DdeI, incubations were continued for 3 min at 30°C and terminated by addition of 0.8 M NaCI/10 mM EDTA, final concentrations. The resulting DNA was processed for sequencing gel analysis as described (28). Lanes 1 and 4, no distamycin; lanes 2 and 5, distamycin present at .1 per basepair; lanes 3 and 6, distamycin present at .6 per basepair. Native topoisomerase I was used in lanes 1-3; heat inactivated (90°C, 10 min) topoisomerase I in lanes 4-6.

1986 Nucleic Acids Research, Vol. 18, No. 8 the protection as evidenced by the dose-dependent increase in cleavage rate in lanes 2 and 3. Ligand binding had little influence on the cleavage rate per se (lanes 5, 6). Distamycin inhibition therefore seems to be exerted at the level of binding.

moderately reduced the overall relaxation rate (Panel C, open symbols). Distamycin Prevents Binding of Topoisomerase I to DNA Binding was monitored by a restriction enzyme protection assay. The core part of the topoisomerase I recognition sequence contains a DdeI-site (see Fig. 6), and prebinding of topoisomerase I impedes DdeI cleavage at the site. This is illustrated in Fig. 3, where the rate of DdeI cleavage in lane 1 (active topoisomerase I present) was clearly reduced relative to lane 4 (heat inactivated topoisomerase I present). The cleavage rate in the presence of heat inactivated topoisomerase I was identical to that obtained with DNA alone (not shown). Addition of distamycin relieved

Mapping of Distamycin Binding Sites Hydroxyl radical generated by reacting EDTA Fe(ll) with hydrogen peroxide (27) was used to footprint distamycin binding sites. Fig. 4A shows an example of such an analysis conducted with a recognition sequence containing fragment, end-labeled in the scissile strand. The most prominent site of protection appeared in the A-tract immediately 5' to the core of the recognition sequence, and therefore overlapped the essential binding region

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on the Wild Type Recognition Sequence. Aliquots of the EcoRI-PvuII fragment of pUC NC 14, single-end labeled strand, were incubated with different amounts of distamycin and reacted with hydroxyl radical as detailed in 'Experimental Procedures'. Panel A shows the scissile strand analyzed on a 12% polyacrylamide/urea gel. Lane 1, A + G sequencing marker (28). Hydroxyl radical footprinting was conducted with the following molar ratios of distamycin per DNA basepair: 0 (lane 2), ol (lane 3), .1 (lane 4), .2 (lane 5), 1 (lane 6), and 10 (lane 7). The bar adjacent to the gel indicates the topoisomerase I binding site (24). Panel B shows densitometer scans of lane 2 (-) and lane 4 (+). Panel C shows the corresponding analysis of the scissile strand in the absence (-) or presence (+) of distamycin (.1 distamycin per basepair).

to visualize either the scissile or the non-scissile

Nucleic Acids Research, Vol. 18, No. 8 1987 of topoisomerase I (24, see summary in Fig. 6). In the stoichiometric range sufficient to inhibit topoisomerase I cleavage, few other sites were seen (Fig. 4A, lanes 2-5), whereas numerous sites were bound at higher concentrations (lanes 6, 7). Fig. 4B shows the aligned densitometer scans of lane 2 (no distamycin) and lane 4 (one distamycin per ten base pairs). The preferred binding site had clearly defined borders and adjoined the 5'-GACTTAG-3' core sequence. The protected region encompassed 4 nucleotides, consistent with a stringently phased binding of a single distamycin molecule (2, 3, 7). A marginal, diffuse protection was observed in the A-tract 3' to the core sequence, indicating that binding in this region was weak and unphased. To exclude a hypothetical, methodology dependent strand bias in the visualization of binding sites (3), the footprinting experiment was repeated with the complementary strand labeled (Fig. 4C). Given the expected dislocation of a binding site mapped on opposite strands (2-3 nucleotides in the 3'-direction, ref.3), the result confirms the binding site observed on the scissile strand. Taken together, the data suggest that inhibition of topoisomerase I action results from binding of distamycin in the upstream Atract in the recognition sequence (shown schematically in Fig. 6A). Distamycin binding in this A-tract did not alter the hydroxyl radical reactivity pattern in the adjacent core sequence. The pattern of hydroxyl radical cleavage in the recognition sequence (Fig. 4C) provides interesting structural information. Both A-tracts show a distinct pattern of smoothly decreasing

reactivity in the 5' to 3' direction. This anomaly is unique to A-tracts and diagnostic of stable helix curvature (29). The reactivity in the core was similar to that of mixed sequence vector DNA, indicating that standard B helical conformation predominates in this region.

Ditamycin Inhibition Does Not Depend on Stable Helix Curvature The observations of Wu and Crothers (8) raises the possibility that the inhibitory effect of distamycin may result from elimination of the stable curvature associated with the upstream A-tract. To address this, a mutant recognition site devoid of curvature in the upstream tract was constructed by replacing the A-tract with a run of alternating A-T's (as depicted in Fig. 6B). The resulting sequence was an efficient substrate for topoisomerase I (cleaved with approximately one third the efficiency of the wild type; S.K., unpublished result), and therefore, the effect of distamycin could be easily monitored by the technique described in Fig. 1. A comparison of distamycin inhibition of topoisomerase I cleavage at the mutant and wild type sequences is shown in Fig. 5A. Cleavage reactions containing different amounts of distamycin were terminated with SDS and subjected to denaturing gel electrophoresis. The cleavage frequencies in the individual lanes were calculated from densitometer scans of the resulting autoradiograms (26) and plotted as a function of the ligand/DNA ratio. Cleavage in the absence of distamycin was arbitrarily set

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A. B. Fig. 5. Distamycin Binding at a Non-Curved Site Inhibits Topoisomerase I. Panel A, inhibition of cleavage. Reaction conditions were as in Fig. 1. The EcoRI-PvuII fragments of pUCNC 14 (wild type) or pUCNC15 (mutant), end-labeled in the scissile strand, were used as substrates. Topoisomerase I was added to preincubation mixtures containing fixed amounts of DNA in varying distamycin concentrations. The reactions were terminated with SDS and electrophoresed on a denaturing gel. Following autoradiography and scanning densitometry, the cleavage frequency in each lane was calculated by dividing the absorbancy in the cleavage band by the total absorbancy in the lane (26). These frequencies were expressed as percentages of the frequency obtained in the absence of distamycin and plotted versus the distamycin/DNA ratio. Circles, wild type; triangles, mutant. Panel B, hydroxyl radical mapping of distamycin binding on the mutant recognition sequence (EcoRIPvuII fragment of pUCNC15, end-labeled in the scissile strand). Lane 1, no distamycin; lane 2, .2 distamycin per basepair; lane 3, .1 distamycin per basepair. The bar adjacent to the gel indicates the topoisomerase I binding site. Panel C, densitometer scans of lane 1 (-) and lane 2 (+).

1988 Nucleic Acids Research, Vol. 18, No. 8

A. 5'- CATGRAA AAAGACT TAGAAAAATTTTTAAA-3T 3'-GTAG'TTTTTTCTGA|ATCTTTTTAAAAATTT-5'

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5'-CATGTATA TAGACTTAGAAAAATTTTTAAA-3' 3'-GTACATATATCTGAATCT TTTTAAAAATT T -5' Fig. 6. Schematic Representation of Distamycin Binding at the Recognition Sequence. Panel A shows the wild type recognition sequence. Arrow, topoisomerase I cleavage site; bar between the strands, the distamycin binding site; wavy lines, the topoisomerase I binding region as deduced from footprinting (24); solid box, the region essential for topoisomerase I cleavage (as deduced from modification interference analysis, (24)); stipled box, a region in which chemical modifications were partially inhibitory to cleavage (24). The DdeI-site is shown in boldface. Panel B, the mutant recognition sequence. Bar between the strands, the distamycin binding site; arrow, the topoisomerase I cleavage site.

at 100 per cent. The two inhibition curves were nearly coincident, demonstrating that distamycin affects topoisomerase I action at the two sequences to the same extent. The distamycin binding site on the mutant sequence was

localized by hydroxyl radical footprinting. Fig. SB and C show that distamycin bound strongly in the alternating A-T-run, and the protected region mapped to the same nucleotide coordinates as on the wild type sequence (compare panels A and B in Fig. 6). The hydroxyl radical pattern recorded in the absence of distamycin (Fig. SC, upper curve) shows normal, 'B-like' reactivity throughout the alternating A-T-run, providing confirmatory evidence that this tract is not curved (29). These results show that distamycin does not inhibit topoisomerase I action by elimination of an essential curvature in the binding site.

DISCUSSION We studied the effect of distamycin on topoisomerase I-DNA interaction. The results demonstrated that this ligand selectively inhibits topoisomerase I action at an A-tract containing recognition sequence. Inhibition was observed under both equilibrium conditions (Fig. 1) and steady-state conditions (Fig. 2). The effect was seen with linear as well as negatively supercoiled substrates and therefore seemed to be independent of the topological state of DNA. Selective inhibition could only be achieved at relatively low distamycin/DNA ratios. Higher ratios tended to override the sequence dependence and cause universal inhibition (our unpublished observations). The sequence dependence rules out the possibility that inhibition could occur by a direct distamycintopoisomerase I interaction causing inactivation of the enzyme. Formally, sequence dependent inhibition could result from a distamycin-topoisomerase interaction altering the sequence specificity of the enzyme. However, the fact that a prominent distamycin binding site maps to the essential part of the topoisomerase I recognition sequence renders this possibility unlikely. Instead, our results suggest that occupation of this site by distamycin prevents topoisomerase I binding at the recognition sequence.

The structural analysis presented in Fig. 4 shows that both Atracts in the recognition sequence are intrinsically bent, raising the possibility that helix curvature is an important affinity

determinant. Examples of protein-DNA recognition that depend critically on helix curvature have been reported (30-32), and in the case of the Crithidia nicking enzyme (31) binding is strongly inhibited by distamycin induced straightening of the DNA. A similar mechanism was recently suggested to be involved in distamycin inhibition of topoisomerase I action in a stably curved segment of kinetoplast DNA (33). We found here, however, that neither topoisomerase I binding nor inhibition thereof depended on curvature in the essential part of the recognition sequence. This apparent discrepancy may relate to the different DNA substrates used. Full clarification of these aspects will have to await mapping of distamycin binding sites in relation to topoisomerase I binding site subdomains on the kinetoplast fragment. Although our results precludes bend straightening as a plausible mechanism of inhibition, distamycin may induce alternative conformational changes deleterious to topoisomerase I binding. If so, the effect is likely to be restricted within the distamycin binding site, since ligand binding did not block Dde I cleavage or impose discernible structural changes elsewhere in the topoisomerase I recognition sequence. Alternatively, inhibition could occur by blockage of normally contacted functional groups, such as backbone phosphates or bases in the minor groove. The latter possibility would be consistent with our observation that an A-tract and a run of alternating A-T's support enzyme binding equally well, since the spatial arrangements of hydrogen bonding potential in the minor groove are indistinguishable in the two polymers (34). Distamycin in some cases induces conformational changes that are propagated into neighboring sequences (35). This property seems to have a dominant influence on topoisomerase II-DNA interaction. Thus, topoisomerase II cleavage was only inhibited at sites coincident with distamycin binding sites. Cleavage was intensified at many sites in the presence of distamycin, presumeably as a result of transmitted structural changes induced by ligand binding at remote positions. As a net result, topoisomerase 11 mediated DNA relaxation was largely unaffected or slightly stimulated, even in the presence of high concentrations of distamycin (12). This behavior contrasts that of topoisomerase I. Although a marginal stimulation of relaxation activity was observed at very low distamycin concentrations, the predominant effect was inhibition (13). This implies that distamycin affects topoisomerase I-DNA interaction in a more direct way than it does topoisomerase II-DNA interaction. Therefore, our conclusion that distamycin acts to displace topoisomerase I from the recognition sequence may apply generally to most topoisomerase I sites on DNA.

ACKNOWLEDGEMENTS This study was supported by the Commission of the European Communities (Contract BI-6-0170-DK), the Danish Cancer Society (grant no. 88-060), the National Agency of Technology (contract no. 1985-133/001-85.521), and the Aarhus University Bioregulation Center.

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Distamycin inhibition of topoisomerase I-DNA interaction: a mechanistic analysis.

Inhibition of eukaryotic DNA topoisomerase I by the minor groove binding ligand, distamycin A, was investigated. Low concentrations of the ligand sele...
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