High resolution hydroxyl radical footprinting of the binding of mithramycin and related antibiotics to DNA.

The preferred binding sites for mithramycin on three different DNA fragments have been determined by hydroxyl radical footprinting. Sequences which appear as one long protected region using DNAase I as a footprinting probe are resolved into several discrete binding domains. Each drug molecule protects three bases from radical attack, though adjacent regions show attenuated cleavage. Mithramycin and the other related compounds induce similar footprinting patterns and appear to recognise GC rich regions with a preference for those containing the dinucleotide step GpG. The ability of each such site to bind the drug depends on the sequence environment in which it is located. The data are consistent with mithramycin binding to the DNA minor groove.     

Methidiumpropyl-EDTA.Fe(II) and DNase I footprinting report different small molecule binding site sizes on DNA.

DNase I and MPE.Fe (II) footprinting both employ partial cleavage of ligand-protected DNA restriction fragments and Maxam-Gilbert sequencing gel methods of analysis. One method utilizes the enzyme, DNase I, as the DNA cleaving agent while the other employs the synthetic molecule, methidium-propyl-EDTA (MPE). For actinomycin D, chromomycin A3 and distamycin A, DNase I footprinting reports larger binding site sizes than MPE.Fe (II). DNase I footprinting appears more sensitive for weakly bound sites. MPE.Fe (II) footprinting appears more accurate in determining the actual size and location of the binding sites for small molecules on DNA, especially in cases where several small molecules are closely spaced on the DNA. MPE.Fe (II) and DNase I report the same sequence and binding site size for lac repressor protein on operator DNA.     

DNA structural variations produced by actinomycin and distamycin as revealed by DNAase I footprinting.

The technique of DNAase I footprinting has been used to investigate preferred binding sites for actinomycin D and distamycin on a 160-base-pair DNA fragment from E. coli containing the tyr T promoter sequence. Only sites containing the dinucleotide step GpC are protected by binding of actinomycin, and all such sites are protected. Distamycin recognizes four major regions rich in A + T residues. Both antibiotics induce enhanced rates of cleavage at certain regions flanking their binding sites. These effects are not restricted to any particular base sequence since they are produced in runs of A and T by actinomycin and in GC-rich sequences by distamycin. The observed increases in susceptibility to nuclease attack are attributed to DNA structural variations induced in the vicinity of the ligand binding site, most probably involving changes in the width of the helical minor groove.     

Hydroxyl radical footprints reveal novel structural features around the NF I binding site in adenovirus DNA.

We have identified a number of as yet unknown structural abnormalities of the NF I-DNA binding site within the inverted terminal repetition of adenovirus DNA by probing it with a hydroxyl radical footprinting technique. NF I binding alters the accessibility of the deoxyribose moieties to hydroxyl radicals both at the 3' and at the 5' side of the recognition sequence 5'-TGG(N)6GCCAA-3'. A smooth bend at the 5' side of the binding sequence is already present in naked linear DNA and it is further enhanced by protein binding. This could be demonstrated not only by hydroxyl radical footprinting but also by studying the temperature dependent mobility during gel electrophoresis of DNA fragments carrying the NF I binding site at circularly permutated positions. We propose that the bent conformation at this site is responsible for facilitating protein/DNA interactions.     

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 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 unessential for efficient binding of topoisomerase I, and (ii) distamycin inhibition did not occur by deformation of a stable band. Alternative modes of inhibition are discussed.     

Interaction of minor groove binding ligands with long AT tracts.

We have used quantitative DNase I footprinting to examine the ability of distamycin and Hoechst 33258 to discriminate between different arrangements of AT residues, using synthetic DNA fragments containing multiple blocks of (A/T)6or (A/T)10in identical sequence environments. Previous studies have shown that these ligands bind less well to (A/T)4sites containing TpA steps. We find that in (A/T)6tracts distamycin shows little discrimination between the various sites, binding approximately 2-fold stronger to TAATTA than (TA)3, T3A3and GAATTC. In contrast, Hoechst 33258 binds approximately 20-fold more tightly to GAATTC and TAATTA than T3A3and (TA)3. Hydroxyl radical footprinting reveals that both ligands bind in similar locations at the centre of each AT tract. At (A/T)10sites distamycin binds with similar affinity to T5A5, (TA)5and AATT, though bands in the centre of (TA)5are protected at approximately 50-fold lower concentration than those towards the edges. Hoechst 33258 shows a similar pattern of preference, with strong binding to AATT, T5A5and the centre of (TA)5. Hydroxyl radical footprinting reveals that at low concentrations both ligands bind at the centre of (TA)5and A5T5, while at higher concentrations ligand molecules bind to each end of the (A/T)10tracts. At T5A5two ligand molecules bind at either end of the site, even at the lowest ligand concentration, consistent with the suggestion that these compounds avoid the TpA step. Similar DNase I footprinting experiments with a DNA fragment containing T n (n = 3-6) tracts reveals that both ligands bind in the order T3< T4 << T5 = T6.     

Footprinting reveals that nogalamycin and actinomycin shuffle between DNA binding sites.

The hypothesis that sequence-selective DNA-binding antibiotics locate their preferred binding sites by a process involving migration from nonspecific sites has been tested by footprinting with DNAase I. Footprinting patterns on the tyrT DNA fragment produced by nogalamycin and actinomycin change with time after mixing the antibiotic with the DNA. Sites of protection as well as enhanced cleavage are seen to develop in a fashion which is both temperature and concentration-dependent. At certain sites cutting is transiently enhanced, then blocked. Limited evidence for slow reaction with echinomycin and mithramycin is presented, but the kinetics of footprinting with daunomycin and distamycin appear instantaneous. The feasibility of adducing direct evidence for shuffling by footprinting seems to be governed by slow dissociation of the antibiotic-DNA complex. It may also be dependent upon the mode of binding, be it intercalative or non-intercalative in character.     

The effects of local DNA sequence on the interaction of ligands with their preferred binding sites

We have examined the effects of local DNA sequence on the interaction of distamycin, Hoechst 33258, echinomycin, actinomycin and mithramycin with their preferred binding sites using a series of DNA fragments that contain every symmetrical hexanucleotide sequence. In several instances we find that the affinity for the ligands' preferred binding sites is affected by the hexanucleotide context in which they are located. The AT-selective minor groove binding ligand Hoechst 33258 shows a 200-fold difference in binding to the 16 different X(A/T)(4)Y sites; the strongest binding is to AAATTT and the weakest is to (G/C)TTAA(C/G). Although TTAA is generally a poor binding site, ATTAAT is better than TTTAAA and they are both much better than GTTAAC and CTTAAG. Similarly, TTATAA and ATATAT are better binding sites than GTATAC and CTATAG. In contrast, distamycin shows less discrimination between the various X(A/T)(4)Y sites, with a 20-fold difference between the best [(A/T)AATT(T/A)] and worst [GATATC and (G/C)TTAA(C/G)] sites. Although actinomycin binds to GpC it shows little or no interaction with any of the GGCC sites, yet shows only a six-fold variation in affinities for the other XYGCXY sites. Echinomycin binds to CpG yet shows no binding to TTCGAA, TGCGCA and AGCGCT, while the best binding is to AACGTT. The tetranucleotides CCGG and ACGT produce consistently good binding sites, irrespective of the surrounding sequences, while the interaction with TCGA and GCGC is sensitive to the hexanucleotide context. Hexanucleotides with a central GCGC, flanked by A and T are weaker echinomycin sites than those flanked by G and C, especially CGCGCG. The best X(G/C)(4)Y binding sites for mithramycin were located at AGCGCT and GGGCCC, and the worst at CCCGGG and TCCGGA. These footprinting fragments are valuable tools for comparing the binding of ligands to all the potential symmetrical hexanucleotides and provide insights into the effects of local DNA sequence on ligand-DNA interactions.     

Distamycin and penta-N-methylpyrrolecarboxamide binding sites on native DNA. A comparison of methidiumpropyl-EDTA-Fe(II) footprinting and DNA affinity cleaving

Using two direct methods we have studied the binding locations and site sizes of distamycin and penta-N-methylpyrrolecarboxamide on three DNA restriction fragments from pBR322 plasmid. We find that methidiumpropyl-EDTA.Fe(II) footprinting and DNA affinity cleaving methods report common binding locations and site sizes for the tri- and pentapeptides bound to heterogeneous DNA. The tripeptide distamycin binds 5-base-pair sites with a preference for poly(dA).poly(dT) regions. The pentapeptide binds 6-7-base-pair sites with a preference for poly(dA).poly(dT) regions. These results are consistent with distamycin binding as an isogeometric helix to the minor groove of DNA with the four carboxamide N-H's hydrogen bonding five A + T base pairs. The data supports a model where each of the carboxamide N-H's can hydrogen bond to two bases, either O(2) of thymine or N(3) of adenine, located on adjacent base pairs on opposite strands of the helix. In most (but not all) cases the tri- and pentapeptide can adopt two orientations at each A + T rich binding site.     

Single-strand DNA binding of actinomycin D with a chromophore 2-amino to 2-hydroxyl substitution

A modified actinomycin D was prepared with a hydroxyl group that replaced the amino group at the chromophore 2-position, a substitution known to strongly reduce affinity for double-stranded DNA. Interactions of the modified drug on single-stranded DNAs of the defined sequence were investigated. Competition assays showed that 2-hydroxyactinomycin D has low affinity for two oligonucleotides that have high affinities (K(a) = 5-10 x 10(6) M(-1) oligomer) for 7-aminoactinomycin D and actinomycin D. Primer extension inhibition assays performed on several single-stranded DNA templates totaling around 1000 nt in length detected a single high affinity site for 2-hydroxyactinomycin D, while many high affinity binding sites of unmodified actinomycin D were found on the same templates. The sequence selectivity of 2-hydroxyactinomycin D binding is unusually high and approximates the selectivity of restriction endonucleases. Binding appears to require a complex structure, including residues well removed from the polymerase pause site.     

Sequence-selective binding of an ellipticine derivative to DNA.

The DNA sequence specificity of an ellipticine derivative bearing an aminoalkyl side chain has been determined by a variety of footprinting methods. The drug exhibits sequence selective binding and discriminates against runs of adenines or thymines. Binding is shown to occur at various sequences with a preference for GC rich regions of DNA. A large enhancement of DNAase I and of hydroxyl radical cleavage in regions rich in A's or T's is observed together with hyperreactivity of adenines towards diethylpyrocarbonate in the presence of drug. This indicates the occurrence of drug-induced changes in critical conformational features of DNA. The total absence of hyperreactivity of guanine residues towards diethylpyrocarbonate appears to be related to the sequence selectivity of drug binding. No alteration of the dimethyl sulphate and methylene blue-induced cleavage of DNA is observed. Irradiation of ellipticine derivative-DNA complexes with UV light followed by alkali treatment leads to selective photocleavage at guanine residues, consistent with the deduced degree of selectivity of the binding reaction.     

Mammalian topoisomerase II activity is modulated by the DNA minor groove binder distamycin in simian virus 40 DNA

DNA topoisomerases II are nuclear enzymes that have been identified recently as targets for some of the most active anticancer drugs. Antitumor topoisomerase II inhibitors such as teniposide (VM-26) produce enzyme-induced DNA cleavage and inhibition of enzyme activity. By adding to such reactions distamycin, a compound whose effects on DNA have been extensively characterized, we investigated the effects of drug binding upon topoisomerase II-mediated DNA cleavage induced by VM-26. We have found a correspondence between distamycin binding (determined by footprinting analysis) and topoisomerase II-mediated cleavage of SV40 DNA (determined by sequencing gel analysis). Distamycin binding potentiated the cleavage of specific sites in the near proximity of distamycin-binding sites (within at least 25 base pairs), which indicates that DNA secondary structure is involved in topoisomerase II-DNA interactions. That distamycin potentiated cleavage only at sites that were recognized in the absence of distamycin and suppressed cleavage directly at distamycin-binding sites indicates that topoisomerase II recognizes DNA on the basis of primary sequence. In addition, distamycin stimulated topoisomerase II-mediated DNA relaxation and antagonized the inhibitory effect of VM-26. These results show that the DNA sequence-specific binding of distamycin produces local and propagated effects in the DNA which markedly affect topoisomerase II activity.     

DNA sequence preferences of several AT-selective minor groove binding ligands.

We have examined the interaction of distamycin, netropsin, Hoechst 33258 and berenil, which are AT-selective minor groove-binding ligands, with synthetic DNA fragments containing different arrangements of AT base pairs by DNase I footprinting. For fragments which contain multiple blocks of (A/T)4 quantitative DNase I footprinting reveals that AATT and AAAA are much better binding sites than TTAA and TATA. Hoechst 33258 shows that greatest discrimination between these sites with a 50-fold difference in affinity between AATT and TATA. Alone amongst these ligands, Hoechst 33258 binds to AATT better than AAAA. These differences in binding to the various AT-tracts are interpreted in terms of variations in DNA minor groove width and suggest that TpA steps within an AT-tract decrease the affinity of these ligands. The behaviour of each site also depends on the flanking sequences; adjacent pyrimidine-purine steps cause a decrease in affinity. The precise ranking order for the various binding sites is not the same for each ligand.     

Determination of equilibrium binding affinity of distamycin and netropsin to the synthetic deoxyoligonucleotide sequence d(GGTATACC)2 by quantitative DNase I footprinting

A new method for determining the equilibrium binding constant of antitumor drugs to specific DNA sequences by quantitative DNase I footprinting is presented. The use of a short synthetic DNA oligomer to define a homogeneous population of DNA binding sites enables the calculation of the free drug concentration and the fraction of DNA sites complexed with drug in solution and is described for the first time. Since a 1:1 stoichiometry is observed for each drug-oligomer DNA complex, it becomes possible to calculate equilibrium binding constants in solution. By use of this technique, the binding affinities of the nonintercalating drugs netropsin and distamycin to the synthetic oligonucleotide d(GGTATACC)2 are determined to be Ka (25 degrees C) = 1.0 X 10(5) and 2.0 X 10(5) M-1, respectively. Quantitation of the temperature dependence associated with complex formation results in a determination of standard enthalpies of -3.75 and -8.48 kcal mol-1 for the binding of netropsin and distamycin, respectively. Calculation of other thermodynamic parameters are found to be in agreement with previous studies and indicate that the DNA binding process for these compounds is predominantly enthalpy driven. This method of quantitative DNase I footprinting is demonstrated to be a useful technique for the measurement of drug affinities to specific binding sites on DNA oligomers which are designed and synthesized expressly for this purpose. Applications of the technique to the determination of drug binding affinities at specific sites within native DNA sequences are discussed.     

Heterogeneity in the actions of drugs that bind in the DNA minor groove.

Distamycin and Hoechst 33258 have long served as the model compounds for biochemical, biophysical, and clinical studies of the drugs that bind in the DNA minor groove. However, the results presented in this investigation clearly show that 4,6-diamidino-2 phenylindole (DAPI) is superior to both of these drugs at negating the effects of intrinsic DNA curvature and anisotropic bendability as measured by electrophoretic and ligation analysis. In addition, DAPI was more effective than distamycin and Hoechst 33258 at inhibiting the assembly of nucleosomes onto synthetic and natural sequences that have multiple closely spaced oligo-AT sequences that serve as drug binding sites. Since these effects may be related to the biological action of the drugs, it was of interest to determine the mechanism that was responsible for the enhanced action of DAPI. The possibility that the differential drug potencies resulted from differential overall affinities of the ligands for A-tract molecules was considered, but drug binding studies suggested that this was not the case. It is also unlikely that the differential drug effects resulted from the binding of the drugs to different DNA sites since the oligo A/T binding sites for DAPI and Hoechst were centered on the same nucleotide positions as revealed by footprinting studies using exonuclease III, DNase I, and hydroxyl radical. However, the footprinting studies with DNase I did uncover a potentially important difference between the drugs. DAPI protected only the AT bp in the binding sites, while distamycin and Hoechst protected these bp as well as flanking Gs and Cs. These results permitted us to advance a preliminary model for the enhanced action DAPI. According to the model, the short length of DAPI and its absolute specificity for A/T bps with narrow minor grooves ensures that only particularly minor grooves that give rise to curvature and anisotropic bendability are occupied by the drug. Consequently, each helical deflection induced by an A-tract in the absence of the drug is countered by an opposite deflection induced by DAPI binding, thus effectively neutralizing intrinsic curvature and bending into the minor groove.     

Visualising the kinetics of dissociation of actinomycin from individual sites in mixed sequence DNA by DNase I footprinting.

We have investigated the kinetics of dissociation of actinomycin D from DNA by a variation of the footprinting technique. Complexes of actinomycin with a radiolabelled DNA fragment (tyrT) were dissociated by addition of a large excess of unlabelled calf thymus DNA and the mixture subjected to DNase I footprinting at subsequent intervals. The rates at which the footprints disappeared varied between the different binding sites. The dissociation was temperature dependent with average time constants of 30 s, 10 mins and 2 hours at temperatures of 37 degrees C, 20 degrees C and 4 degrees C respectively. The dissociation from a DNA fragment containing the synthetic insert T9GCA9 was significantly faster, with a half-life of about 1 min at 20 degrees C. In contrast, the dissociation of distamycin was too fast to measure (< 5 s) even at 4 degrees C.     

Specific protection of DNA by distamycin A, netropsin and bis-netropsins against the action of DNAse I

Interaction of netropsin, distamycin A and a number of bis-netropsins with DNA fragments of definite nucleotide sequence was studied by footprinting technique. The nuclease protection experiments were made at fixed DNA concentration and varying ligand concentrations. The affinity of ligand for a DNA site was estimated from measurements of ligand concentration that causes 50% protection of the DNA site. Distribution pattern of the protected and unprotected regions along the DNA fragment was compared with the theoretically expected arrangement of the ligand along the same DNA. The comparison led us to the following conclusions: 1. Footprinting experiments show that at high levels of binding the arrangement of netropsin molecules along the DNA corresponds closely to the distribution pattern expected from theoretical calculations based on the known geometry of netropsin--DNA complex. However, the observed differences in the affinity of netropsin for various DNA sequences is markedly greater than that expected from theoretical calculations. 2. Netropsin exhibits a greater selectivity of binding than that expected for a ligand with three specific reaction centers associated with the antibiotic amide groups. It binds preferentially to DNA regions containing four or more successive AT pairs. Among 13 putative binding sites for netropsin with four or more successive AT pairs there are 11 strong binding sites and two weaker sites which are occupied at 2 D/P less than or equal to 1/9 and 2 D/P = 1/4, respectively. 3. The extent of specificity manifested by distamycin A is comparable to that shown by netropsin although the molecule of distamycin A contains four rather than three amide groups. At high levels of binding distamycin A occupies the same binding sites on DNA as netropsin does. 4. The binding specificity of bis-netropsins is greater than that of netropsin. Bis-netropsins can bind to DNA in such a way that the two netropsin-like fragments are implicated in specific interaction with DNA base pairs. However, the apparent affinity of bis-netropsins estimated from footprinting experiments is comparable with that of netropsin for the same DNA region. 5. At high levels of binding bis-netropsins and distamycin A (but not netropsin) can occupy any potential site on DNA irrespectively of the DNA sequence. 6. Complex formation with netropsin increases sensitivity to DNase I at certain DNA sites along with the protection effect observed at neighboring sites.     

Characterization of distamycin A binding to damaged DNA

We previously reported that distamycin A, a natural antibiotic known as a minor groove binder, could bind to DNA duplexes containing the (6-4) photoproduct formed at its target site, whereas the binding was not observed for duplexes containing the cis-syn cyclobutane pyrimidine dimer in the same sequence context. In this study, we have further analyzed the binding of this drug to lesion-containing duplexes to elucidate its damaged-DNA recognition mechanism. Surface plasmon resonance measurements using various types of DNA showed that distamycin A could bind to several types of lesion-containing DNA. Curve fitting of the CD titration data revealed that the complex formation occurred with K(d) values around 10(-6) and a stoichiometry of 1:1. The results obtained in this study suggested that distamycin A binds to damaged DNA in the same way as to the normal target site, by recognizing the chemical structure of the minor groove.