Interactions between distamycin A analogs bound to DNA

The experimental binding isotherms of the distamycin A analog to 8 natural and synthetic DNAs were analyzed. The shapes of binding isotherms suggest that the bound ligand molecule induces transitions of DNA (B-form) into two perturbated conformation states. These transitions are responsible for the existence of positive and negative cooperative effects on binding of distamycin analogs to DNA. At low levels of binding positive cooperative effects play a dominating role whereas at high levels of binding negative cooperative effects are observed. These cooperative effects can be described by the aid of a potential of pairwise interactions between nearest neighbour bound antibiotic molecules. A detailed analysis of experimental binding isotherms shows that characteristic distances over which these interactions are extended depend on the AT content of DNA. The energetical and structural parameters characterising the allosteric transitions of DNA to the perturbated states are obtained.     

Rates of heat-induced DNA purine alterations in synthetic polydeoxyribonucleotides

Damage to DNA by heat can occur at physiological conditions. The effects of the varying conformational states adopted by double-stranded DNA on the incidences and distributions of thermally induced hydrolytic purine alterations are unknown. The possible role of conformational changes on damage by heat to purines in DNA polymers was therefore investigated. Model compounds used were the synthetic alternating copolymer poly(dG-dC):poly(dG-dC) and the homopolymer poly(dG):poly(dC). Base damages were assayed by high performance liquid chromatography using polymers radioactively labeled in guanine. Conformational states were assayed by circular dichroic spectral changes. Incubation and heating of the polymers in 1 mM Mn2+ caused the spectral shift reported for the left-handed Z-DNA conformation in the alternating copolymer and the change reported for the triple helix in the homopolymer. After incubation at 85 degrees C., incidences of base damages were compared between the polymers. No deamination of guanine to xanthine was observed under any conditions. The presence of manganese reduced depurination in both polymers. Rates of guanine imidazole ring openings to yield 2,6-diamino-4-hydroxy-5-formamidopyrimidine were increased in the presence of the cation and constituted the chief form of purine damage in the homopolymer. Therefore, the distribution of heat-induced DNA alterations within the genome may be determined by DNA conformational states. This observed opening of purine imidazole rings in the presence of manganese ions may have mutagenic consequences and may be involved in carcinogenesis by metals.     

Interaction of the peptide antibiotic bacitracin with DNA and synthetic polynucleotides

The aim of the present paper was to study the specific character of interaction of peptide antibiotic bacitracin with DNA and to estimate the interaction constant. The influence of bacitracin on bacteriophage DNA restriction by HindIII and SmaI endonucleases was studied. The possibility of arranging the polynucleotide template by small ligands was shown.     

Design, synthesis, and intracellular localization of a fluorescently labeled DNA binding polyamide related to the antibiotic distamycin

The design and synthesis of the lipophilic (9) and fluorescent (10) conjugates of a structural analogue of distamycin and their in vitro cellular localization studies are reported. Confocal laser scanning microscopy (CLSM) indicates that 10 rapidly enters human ovarian adenocarcinoma (SKOV-3) cells with principal uptake in mitochondria and uniform cytoplasmic distribution.     

Specific interactions of distamycin with G-quadruplex DNA

Distamycin binds the minor groove of duplex DNA at AT-rich regions and has been a valuable probe of protein interactions with double-stranded DNA. We find that distamycin can also inhibit protein interactions with G-quadruplex (G4) DNA, a stable four-stranded structure in which the repeating unit is a G-quartet. Using NMR, we show that distamycin binds specifically to G4 DNA, stacking on the terminal G-quartets and contacting the flanking bases. These results demonstrate the utility of distamycin as a probe of G4 DNA–protein interactions and show that there are (at least) two distinct modes of protein–G4 DNA recognition which can be distinguished by sensitivity to distamycin.     

Interaction of distamycin A and netropsin with quadruplex and duplex structures: a comparative 1H-NMR study

Homonuclear NMR techniques have been used to investigate the interactions of the minor groove binding agents distamycin A (Dist-A) and the related drug netropsin (Net) with three quadruplexes characterized by different groove widths: [d(TGGGGT)]4 (Q1), [d(GGGGTTTTGGGG)]2 (Q2), and d(GGGGTTGGGGTGTGGGGTTGGGG) (Q3). Netropsin has been found to be in a fast chemical exchange with all three kinds of quadruplexes, whereas Dist-A interacts tightly with Q1 and, at a less extent, with Q2. In order to determine the degree of selectivity of Dist-A for two- rather than four-stranded DNA, we titrated with Dist-A an equimolar solution of Ql and the duplex d(CGCAAATTTGCG)2 (D). This comparative 1H-NMR study allowed us to conclude that Dist-A and, consequently, Net possess higher affinity for duplex DNA.     

Specific interactions of distamycin A and its analogs with (A-T) rich and (G-C) rich duplex regions of DNA and deoxypolynucleotides.

The specific interaction of distamycin A and analogs with DNA's and synthetic deoxypolynucleotide duplexes were studied in detail by means of circular dichroism and the data were analyzed together with viscosity results of several natural DNA's. At low ligand to nucleotide ratio the previously reported specific binding to (A-T) pairs of DNA is verified by a highly favoured interaction with (A-T)-enriched segments of distamycins containing four and five methylpyrrole carboxamide units. At higher distamycin concentration a second specific binding to (G-C) pairs most probably through hydrogen bonding is established. Viscometric results suggest a distamycin-induced local bending of the helix and could support the idea of a preferential alignment of the ligand molecule along only one strand in the groove which differs from the netropsin interaction mechanism. The possibility of an overlapping binding of the oligopeptides in the small groove is discussed.     

Modification of DAPI banding on human chromosomes by prestaining with a DNA-binding oligopeptide antibiotic, distamycin A

A DNA-binding AT-specific oligopeptide antibiotic, distamycin A, was used as non-fluorescent counterstain in conjunction with the DNA-binding AT-specific fluorochrome 4′-6-diamidino-2-phenylindole (DAPI) to investigate the effect of the antibiotic on DAPI fluorescent banding of human chromosomes. Distamycin A-pretreated metaphases and interphase nuclei exhibited a significantly lower overall fluorescence intensity than DAPI controls. Chromosome arms were pale and intercalary DAPI bands (Q bands) were obliterated, but some specific regions of constitutive heterochromatin remained brightly fluorescent. These were mainly the constrictions of chromosomes 1, 9 and 16, the short arm of chromosome 15, and the distal part of the Y. The distamycin A/DAPI banding pattern appears to be comparable to that reported for anti-5-methylcytosine binding [11]. The observations are discussed as they relate to the roles of chromosomal DNAs and proteins in chromosome banding.     

Interaction of synthetic analogs of distamycin with DNA. Role of the conjugated N-methylpyrrole system in specificity of binding

Interaction of two synthetic analogs of distamycin (Dst), PPA and PAP, containing a saturated beta-alanine moiety replacing one N-methylpyrrole ring, with different polynucleotides and natural DNAs were studied using UV and CD spectroscopy. The results indicate that, similar to Dst, these analogs bind to DNA via the minor groove with a specificity towards AT-base pairs. It may be proposed that pyrrole chromophores in Dst probably do not play a role in the AT-base selectivity exhibited by Dst.     

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.     

Adduct formation between the carcinogen N-acetoxy-2-acetylaminofluorene and synthetic polydeoxyribonucleotides.

The chemical carcinogen N-acetoxy-2-acetylaminofluorene (NA-AAF) was reacted with poly(dG-dC) - poly(dG-dC); poly dG - poly dC; poly(dA-dT) - poly (dA-dT); and poly dA - poly dT under a variety of conditions. Poly (dG-homo GC polymer and 10--20 more reactive the A + T polymers. Lowering the ionic strength increased the extent of reaction, while pH change (8.9 vs. 5.5) had only a small effect. If ionic strength was adjusted so that the two guanine-containing polymers showed equal thermal stability (as judged by Tm) then the alternating copolymer was 7 times as reactive as the homopolymer. In aggreement with previous investigators, the major product was found to be 8-(N-2-fluorenylacetamido) deoxyguanosine.     

Interactions of meso-tetra-(4-N-oxyethylpyridyl) porphyrin, its 3-N analog and their metallocomplexes with duplex DNA

Interactions of meso-tetra-(4-N-oxyethylpyridyl) porphyrin (TOEPyP(4)), its 3-N analog (TOEPyP(3)) and their Co, Cu, Ni, Zn metallocomplexes with duplex DNA have been investigated by uv/visible absorbance and circular dichrosim spectroscopies. Results reveal the interactions of these complexes with duplex DNA are of two types. (1) External binding of duplex DNA by metalloporphyrins containing Zn and Co, and (2) Binding of duplex DNA both externally and internally (by intercalation) by porphyrins not containing metals, and metalloporphyrins containing Cu and Ni. Results indicate that (4N-oxyethylpyridyl) porphyrins intercalate more preferably in the structure of duplex DNA and have weaker external binding than 3N-porphyrins.     

Nucleotide sequences in DNA showing preferential binding with distamycin A and acridine derivatives

The specificity of DNA X dye binding was studied. Antibiotic distamycin A was bound most strongly to the DNA sequences composed of three or more neighboring A X T pairs. Acrichin and 7-aminoacrichin proved to be weak specific inhibitors binding predominantly within the A X T regions.     

Effect of DNA groove binder distamycin A upon chromatin structure

Background

Distamycin A is a prototype minor groove binder, which binds to B-form DNA, preferentially at A/T rich sites. Extensive work in the past few decades has characterized the binding at the level of double stranded DNA. However, effect of the same on physiological DNA, i.e. DNA complexed in chromatin, has not been well studied. Here we elucidate from a structural perspective, the interaction of distamycin with soluble chromatin, isolated from Sprague-Dawley rat.

Methodology/Principal Findings

Chromatin is a hierarchical assemblage of DNA and protein. Therefore, in order to characterize the interaction of the same with distamycin, we have classified the system into various levels, according to the requirements of the method adopted, and the information to be obtained. Isothermal titration calorimetry has been employed to characterize the binding at the levels of chromatin, chromatosome and chromosomal DNA. Thermodynamic parameters obtained thereof, identify enthalpy as the driving force for the association, with comparable binding affinity and free energy for chromatin and chromosomal DNA. Reaction enthalpies at different temperatures were utilized to evaluate the change in specific heat capacity (ΔCp), which, in turn, indicated a possible binding associated structural change. Ligand induced structural alterations have been monitored by two complementary methods - dynamic light scattering, and transmission electron microscopy. They indicate compaction of chromatin. Using transmission electron microscopy, we have visualized the effect of distamycin upon chromatin architecture at di- and trinucleosome levels. Our results elucidate the simultaneous involvement of linker bending and internucleosomal angle contraction in compaction process induced by distamycin.

Conclusions/Significance

We summarize here, for the first time, the thermodynamic parameters for the interaction of distamycin with soluble chromatin, and elucidate its effect on chromatin architecture. The study provides insight into a ligand induced compaction phenomenon, and suggests new mechanisms of chromatin architectural alteration.     

Quantitative estimation of the contribution of pyrrolcarboxamide groups of the antibiotic distamycin A into specificity of its binding to DNA AT pairs.

Interaction of DNA with the analogs of the antibiotic distamycin A having different numbers of pyrrolcarboxamide groups and labeled with dansyl was studied. The binding isoterms of the analogs to synthetic polydeoxyribonucleotides were obtained. Analysis of the experimental data leads to the following conclusions: (1) the free energy of binding of the analogs to poly(dA).poly(dT) depends linearly on the number of amide groups in the molecule of the analog whereas attachment of each pyrrolcarboxamide group produces changes of 2 kcal/mole in the free energy; (2) attachment of a pyrrolcarboxamide unit to the GC pair results in the free energy change of 0.95 kcal/mole; (3) the binding of analogs to poly(dA).poly(dT) is a cooperative process, presumbly, dependent on conformational changes induced by the binding of analogs to DNA.     

Selective binding of actinomycin D and distamycin A to DNA.

The exact sites at which a number of drugs inhibit the nick translation of DNA by E.coli DNA polymerase-I have been pinpointed. In order to do this, a method has been developed for sequencing double-stranded plasmid DNA from the site of a specifically induced nick. The initial experiments have concentrated on analysis of drug inhibition of nick translation in a 200 nucleotide region near the Eco Rl origin of pBR313. Many drugs were found to inhibit nick translation in a highly sequence specific manner. For actinomycin D, significant inhibition occurred at just four sites in the nucleotide sequence under test and only one sequence (pGpCpGpCpGpGp) gave really strong inhibition. Distamycin A gave a different pattern of inhibition with particularly strong stops in just two of the many A-T rich regions in the DNA. Experiments with caffeine suggest that factors in addition to primary sequence are important in determining where major inhibition occurs.     

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.