Netropsin Binding Research Articles

Netropsin, a DNA-binding oligopeptide structural and binding studies

The crystal structure of netropsin, an oligopeptide which binds to DNA, has been determined. The molecule is bowed with the amide groups on the concave side, and the carbonyl and methyl groups on the convex side. The amide groups participate in extensive hydrogen bonding with water molecules; the charged amino end groups interact with the sulfate anions. Binding of netropsin to poly(dA) · poly(dT) under conditions of different ionic strength was also studied. Utilizing the crystallographic as well as the binding data, it is possible to build a model which explains the specificity of this antibiotic.

Influence of nucleotide sequence on dA.dT-specific binding of Netropsin to double stranded DNA.

Using CD measurements the complex formation of Netropsin (Nt) with poly(dA-dC).poly(dT-dG) and its stability against high salt concentrations is compared with that of poly(dA).poly(dT) and poly(dA-dT).POLY(DT-dA). It is experimentally shown that the insertion of a dG.dC pair in dA.dT sequences strongly reduces the specific interaction of Nt with DNA duplexes. The specificity of the interaction is strongly increased by two or more consecutive thymine residues as present in thymine isostichs of double stranded DNA's.

Conformational features of distamycin-DNA and netropsin-DNA complexes by Raman spectroscopy.

The binding of distamycin and netropsin to duplex DNA has been studied by Raman spectroscopy. Several changes occur in the Raman spectra of these drugs upon binding DNA. These changes were analyzed by assigning specific motions to the observed Raman bands through the use of molecular subunits of the drugs and normal mode calculations. Our analysis indicates that pyrrole ring and peptide group vibrations are altered upon binding to DNA. The environments of the pyrrole ring methyl groups are not affected by the binding. These data provide physical evidence consistent with a binding model in which the methyl groups on the pyrroles project away from the DNA and the peptide N-H groups form hydrogen bonds with the DNA.

Photoreaction of psoralen and other furocoumarins with nucleic acids

The 360-nm photoinitiated reactions of certain furo[3,2-g]coumarins with DNA have been examined using ethidium fluorescence assays. Psoralent at 1.85 × 10 −4 M gives 3.3 × 10 −5, 1.8 × 10 −5, and 4.5 × 10 −6 interstrand cross-links/nucleotide with DNAs of (A + T) content 70, 60, and 50%, respectively. The relative rates of cross-linking of λ-DNA are 4-methylpsoralen > psoralen > angelicin ⪢ 4-phenylpsoralen. Angelicin (isopsoralen) gives a small (12–14%) but reproducible amount of DNA interstrand cross-links. Addition of netropsin, an antibiotic that binds preferentially to (A + T)-rich regions, to Clostridium perfringens DNA reduces the extent of cross-linking by psoralen from 66 to 10% in 50 min. In contrast, pretreatment of DNA with olivomycin or chromomycin A 3 [which bind to (G + C)-rich regions] has little effect on psoralen cross-linking. Relative rates of monoadduction of furocoumarins to PM2-CCC-DNA detected by thermal depyrimidation and alkaline strand scission is angelicin > 4-methyl-4′,5′-dihydropsoralen > 4′,5′-dihydropsoralen > 3,4-dihydropsoralen (no monoadduction), indicating angelicin is suitable for photolabeling of chromatin. Binding of netropsin to the PM2-DNA prevents cross-linking by angelicin but permits and enhances monoadduction. In contrast neither olivomycin nor chromomycin affects the reaction of angelicin with DNA. In the frozen solution, where the photoinduced cross-linking of DNA by psoralen may be suppressed, psoralen forms monoadducts about twice as readily as angelicin. Subsequent 360-nm irradiation of the psoralen monoadducts at ambient temperatures (and in separate experiments after dialysis to remove unreacted psoralen) completes the cross-links.

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.

Accessibility of the minor groove of DNA in chromatin to the binding of antibiotics netropsin and distamycin A.

The interaction of the antibiotics distamycin A, distamycin analogue and netropsin with chromatin of calf thymus has been studied by circular dichroism measurements and by gel filtration. The minor groove of DNA in chromatin is accessible by 83-89% to the binding of these antibiotics as compared with that of free DNA. The present results combined with our data on the methylation of chromatin with dimethylsulphate [3] strongly suggest that the minor groove of DNA in chromatin is not occupied by chromatin proteins.

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.

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.

Netropsin: A SPECIFIC PROBE FOR A-T REGIONS OF DUPLEX DEOXYRIBONUCLEIC ACID

Abstract The binding of netropsin to 31 different natural and biosynthetic DNAs, DNA-RNA hybrids, and RNAs was studied in order to delineate the nucleic acid structural features necessary for binding. The measurements employed were spectral titrations, analytical density gradient centrifugation, thermal denaturation, equilibrium dialysis, and inhibition of in vitro replication and in vitro transcription. The major conclusions are: (a) in 0.1 m or higher salt concentrations, netropsin has a marked specificity for DNAs which contain A-T (or I-C) pairs. It binds tightly to two DNAs which contain only A-T pairs and to two DNAs which contain only I-C pairs. However, no measurable binding was found for two DNAs which contain only G-C pairs. (b) Netropsin's inability to bind to G-C paired regions is a consequence of the 2-amino group of guanine. (c) Netropsin is specific for duplex DNA; no binding was observed to five single-stranded DNAs, three helical RNAs, or two of the three DNA-RNA hybrids studied. (d) Netropsin binds to DNA by a nonintercalating mechanism, since it does not cause unwinding of supercoiled DNA. (e) Netropsin inhibits in vitro DNA and RNA synthesis by binding to the template or to the primer, or to both. (f) The closest distance between bound netropsin molecules is three base pairs. A molecular model for netropsin binding in the minor groove of DNA is proposed.

Conformation dependent binding of netropsin and distamycin to DNA and DNA model polymers.

The binding of the antibiotics netropsin and distamycin A to DNA has been studied by thermal melting, CD and sedimentation analysis. Netropsin binds strongly at antibiotic/nucleotide ratios up to at least 0.05. CD spectra obtained using DNA model polymers reveal that netropsin binds tightly to poly (dA) . poly (dT), poly (dA-dT) . poly(dA-dT) and poly (dI-dC) . poly (dI-dC) but poorly, if at all, to poly (dG) . poly (dC). Binding curves obtained with calf thymus DNA reveal one netropsin-binding site per 6.0 nucleotides (K(a)=2.9 . 10(5) M(-1)); corresponding values for distamycin A are one site per 6.1 nucleotides with K(a)= 11.6 . 10(5) M(-1). Binding sites apparently involve predominantly A.T-rich sequences whose specific conformation determines their high affinity for the two antibiotics. It is suggested that the binding is stabilized primarily by hydrogen bonding and electrostatic interactions probably in the narrow groove of the DNA helix, but without intercalation. Any local structural deformation of the helix does not involve unwinding greater than approximately 3 degrees per bound antibiotic molecule.

Adenosine thymidine cluster-specific elongation and stiffening of DNA induced by the oligopeptide antibiotic netropsin

Conformationsl changes of DNA induced by netropsin binding have been studied by way of viscosity measurements at different DNA molecular weights. On the basis of equations derived in the Appendix for semi-rigid polymers and specialized for DNA at standard conditions, the viscosity changes are quantitatively interpreted in terms of changes of contour length and persistence length. Whereas the stiffening per bound netropsin molecule is relatively independent of the degree of binding, the elongation per one netropsin molecule bound decreases from approximately 1.5 A to zero at nearly 4 netropsin molecules/100 base pairs. A.T clusters are the sites most responsible for netropsin-induced conformationsl changes, as follows from former viscosity measurements carried out at different A + T contents. A possible role of similar conformational changes in specificity and selectivity of DNA-protein interaction is discussed.

Influence of netropsin and distamycin A on the secondary structure and template activity of DNA.

The action of netropsin and distamycin A on the DNA secondary structure and on the template properties of DNA in the DNA-and RNA-polymerase systems have been studied. Ultraviolet absorption measurements indicate characteristic hypochromic effects for the DNA-antibiotic complexes due to binding of netropsin and distamycin A to DNA. These are accompanied by the appearance of an absorption peak beyond 320 nm. This ultraviolet absorption maximum depends on the (A+T) content of DNA and disappears upon heat denaturation. These results together with earlier observations indicate an A and T specificity of the binding of the oligopeptides netropsin and distamycin A. As demonstrated by absorbance-temperature measurements netropsin increases considerably the thermal stability of DNA. Drastic changes can even be obtained with ratios below 5 moles netropsin per 100 DNA phosphate groups. The antibiotic dependent increase in the melting temperature is more pronounced for the DNA · netropsin than for the DNA · distamycin A complex. The structure of denatured DNA is also affected markedly by both antibiotics. From the results it is concluded that binding of netropsin and distamycin A to DNA is the result of their affinity to double-helical and base stacked regions. Comparison of the effect of these antibiotics on DNA-and RNA-synthesis in vitro shows that both netropsin and distamycin A inhibit the DNA- and RNA-polymerase reactions. In both systems the template activity of native as well as of denatured DNA is affected by these antibiotics. Using chromatin of Ehrlich ascites tumor cells as template it could be shown, that the nucleoprotein complex is as sensitive to the action of distamycin A as pure DNA.

Interaction of the oligopeptide antibiotics netropsin and distamycin a with nucleic acids

The influence of the oligopeptide antibiotics netropsin and distamycin A on the structure of DNA, ribosomal RNA and transfer RNA have been investigated by thermal melting, ultraviolet spectrophotometry, viscosity and optical rotatory dispersion measurements. The binding of both oligopeptides is accompanied by a pronounced stabilization of the DNA helix structure which persists up to high ionic strength (1 m-NaClO 4) while RNA is relatively unaffected at comparable ionic conditions. A · T-rich DNA exhibits a much higher increase of the melting temperature than A · T-poor DNA: in the presence of netropsin ΔT m was found to be 33 deg.C for 71 mole % A + T compared to 8 deg.C for 28 mole % A + T. A pronounced increase in the viscosity of DNA-netropsin complexes occurs up to 0.05 netropsin/ DNA-P depending on the A + T-content of the DNA. On the other hand In η rel c decreases immediately upon addition of distamycin A up to 0.05 distamycin A/DNA-P. Drastic changes were also observed in the shape of the optical rotatory dispersion curve of DNA in the presence of netropsin or distamycin A beyond 250 nm associated with an optically induced transition in the regions of 315 and 330 nm. RNA does not show a similar effect. The ultraviolet-absorption maximum of the oligopeptides is displaced towards longer wavelength on interaction with DNA. The results clearly demonstrate that the DNA structure is very sensitive to the binding of both oligopeptides while RNA is relatively insensitive. The DNA-antibiotic complexes formed are very stable due to the high affinity and specificity of netropsin and distamycin A for A · T-rich domains. Two classes of binding sites are discussed for the netropsin interaction with DNA. Distamycin A apparently seems to form intra- and intermolecular cross-links between DNA helices. The binding of netropsin and distamycin A to DNA causes a perturbation of the DNA B-conformation. The results are interpreted in terms of a contribution of the chromophore system of netropsin and distamycin A to the pronounced conformational changes observed. A tentative model for the interaction of these oligopeptides with DNA is briefly discussed.