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.     

DNA-sequence specific recognition by a thiazole analogue of netropsin: a comparative footprinting study.

Four different footprinting techniques have been used to probe the DNA sequence selectivity of Thia-Net, a bis-cationic analogue of the minor groove binder netropsin in which the N-methylpyrrole moieties are replaced by thiazole groups. In Thia-Net the ring nitrogen atoms are directed into the minor groove where they could accept hydrogen bonds from the exocyclic 2-amino group of guanine. Three nucleases (DNAase I, DNAase II, and micrococcal nuclease) were employed to detect binding sites on the 160bp tyr T fragment obtained from plasmid pKM delta-98, and further experiments were performed with 117mer and 253mer fragments cut out of the plasmid pBS. MPE.Fe(II) was used to footprint binding sites on an EcoRI/HindIII fragment from pBR322. Thia-Net binds to sites in the minor groove containing 4 or 5 base pairs which are predominantly composed of alternating A and T residues, but with significant acceptance of intrusive GC base pairs. Unlike the parent antibiotic netropsin, Thia-Net discriminates against homooligomeric runs of A and T. The evident preference of Thia-Net for AT-rich sites, despite its containing thiazole nitrogens capable of accepting GC sites by hydrogen bonding, supports the view that the biscationic nature of the ligand imposes a bias due to the electrostatic potential differences in the receptor which favour the ligand reading alternating AT sequences.     

Quantitative footprinting analysis using a DNA-cleaving metalloporphyrin complex

The results of quantitative footprinting studies involving the antiviral agent netropsin and a DNA-cleaving cationic metalloporphyrin complex are presented. An analysis of the footprinting autoradiographic spot intensities using a model previously applied to footprinting studies involving the enzyme DNase I [Ward, B., Rehfuss, R., Goodisman, J., & Dabrowiak, J. C. (1988) Biochemistry 27, 1198-1205] led to very low values for netropsin binding constants on a restriction fragment from pBR-322 DNA. In this work, we show that, because the porphyrin binds with high specificity to DNA, it does not report site loading information in the same manner as does DNase I. We elucidate a model involving binding equilibria for individual sites and include competitive binding of drug and porphyrin for the same site. The free porphyrin and free drug concentrations are determined by binding equilibria with the carrier (calf thymus DNA) which is present in excess and acts as a buffer for both. Given free porphyrin and free netropsin concentrations for each total drug concentration in a series of footprinting experiments, one can calculate autoradiographic spot intensities in terms of the binding constants of netropsin to the various sites on the 139 base pair restriction fragment. The best values of these binding constants are determined by minimizing the sum of the squared differences between calculated and experimental footprinting autoradiographic spot intensities. Although the determined netropsin binding constants are insensitive to the value assumed for the porphyrin binding constant toward its highest affinity sites, the best mean-square deviation between observed and calculated values, D, depends on the choice of (average) drug binding constant to carrier DNA, Kd.(ABSTRACT TRUNCATED AT 250 WORDS)     

Binding properties and DNA sequence-specific recognition of two bithiazole-linked netropsin hybrid molecules.

We report the DNA binding properties of two hybrid molecules which result from the combination of the DNA sequence-specific minor groove ligand netropsin with the bithiazole moiety of the antitumor drug bleomycin. The drug-DNA interaction has been investigated by means of electric linear dichroism (ELD) spectroscopy and DNase I footprinting. In compound 1 the two moieties are linked by a flexible aliphatic tether while in compound 2 the two aromatic ring systems are directly coupled by a rigid peptide bond. The results are consistent with a model in which the netropsin moiety of compound 1 resides in the minor groove of DNA and where the appended bithiazole moiety is projected away from the DNA groove. This monocationic hybrid compound has a weak affinity for DNA and shows a strict preference for A and T stretches. ELD measurements indicate that in the presence of DNA compound 2 has an orientation typical of a minor groove binder. Similar orientation angles were measured for netropsin and compound 2. This ligand which has a biscationic nature tightly binds to DNA (Ka = 6.3 x 10(5) M-1) and is mainly an AT-specific groove binder. But, depending on the nature of the sequence flanking the AT site first targeted by its netropsin moiety, the bithiazole moiety of 2 can accommodate various types of nucleotide motifs with the exception of homooligomeric sequences. As evidenced by footprinting data, the bithiazole group of bleomycin acts as a DNA recognition element, offering opportunities to recognize GC bp-containing DNA sequences with apparently a preference (although not absolute) for a pyrimidine-G-pyrimidine motif. Thus, the bithiazole unit of bleomycin provides an additional anchor for DNA binding and is also capable of specifically recognizing particular DNA sequences when it is appended to a strongly sequence selective groove binding entity. Finally, a model which schematizes the binding of compound 2 to the sequence 5'-TATGC is proposed. This model readily explains the experimentally observed specificity of this netropsin-bithiazole conjugate.     

Transmission of stability (telestability) in deoxyribonucleic acid. Physical and enzymatic studies on the duplex block polymer d(C15A15) - d(T15G15).

The properties of the duplex block polymer d(C15A15) - d(T15G15) were examined by thermal denaturation and nuclease susceptibility studies in the absence and presence of drugs (actinomycin and netropsin) which bind specifically to only one end of the block polymer. The nucleotide composition of one region of this synthetic double-helical DNA affected the properties of a contiguous but remote region. Furthermore, the binding of actinomycin influenced the properties of both the binding and nonbinding regions. These findings suggest a mechanism for gene regulation at a distance.     

Photoaffinity approaches to determining the sequence selectivities of DNA-small molecule interactions: actinomycin D and ethidium.

The DNA photoaffinity ligands, 7-azidoactinomycin D and 8-azidoethidium, form DNA adducts that cause chain cleavage upon treatment with piperidine. Chemical DNA sequencing techniques were used to detect covalent binding. The relative preferences for modifications of all possible sites defined by a base pair step (e.g. GC) were determined within all quartet contexts such as (IGCJ). These preferences are described in terms of 'effective site occupations', which express the ability of a ligand to covalently modify some base in the binding site. Ideally, the effective site occupations measured for photoaffinity agents can also be related to site-specific, non-covalent association constants of the ligand. The sites most reactive with 7-azidoactinomycin D were those preferred for non-covalent binding of unsubstituted actinomycin D. GC sites were most reactive, but next-nearest neighbors exerted significant influences on reactivity. GC sites in 5'-(pyrimidine)GC(purine)-3' contexts, particularly TGCA, were most reactive, while reactivity was strongly suppressed for GC sites with a 5'-flanking G, or a 3'-flanking C. High reactivities were also observed for bases in the first (5') GG steps in TGGT, TGGG and TGGGT sequences recently shown to bind actinomycin D with high affinity. Pyrimidine-3',5'-purine steps and GG steps flanked by a T were most preferred by 8-azidoethidium, in agreement with the behavior of unsubstituted ethidium. The good correspondence between expected and observed covalent binding preferences of these two azide analogs demonstrates that photoaffinity labeling can identify highly preferred sites of non-covalent DNA binding by small molecules.     

Fluorescent intercalator displacement analyses of DNA binding by the peptide-derived natural products netropsin, actinomycin, and bleomycin

The response of the high-throughput fluorescent intercalator displacement (HT-FID) assay reported recently by Boger et al. to peptide-based DNA binding intercalators and metal complexes was examined through the study of actinomycin and Co(III).bleomycin-B2. Along with a validation of netropsin that illustrated the good laboratory-to-laboratory reproducibility of the assay, our examination of actinomycin revealed results for a four base pair cassette library of DNA hairpins that paralleled the known DNA site-selectivity of this agent and also indicated the involvement of the flanking sequences of the hairpin oligonucleotide. In addition, for Co(III).bleomycin-B2 the established cleavage site-selectivity for 5'-GT and 5'-GC sites was correlated to drug-DNA association in this binding-only assay; our results also suggest a tetranucleotide site-selectivity for metallobleomycin involving cross-strand, 'back-to-back' 5'-GT and 5'-GC sites such as 5'-ACGT and 5'-ACGC.     

Specific DNA cleavage by an analog of netropsin containing a copper(II) chelating peptide Gly-Gly-His]

Experimental data are reported on DNA-cleaving activity of the synthetic netropsin analogs consisting of the two N-propylpyrrole carboxamide units linked covalently through two or three glycine residues to a copper-chelating tripeptide glycyl-glycyl-L-histidine. Incubation of DNA restriction fragment and netropsin analog in the presence of ascorbate, hydrogen peroxide and Cu2+ ions resulted in selective cleavage of the DNA at or near the preferred sites for binding of netropsin analog. A similar cleavage pattern is observed after X-ray irradiation of DNA complexes with netropsin analogs tethered with Cu2+ ions. The cleavage patterns are found to be dependent on the length of the connecting chain between the histidine-containing tripeptide and netropsin analog. The netropsin analog containing three glycine residues in the connecting chain, but not the analog with a shorter linker chain, can generate an intense cleavage of one of the two polynucleotide chains at a position corresponding to the presumed binding site for the dimeric ligand species. More than 50% of the total DNA can be cleaved at this position after X-ray irradiation. From analysis of the nucleotide sequences surrounding the preferred cleavage site on several DNA fragments we found that the consensus is 5'-TTTTNCA*AAA-3', where N is an arbitrary nucleotide. The Cu(2+)-mediated cleavage of DNA occurs at the second adenine (indicated by an asterisk) from the 5'-end of the sequence. The greatest cleavage activity is observed when the molar ratio of Cu2+ to the netropsin analog is equal to 0.5. Evidently, the Cu(2+)-ligated and unligated oligopeptide species interacts with each other to form a heterodimer bound to DNA at the cleavage site. To test the validity of this model we have studied the binding of unligated netropsin analog and netropsin analog complexed with Cu2+ ion to a self-complementary oligonucleotide 5'-GCGTTTTGCAAAACGC-3'. It is found that binding of Cu(2+)-ligated netropsin analog to the DNA oligomer preincubated with unligated form of the oligopeptide is a cooperative process for which interactions between the two bound ligands are responsible. The cooperativity parameter is estimated to be on the order of factor 6. Finally, a model is proposed in which a heterodimer stabilized by interligand beta-sheet binds in the minor DNA groove.     

Daunomycin modifies the sequence-selective recognition of DNA by actinomycin.

The antitumour antibiotic actinomycin D normally binds to DNA by intercalation at sequences containing the CpG step, but in the presence of daunomycin it has been reported to interact with poly(dA-dT). This observation has neither been confirmed nor explained. Here we have used a photoreactive 7-azido derivative of actinomycin to study the effect of daunomycin on its binding to three DNA fragments. Daunomycin did indeed alter the binding of actinomycin to the DNA, such that the antibiotic was displaced from its primary GpC sites onto secondary sites in the DNA, though not to AT regions especially. These findings suggest a possible scientific explanation for the increased toxicity seen during combination chemotherapy with these two drugs.     

Drug-DNA sequence-dependent interactions analysed by electric linear dichroism.

The interactions between 20 drugs and a variety of synthetic DNA polymers and natural DNAs were studied by electric linear dichroism (ELD). All compounds tested, including several clinically used antitumour agents, are thought to exert their biological activities mainly by virtue of their abilities to bind to DNA. The selected drugs include intercalating agents with fused and unfused aromatic structures and several groove binders. To examine the role of base composition and base sequence in the binding of these drugs to DNA, ELD experiments were carried out with natural DNAs of widely differing base composition as well as with polynucleotides containing defined alternating and non-alternating repeating sequences, poly(dA).poly(dT), poly(dA-dT).poly(dA-dT),poly(dG).poly(dC) and poly(dG-dC).poly(dG-dC). Among intercalating agents, actinomycin D was found to be by far the most GC-selective. GC selectivity was also observed with an amsacrine-4-carboxamide derivative and to a lesser extent with methylene blue. In contrast, the binding of amsacrine and 9-aminoacridine was practically unaffected by varying the GC content of the DNAs. Ethidium bromide, proflavine, mitoxantrone, daunomycin and an ellipticine derivative were found to bind best to alternating purine-pyrimidine sequences regardless of their nature. ELD measurements provided evidence for non-specific intercalation of amiloride. A significant AT selectivity was observed with hycanthone and lucanthone. The triphenyl methane dye methyl green was found to exhibit positive and negative dichroism signals at AT and GC sites, respectively, showing that the mode of binding of a drug can change markedly with the DNA base composition. Among minor groove binders, the N-methylpyrrole carboxamide-containing antibiotics netropsin and distamycin bound to DNA with very pronounced AT specificity, as expected. More interestingly the dye Hoechst 33258, berenil and a thiazole-containing lexitropsin elicited negative reduced dichroism in the presence of GC-rich DNA which is totally inconsistent with a groove binding process. We postulate that these three drugs share with the trypanocide 4',6-diamidino-2-phenylindole (DAPI) the property of intercalating at GC-rich sites and binding to the minor groove of DNA at other sites. Replacement of guanines by inosines (i.e., removal of the protruding exocyclic C-2 amino group of guanine) restored minor groove binding of DAPI, Hoechst 33258 and berenil. Thus there are several cases where the mode of binding to DNA is directly dependent on the base composition of the polymer. Consequently the ELD technique appears uniquely valuable as a means of investigating the possibility of sequence-dependent recognition of DNA by drugs.     

A high buoyant density fraction in mammalian DNA: Characterization and impact on the detection of heteroduplex DNA

DNA from both Chinese hamster ovary (CHO) cells and human fibroblasts contains a high buoyant density fraction. This fraction of DNA was purified from CHO cells and characterized. Compared with mainband CHO DNA, this high buoyant density DNA binds more of a GC-specific dye, actinomycin D (actD), and less of an AT-specific dye, netropsin, which suggests that its increased density is due to an increase in clusters of GC base pairs. The detection of heteroduplex DNA which has been hypothesized to occur during sister chromatid exchange formation is considerably complicated by the presence of this high density DNA. Experiments to detect heteroduplex DNA in CHO cells, using actD to shift the position of the high density DNA, did not reveal any underlying heteroduplex material, thus placing an upper limit on the size of the hypothesized heteroduplex regions. Experiments with both CHO cells and with human fibroblasts indicated that the amount of the high buoyant density DNA did not consistently increase when the sister chromatid exchange frequency increased.     

Antibiotic induced electrophoretic mobility shifts of DNA restriction fragments.

Several antibiotics, netropsin, distamycin A, actinomycin D, Hoechst 33258 and olivomycin, which demonstrate base specificity in their DNA binding properties have been found to alter the electrophoretic mobility of DNA restriction fragments in native polyacrylamide gels. The antibiotics mostly reduced the migration of larger DNA fragments, but netropsin and Hoechst 33258 were observed to increase the migration rate of several DNA fragments of intermediate size. DNA fragments of similar molecular weight which comigrate as a single gel band can at times be separated as the result of differential mobility shifts promoted by antibiotic DNA complex formations.     

Netropsin specifically recognizes one of the two conformationally equivalent strands of poly(dA).poly(dT). One dimensional NMR study at 500 MHz involving NOE transfer between netropsin and DNA protons

Recent observations that the heteronomous structural model for poly(dA).poly(dT) is not found in solution and that in this DNA, the two strands are conformationally equivalent (J. Biomole. Str. Dyns. 2, 1057 (1985], has added a new dimension to the structural dynamics of DNA-netropsin complex. Does the antibiotic somehow distinguish between the two strands and specifically interact with only one of the conformationally equivalent strands? Model-building studies suggest that netropsin can either bind to the dA-strand in the minor groove such that H-bonds are formed between the imino protons N4-H, N6-H, N8-H of netropsin and N3 atoms of A or can bind to the dT-strand in the minor groove and form H-bonds between the imino-protons N4-H, N6-H, N8-H of netropsin and O2 atoms of T. If netropsin binds to the dA-strand, AH2 atoms of poly(dA).poly(dT) would be in closer proximity to the imino protons N4-H, N6-H, N8-H and pyrrole ring protons C5-H, C11-H of netropsin than they would be, if netropsin binds to the dT-strand. In order to distinguish these possibilities experiments were conducted which involved NOE energy transfer between netropsin and DNA protons in the drug-DNA complex. Difference NOE spectra of netropsin-poly(dA).poly(dT) complex in which AH2 was irradiated indicate that dominant NOEs were observed at the imino and pyrrole ring protons of netropsin. When the netropsin pyrrole ring protons were irradiated, the magnetization transfer was at AH2 of DNA. These observations suggest that netropsin binds to the dA-strand of poly(dA).poly(dT) even though dA/dT strands are conformationally equivalent.     

Binding of netropsin to DNA in complexes with polypeptides containing repetitive lysine sequences

The interaction of the antibiotic netropsin with calf thymus DNA, T4 DNA and poly(dA-dT) . poly(dA-dT) in complexes with sequential polypeptides containing repetitive lysine sequences and histone H1 was investigated using circular dichroism spectroscopy and equilibrium dialysis. Both soluble DNA-polypeptide complexes and insoluble complexes showed binding of netropsin. The possibility of displacement of polypeptides from DNA binding sites by competition with netropsin molecules was eliminated by experiments using 14C-labelled polypeptides. From the analysis of CD titration behavior as well as from the results of equilibrium dialysis studies it follows that netropsin does not compete with polypeptides for DNA binding sites, which suggests that these two ligands occupy different sites. Various explanations for minor differences in the CD behavior of the bound netropsin in the saturation region are also discussed.     

Binding of mithramycin to DNA in the presence of second drugs

Comparative DNA equilibrium binding studies with mithramycin (MTR) and ethidium bromide in the presence and in the absence of second drugs were investigated by spectral titrations. Unusual curvatures (in contrast to those due to neighbor exclusion or anticooperativity) are found in the Scatchard plots of MTR-DNA titrations in the presence of netropsin, a minor-groove binder. Parallel studies with ethidium bromide indicate that although the presence of netropsin significantly reduces the binding ability of ethidium, no unusually curved Scatchard plots are obtained. The unusual curvature exhibited by the Scatchard plots of MTR titrations in the presence of netropsin indicates that the binding of netropsin greatly affects the MTR binding to DNA and can be simulated by an explicit incorporation of the second drug-DNA interaction in the binding formalism. Since netropsin is a minor-groove binder, its interference with the binding of MTR is in accord with the notion that MTR also binds at this groove. The observation of negligible effects on the DNA binding ability of MTR in the presence of either a major-groove or a phosphate group binder lends further support to this conclusion. Consistent with its guanine specificity, studies with synthetic polynucleotides suggest that MTR exhibits negligible affinity for poly(dA-dT).poly(dA-dT) or poly(dA).poly(dT).(ABSTRACT TRUNCATED AT 250 WORDS)     

Rate enhancements in the DNase I footprinting experiment.

Footprinting experiments for DNase I digests of a 139-base-pair segment of pBR-322 DNA in the presence of either netropsin or actinomycin D were carried out. Plots of oligonucleotide concentration as a function of drug concentration were analyzed to study the enhancement in cleavage rates at approximately 30 sites, accompanying drug binding at other sites. The pattern of enhancements is not consistent with drug-induced DNA structural changes, but agrees with a redistribution mechanism involving DNase I. Since the total number of enzyme molecules per fragment remains unchanged, drug binding at some sites increases the enzyme concentration at other sites, giving rise to increased cleavage. The consequences of the redistribution mechanism for analysis of footprinting experiments are indicated.     

Studies on the Action of the Nuclear Factor Promoting Actinomycin D-Binding Capacity of Chromatin

It is well known that actinomycin D binds to C-G pairs of DNA. The amount of actinomycin D bound to chromatin thus depends directly on the demasked sites of chromatin DNA. The actinomycin D binding of rat liver chromatin, obtained by the method of Dingman and Sporn, was studied in the presence and absence of liver and kidney nuclear extracts (NE). The actinomycin D binding of liver chromatin increases greatly under the action of liver nuclear extract. No changes occur in liver chromatin actinomycin D binding capacity after the action of kidney NE. The removal of protein or RNA from liver NE removes its ability to change the actinomycin D binding capacity of the liver chromatin. According to the obtained results it may be assumed that the nuclear extract contains the factor which plays a role in controlling cell differentiation.     

Studies on the action of the nuclear factor promoting actinomycin D-binding capacity of chromatin

It is well known that actinomycin D binds to C-G pairs of DNA. The amount of actinomycin D bound to chromatin thus depends directly on the demasked sites of chromatin DNA. The actinomycin D binding of rat liver chromatin, obtained by the method of Dingman and Sporn, was studied in the presence and absence of liver and kidney nuclear extracts (NE). The actinomycin D binding of liver chromatin increases greatly under the action of liver nuclear extract. No changes occur in liver chromatin actinomycin D binding capacity after the action of kidney NE. The removal of protein or RNA from liver NE removes its ability to change the actinomycin D binding capacity of the liver chromatin. According to the obtained results it may be assumed that the nuclear extract contains the factor which plays a role in controlling cell differentiation.     

Changes in drug 13C NMR chemical shifts as a tool for monitoring interactions with DNA

The antibiotic drug, netropsin, was complexed with the DNA oligonucleotide duplex [d(GGTATACC)]2 to monitor drug 13C NMR chemical shifts changes. The binding mode of netropsin to the minor groove of DNA is well-known, and served as a good model for evaluating the relative sensitivity of 13C chemical shifts to hydrogen bonding. Large downfield shifts were observed for four resonances of carbons that neighbor sites which are known to form hydrogen bond interactions with the DNA minor groove. Many of the remaining resonances of netropsin exhibit shielding or relatively smaller deshielding changes. Based on the model system presented here, large deshielding NMR shift changes of a ligand upon macromolecule binding can likely be attributed to hydrogen bond formation at nearby sites.     

Transferring the purine 2-amino group from guanines to adenines in DNA changes the sequence-specific binding of antibiotics.

The proposition that the 2-amino group of guanine plays a critical role in determining how antibiotics recognise their binding sites in DNA has been tested by relocating it, using tyrT DNA derivative molecules substituted with inosine plus 2,6-diaminopurine (DAP). Irrespective of their mode of interaction with DNA, such GC-specific antibiotics as actinomycin, echinomycin, mithramycin and chromomycin find new binding sites associated with DAP-containing sequences and are excluded from former canonical sites containing I.C base pairs. The converse is found to be the case for a group of normally AT-selective ligands which bind in the minor groove of the helix, such as netropsin: their preferred sites become shifted to IC-rich clusters. Thus the binding sites of all these antibiotics strictly follow the placement of the purine 2-amino group, which accordingly must serve as both a positive and negative effector. The footprinting profile of the 'threading' intercalator nogalamycin is potentiated in DAP plus inosine-substituted DNA but otherwise remains much the same as seen with natural DNA. The interaction of echinomycin with sites containing the TpDAP step in doubly substituted DNA appears much stronger than its interaction with CpG-containing sites in natural DNA.