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At least 19 recordsLinked to original sources

PubMed1977

Netropsin-poly(dA-dT) complex in solution: structure and dynamics of antibiotic-free base pair regions and those centered on bound netropsin.

The biphasic duplex-to-strand transition for the netropsin.poly(dA-dT) complex, phosphate/drug mole ratio (P/D) = 50, has been investigated by high-resolution proton nuclear magnetic resonance (NMR) spectroscopy at the nonexchangeable base and sugar protons in 0.1 M cacodylate solution. The NMR spectral parameters monitor the structure and dynamics of the opening of antibiotic-free base pair regions (55 degrees-65 degrees) and the opening of base regions centered on bound netropsin (90 degrees-100 degrees). The gradual addition of netropsin to poly(dA-dT) results in structural perturbations extending into the antibiotic-free base pair regions that begin to level off above 0.02 antibiotic molecules per polynucleotide phosphate (P/D = 50). The NMR chemical shift parameters at the antibiotic-free base pair regions in the P/D = 50 complex suggest changes in the glycosidic torsion angles of the deoxyadenosine and thymidine residues and less pronounced changes in the base pair overlap geometries. The dissociation rates of the antibiotic-free base pair regions are at least an order of magnitude slower in the P/D = 50 netropsin.poly(dA-dT) complex compared to related parameters for poly(dA-dT) and the P/D = 50 ethidium bromide-poly(dA-dT) complex. There is decreased segmental mobility at the antibiotic-free strand regions in the temperature range (65 degrees-90 degrees) between the two transitions in the biphasic melting curve of the P/D = 50 netropsin-poly(dA-dT) complex. Netropsin stabilizes at least five base pairs, with their center at its binding site.

Guanidines↗

PubMed1978

Magnetic circular dichroism of netropsin and natural circular dichroism of the netropsin-DNA complex.

We report the first measurement of the magnetic circular dichroism (MCD) of the basic polypeptide antibiotic netropsin (Nt). The MCD shows that the longest wavelength absorption band of Nt is the sum of more than one component and permits a radically new interpretation of the circular dichroism of the complex which Nt forms with DNA. We conclude that Nt has no major effect on the CD and thus the helical structure of the bases of the DNA to which it is bound. Thus the ability of Nt to inhibit the function of DNA polymerase, RNA polymerase, and the photoreactivating enzyme must be mediated by factors other than a distortion of the helical structure of the bases.

Animals↗

PubMed1992

Drug binding to higher ordered DNA structures: netropsin complexation with a nucleic acid triple helix.

We have used a combination of spectroscopic and calorimetric techniques to characterize how netropsin, a ligand that binds in the minor groove of DNA, influences the properties of a DNA triple helix. Specifically, our data allow us to reach the following conclusions: (i) netropsin binds to the triplex without displacing the major-groove-bound third strand; (ii) netropsin binding to the triplex exhibits a lower saturation binding density (7.0 base triplets per netropsin bound) than netropsin binding to the corresponding duplex (5.5 base pairs per netropsin bound); (iii) the netropsin-free and the netropsin-bound triplexes each melt in two well-resolved transitions, initial conversion of the triplex to the duplex state followed by duplex melting to the component single-stranded states; (iv) netropsin remains bound to DNA as the triplex melts to the duplex state; (v) netropsin binding thermally destabilizes the triplex in equilibrium with duplex equilibrium dramatically, while thermally stabilizing the duplex to single-strand equilibrium; (vi) netropsin binding to the triplex is enthalpically 4 times more favorable (more exothermic) than netropsin binding to the corresponding duplex; (vii) netropsin binding to the triplex decreases the cooperativity of the triplex----duplex melting event. These results demonstrate that occupancy of the minor groove of a triplex by a ligand such as netropsin can exert a profound impact on the properties of the host triplex, particularly with regard to the equilibrium in which the third strand is expelled from the major groove. Thus, our results reveal considerable major groove/minor groove crosstalk. Such knowledge may prove of practical importance by providing an approach for modulating the affinity and specificity of major-groove-binding third strands in triplex-forming protocols designed to target specific duplex domains. Fundamentally, our results provide insights into the crosstalk that can result when ligands bind to the two major receptor sites of duplex DNA--namely, the major and minor grooves.

Calorimetry, Differential Scanning↗

PubMedDate not supplied

[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.

Amino Acid Sequence↗

PubMed1992

Structural consequences of a carcinogenic alkylation lesion on DNA: effect of O6-ethylguanine on the molecular structure of the d(CGC[e6G]AATTCGCG)-netropsin complex.

Exposure of cells to alkylating agents produces DNA lesions, most of which are repaired. However some alkyl lesions persist and play a role in inducing point mutations and the subsequent carcinogenic conversion. O6-Ethylguanine (e6G) is a relatively persistent alkylation lesion caused by the exposure of DNA to N-ethyl-N-nitrosourea. We study the consequence of the e6G incorporation in DNA by X-ray crystallography. We have obtained crystals of the modified DNA dodecamer d(CGC[e6G]AATTCGCG) and the unmodified d(CGCGAATTCGCG), complexed to the minor groove binding drug netropsin. The space group of both crystals is P2(1)2(1)2(1), isomorphous to other related dodecamer DNA crystals. The structures have been solved by the molecular replacement method and refined by the constrained least-squares procedure to R-factors of approximately 16% at resolution of approximately 2.5 A. The two independent e6G-C base pairs in the DNA duplex adopt different base-pairing schemes. The e6G4-C21 base pair has a configuration similar to a normal Watson-Crick base pair, except with one three-centered hydrogen bond pair and one direct hydrogen bond between e6G4 and C21. In contrast, the e6G16-C9 base pair adopts a wobble configuration. The ethyl group is in the proximal orientation (to N7) in both base pairs. These observations enrich and support those found in the crystal structure of d(CGC[e6G]AATTCGCG), complexed to minor groove binding drugs Hoechst 33258 and Hoechst 33342 [Sriram et al. (1992) EMBO J. 11, 225-232]. We suggest that a dynamic equilibrium between these two configurations for the e6G-C base pair is likely and would present an ambiguous signal to the cellular transcription, replication, or repair mechanisms. In contrast, thymine can pair with e6G in only one way, albeit imperfect, mimicking a Watson-Crick base pair. This may be a plausible explanation of why thymine is found preferentially incorporated across the e6G during replication. In addition, we analyze the influence of the alkylation lesion on DNA and the molecular details of netropsin-DNA interaction. In the present two new netropsin complexes, the netropsin spans across five base pairs (starting halfway between C3-G22 and e6G4-C21 base pairs and ending at T8-A17 base pair) in the narrow minor groove. This is in contrast to the earlier crystal structure of netropsin complexed with another DNA dodecamer having the same AATT central core sequence, d(CGCGAATT[br5C]GCG) [Kopka et al. (1985) J. Mol. Biol. 272, 390-395]. In the latter structure, the netropsin lies between G4-br5C21 and br5C9-G16 base pairs.(ABSTRACT TRUNCATED AT 400 WORDS)

Alkylation↗

PubMed1992

Thermodynamics of interaction of a fluorescent DNA oligomer with the anti-tumour drug netropsin.

Fluorescence spectroscopy was used to study the interaction between the minor-groove-binding drug netropsin and the self-complementary oligonucleotide d(CTGAnPTTCAG)2 containing the fluorescent base analogue 2-aminopurine (nP). The binding of netropsin to this oligonucleotide causes strong quenching of the 2-aminopurine fluorescence, observed by steady-state as well as time-resolved spectroscopy. From fluorescence titrations, binding isotherms were recorded and evaluated. The parameters showed one netropsin binding site/oligonucleotide duplex and an association constant of about 10(5) M-1 at 25 degrees C, 3-4 orders of magnitude weaker than for an exclusive adenine/thymine host sequence. From the temperature dependence of the association constant the thermodynamic parameters were obtained as delta G = -29 kJ/mol, delta H = -12 kJ/mol and delta S = +55 J.mol-1.K-1 at 25 degrees C. These parameters resemble those of the interaction of poly[(dG-dC).(dG-dC)] with netropsin, indicating a mainly entropy-driven reaction. The amino group of 2-aminopurine, like that of guanine, resides in the minor groove of DNA. Therefore the relatively weak binding of netropsin to d(CTGAnPTTCAG)2 is probably related to partial blockage of the tight fit of netropsin into the preferred minor groove of an exclusive adenine/thymine host sequence.

2-Aminopurine↗

PubMed1975

Identification and separation of components of calf thymus DNA using a CsC1-netropsin density gradient.

Calf thymus DNA containing satellite components of various densities was used as a model to study the effect of netropsin on the density of DNA in a CsCl gradient. The binding of netropsin resulted in a decrease in density which depended upon the quantity of netropsin added and on the average composition of the DNA. Differences in density of DNA components were higher in CsCl - netropsin gradients than in simple CsCl gradients. By use of netropsin a main band and four satellite bands could be differentiated in calf thymus DNA. Satellite DNA's were isolated using preparativeCsCl - netropsin gradient centrifugation and were characterised by density and homogeneity in native and in reassociated state. Two of the satellite components, with densities of 1.722 and 1.714 g/cm minus 3, are probably of homogenous sequence, the other two components of densities 1.709 and 1.705 g/cm minus 3 appear to be heterogeneous.

Animals↗

PubMed1992

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.

Base Sequence↗

PubMed1992

The formation of A-DNA in NaDNA films is suppressed by netropsin.

Oriented films of NaDNA complexed with netropsin were studied with deuterium nuclear magnetic resonance (2H NMR), X-ray diffraction and ultraviolet (UV) linear dichroism to obtain information about the influence of netropsin on the structural arrangement of the DNA bases and on the B-A transition. The results of these studies clearly demonstrate a strong suppression of the formation of A-DNA at relative humidities (RHs) down to about 50%. The suppression was complete in the NaDNA-netropsin complex studied with 2H NMR which had a netropsin input ratio, r, of 0.22 drug/base pair. The sample used for UV linear dichroism had a similar input ratio while the X-ray diffraction samples had input ratios between 0.033 and 0.39 drug/base pair. Together, the results of these studies are in agreement with previous infrared (IR) linear dichroism studies of the conformation of the sugar-phosphate backbone in NaDNA-netropsin complexes, which showed that the B-A transition is suppressed for r-values down to approximately 0.1 drug/base pair (Fritzsche, H., Rupprecht, A. and Richter, M., Nucleic Acids Res. 12 (1984) 9165-9177).

Animals↗

PubMed1975

The compatibility of netropsin and actinomycin binding to natural deoxyribonucleic acid.

The simultaneous binding of netropsin and actinomycin to four natural DNAs was studied to determine the influence of one ligand on the binding of the other. Actinomycin binds specifically to GC sites, whereas netropsin binds specifically to AT sites. Spectral titrations, thermal denaturation, and analytical buoyant density centrifugation were employed to measure the binding interference of these drugs. The binding of actinomycin to DNA was decreased by the presence of netropsin. Increasing the GC content of the DNA resulted in a decreased effect of netropsin on actinomycin binding. Quantitative analysis of the binding parameters indicated that netropsin and actinomycin can bind in close proximity along the DNA chain. Supercoiled DNA gave the same result as linear DNA. These results imply that DNA can absorb alterations in conformation within a short distance.

Anti-Bacterial Agents↗

PubMed1978

The use of netropsin with CsCl gradients for the analysis of DNA and its application to restriction nuclease fragments of ribosomal DNA from Physarum polycephalum.

Netropsin binds to DNA in caesium chloride density gradients and reduces the density of the DNA. The DNA is saturated at a netropsin/DNA weight ratio of about 6 and the change in density, deltarho, at saturation is given by deltarho = -109 (dA + dT content)1.87 mg/ml for the six DNAs tested covering dA + dT contents from 0.28 to 0.69. At lower netropsin/DNA ratios the observed density shifts are consistent with a two-site model for netropsin binding to DNA. Netropsin approximately doubles the resolution of Physarum polycephalum nucleolar satellite DNA from main-band DNA. The fragments of P. polycephalum nucleolar satellite DNA obtained with the restriction endonuclease HindIII do not separate on CsCl gradients, even in the presence of netropsin, which shows that the transcribed and non-transcribed sequences in this DNA have similar nucleotide compositions.

Centrifugation, Density Gradient↗

PubMed1979

Netropsin . dG-dG-dA-dA-dT-dT-dC-dC complex. Antibiotic binding at adenine . thymine base pairs in the minor groove of the self-complementary octanucleotide duplex.

The structure of the netropsin . dG-dG-dA-dA-dT-dT-dC-dC complex (one antibiotic molecule/self-complementary octanucleodide duplex) and its dynamics as a function of temperature have been monitored by the nuclear magnetic resonances of the Watson-Crick protons, the nonexchangeable base and sugar protons and the backbone phosphates. The antibiotic forms a complex with the nucleic acid duplex at the dA . dT-containing tetranucleotide segment dA-dA-dT-dT, with slow migration amongst potential binding sites at low temperature. The downfield shifts in the exchangeable protons of netropsin on complex formation demonstrate the contributions of hydrogen-bonding interactions between the antibiotic and the nucleic acid to the stability of the complex. Complex formation results in changes in the glycosidic torsion angles of both thymidine residues and one deoxyadenosine residue as monitored by chemical shift changes in the thymine C-6 and adenine C-8 protons. The close proximity of the pyrrole rings of the antibiotic and the base-pair edges in the minor groove is manifested in the downfield shifts (0.3--0.5 ppm) of the pyrrole C-3 protons of netropsin and one adenine C-2 proton and one thymine N-3 base-pair proton on complex formation. The internucleotide phosphates of the octanucleotide undergo 31P chemical shift changes on addition of netropsin and these may reflect, in part, contributions from electrostatic interactions between the charged ends of the antibiotic and the backbone phosphates of the nucleic acid.

Base Composition↗

PubMed1976

Effect of netropsin on the derepression of enzymes during growth and sporulation of Bacillus subtilis.

Netropsin, a polypeptide antibiotic which binds specifically to adenylate-thymidylate-rich regions of deoxyribonucleic acid, inhibitis sporulation at about stage II, but does not inhibit growth of Bacillys subtilis. An analysis of the sporulation-associated enzymes aconitase, alkaline phosphatase, and glucose dehydrogenase revealed that their rates of expression were not affected by the presence of the antibiotic. The derepression of histidase, a vegetatively induced enzyme was stimulated by netropsin. Oxygen utilization by the cells during sporulation was not effected nor was spore germination prevented by the drug. Netropsin, however, did prevent the formation of dipicolinic acid. These and earlier results suggest that netropsin may be affecting the transcription of only select sporulation genes that are particularly rich in adenylate-thymidylate base pairs.

Aconitate Hydratase↗

PubMed1975

(dA-dT) dependent inactivation of the DNA template properties by interaction with netropsin and distamycin A.

The inhibitory effect of the polypeptide antibiotics netropsin and distamycin A on DNA dependent nucleic acid synthesis has been shown to be related to the base composition of the template DNA. A number of natural DNA's of quite different dA-dT content as well as poly (dI-dC)-poly (dI-dC), poly (dA-dT)-poly (dA-dT), poly (dA) - poly (dT) and poly (dG) - poly (dC) has been studied as templates in DNA and in part in RNA polymerase reaction. The highest binding efficiency of netropsin existing for (dA-dT) - containing DNA polymers and the less pronounced interaction with the (dI-dC)-containing polymer shown by the melting and CD spectrral behaviour of the complexes are entirely reflected in the template inactivation. The same is evident for distamycin A. However, in contrast to netropsin the antibiotic distamycin A exhibits some binding tendency to poly (dG) - poly (dC). Binding effects of a netropsin derivative to DNA and (dA-dT) -containing polymers suggest the importance of hydrogen bonds of the peptide groups in the complex formation.

Adenine Nucleotides↗

PubMed1976

Base composition heterogeneity of mammalian DNAs in CsCl-netropsin density gradient.

DNAs of 15 mammals and some lower organisms were analysed by CsCl-netropsin density gradient centrifugation. Increased resolving power of this method enabled to detect many new components in mammalian DNAs. Distinct components were detected in the density range of the main band. These components found in different mammalian DNAs have probably limited variation in the G+C content. Most of other components seems to be species specific. The DNAs of lower organisms form homogeneous band even in the presence of netropsin. The relation between densities in CsCl-netropsin and CsCl density gradient is nonlinear. This result supports a hypothesis that in high ionic strength netropsin is preferentially bound to (dA.dT) clusters.

Animals↗

PubMed1992

[Ligands possessing affinity to specific DNA base pair sequences. IX. Synthesis of netropsin and distamycin A analogs having sarcolysin residues or a platinum(II) atom].

In search for compounds capable of forming covalent bonds with DNA AT-pair clusters, distamycin A and netropsin analogues containing DL-sarcolysin or platinum (II) atom at the N-terminus of the molecule were synthesized, as well as bis-netropsin and bis-distamycin in which two netropsin- or distamycin-like fragments are bound via a cis-diammineplatinum (II) residue. It is shown that these substances can be used for the DNA selective cleavage.

Base Composition↗

PubMed1992

Binding of netropsin to a DNA triple helix.

The interaction of netropsin, a minor groove binding drug, with T-A-T triple helix and A-T double helix was studied using circular dichroism spectroscopy and thermal denaturation. The triple helix was made by an oligonucleotide (dA)12-x-(dT)12-x-(dT)12, where x is a hexaethylene glycol chain bridged between the 3' phosphate of one strand and the 5' phosphate of the following strand. This oligonucleotide is able to fold back on itself to form a very stable triplex. Changing the conditions allows the same oligonucleotide in a duplex form with a (dT)12 dangling arm. Circular dichroism spectroscopy demonstrates that netropsin can bind to the triple helical structure. Spectral analysis shows that the bound drug exhibits a conformation and an environment similar in double-stranded and in triple-stranded structure. However, the binding constant to the triple-stranded structure is found smaller than the binding constant to the double-stranded one. Thermal denaturation experiments demonstrate that netropsin destabilizes the triplex whereas it stabilizes the duplex.

Base Sequence↗

PubMed1976

Duplex structure formation between oligo(dA)'s and oligo(dT)'s generated by thymine-specific interaction with netropsin.

The formation of oligomeric duplex molecules in the presence of the antibiotic netropsin in the series p(dA)n-p(dT)n is demonstrated using low-temperature CD measurements. Addition of Netropsin to mixtures of oligomers generates the same type of CD spectra as observed for poly(dA)-poly(dT) and maintains the duplex structure at temperatures at which base pairing of free oligomers is thermodynamically unstable. The shortest chain length forming a netropsin complex by thymine-specific interaction with the oligopeptide is represented by p(dA)4-p(dt)4. Studies with sequence isomers show that adjacent thymine residues strongly favour the complex formation with the oligopeptide.

Adenine Nucleotides↗