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PubMed1982

Differential stabilization by netropsin of inducible B-like conformations in deoxyribo-, ribo- and 2'-deoxy-2'-fluororibo-adenosine containing duplexes of (dA)n . (dT)n and (dA)n . (dU)na.

Six polynucleotide duplexes containing polydeoxyadenylic acid, polyadenylic acid or poly-2'-deoxy-2'-fluoro-adenylic acid in one strand, and polydeoxyuridylic acid or polydeoxythymidylic acid in the other strand have been studied by circular dichroism, ionic strength dependence of melting temperatures and binding of the DNA specific antibiotic netropsin. Circular dichroism spectra of (dA)n . (dT)n and (dA)n . (dU)n indicated the presence of the B-form of DNA, while those of (dAfl)n . (dT)n and (rA)n . (dT)n (and the corresponding (dU)n hybrids) indicated the presence of the A-form. (dAfl)n . (dT)n and (dAfl)n . (dU)n bound netropsin only slightly less than the (dA)n containing duplexes, while replacement by (rA)n decreased netropsin binding to a large degree. Since netropsin requires B-DNA for binding, it is concluded that the A to B transition is facilitated in the case of fluorine substitution in the sugar moiety, while the 2'-OH group greatly limits this conformational change.

Adenosine↗

PubMed1980

Protection of (dA.dT) cluster regions in the DNAase I cleavage of DNA by specific interaction with netropsin.

The specific DNA binding ligand netropsin selectively blocks dA-dT base pairs in clusters containing two or more consecutive thymine residues at the dNAase I cleavage sites of DNA. Using CD and UV absorption measurements it is shown, that at various ratios of netropsin to nucleotide concentrations and even at satuation of ligand interaction the enzyme cuts along regions containing dG-dC pairs sandwiched between dA-dT pairs. This follows a slow kinetics and is associated with a release of netropsin from those segments. These facts suggests the usefulness of the partial protection of certain DNA sequences in DNAase I cleavage sites in producing DNA fragments in structural studies of the genome. A possible interpretation of the effect of netropsin binding on the enzymatic hydrolysis of phosphodiester bonds of the helix is discussed.

Animals↗

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↗

PubMed1980

Ultrastructural analysis of the effect of netropsin on sporulation of Bacillus subtilis.

An electron microscopic analysis of Bacillus subtilis cells revealed that netropsin blocked sporulation ultrastructurally at stages 0-I. These observations are consistent with previous results which indicated that cells were not committed to sporulation in the presence of the drug. Further, the addition of netropsin up to stage III of sporulation prevented the normal sharp increase in dihydrodipicolinate synthase activity which results in dipicolinic acid accumulation of stage IV. The addition of netropsin after stage III had much less effect on the synthesis of dihydrodipicolinate synthase and on sporulation. Thus, morphological events in sporulation are blocked early whereas a sporulation-associated enzyme may be affected at later stages. These data indicate than netropsin affects the expression of sporulation-associated genes in a differential manner.

Bacillus subtilis↗

PubMed2006

Configurational entropy change of netropsin and distamycin upon DNA minor-groove binding.

Binding of a small molecule to a macromolecular target reduces its conformational freedom, resulting in a negative entropy change that opposes the binding. The goal of this study is to estimate the configurational entropy change of two minor-groove-binding ligands, netropsin and distamycin, upon binding to the DNA duplex d(CGCGAAAAACGCG).d(CGCGTTTTTCGCG). Configurational entropy upper bounds based on 10-ns molecular dynamics simulations of netropsin and distamycin in solution and in complex with DNA in solution were estimated using the covariance matrix of atom-positional fluctuations. The results suggest that netropsin and distamycin lose a significant amount of configurational entropy upon binding to the DNA minor groove. The estimated changes in configurational entropy for netropsin and distamycin are -127 J K(-1) mol(-1) and -104 J K(-1) mol(-1), respectively. Estimates of the configurational entropy contributions of parts of the ligands are presented, showing that the loss of configurational entropy is comparatively more pronounced for the flexible tails than for the relatively rigid central body.

Binding Sites↗

PubMed1983

Studies on histone H1 condensing properties in complexes with DNA and polydeoxyribonucleotides using netropsin as a probe.

The binding of histone H1 with DNA and synthetic DNA duplex polymers with respect to its property to induce higher ordered structures has been studied using the DNA binding antibiotic netropsin as a probe. It was shown that the formation of distinct steps of different condensed structures (double-fibers, cable- and stem-like forms) is influenced by the ionic strength. CD titration data of DNA-H1 complexes with netropsin at 20 mM NaCl indicated no change in the binding to strong affinity sites (dA X dT clusters) as compared to free DNA's, while weak netropsin binding regions are strongly affected by competition interaction with H1. At low histone concentration the presence of netropsin favours the formation of double fibers. CD and electron microscopic findings indicated that at 20 mM NaCl the occurrence of condensed structures of DNA histone H1 complexes is not dependent on the base content. The major groove interaction of H1 most probably plays the major role in the formation of higher ordered structures. However, the minor groove binding might be involved as a secondary event. A hierarchy of relevant morphological structures observed for DNA-H1 complexes is presented.

Chemical Phenomena↗

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↗

PubMed1988

Netropsin increases formation of mRNA coding for a neutral metalloproteinase in Bacillus megaterium.

The anticancer drug netropsin increases the synthesis of an exocellular metalloproteinase during exponential growth as well as in the stationary phase of a sporulating strain of Bacillus megaterium. Its effect is due to a stimulation of the synthesis of the mRNA coding for the proteinase, determined as a residual synthesis of the enzyme in the presence of actinomycin D. The half-life of the proteinase mRNA (5-6 min at 35 degrees C) is not affected by netropsin. Netropsin relieves partially the repression of the proteinase mRNA caused by amino acids, whereas the repression brought about by an increased temperature is almost unaffected by the drug.

Amino Acids↗

PubMed1980

Cat satellite DNA. Isolation using netropsin with CsCl gradients.

The absence of centromeric bands in the karyotype of Felis catus is confirmed. It is also confirmed that no satellite band is visible in CsCl density gradients. However, a satellite is observed both by recentrifuging the fraction of the DNA that bands at high density in CsCl and by using netropsin to enhance the resolution of a CsCl gradient containing total F. catus DNA. The satellite, about 0.5% of total DNA, was isolated by repeated centrifugation in CsCl alone and in CsCl with netropsin. Netropsin was removed and a pure satellite DNA obtained. The reassociation kinetics (C0t1/2 less than 10(-3) M . s) show that the satellite is of the simple sequence type and hence a candidate for centromeric heterochromatin. Its cytological localisation awaits in situ hybridisation experiments.

Animals↗

PubMed1980

Magnetic circular dichroism study of the binding of netropsin and distamycin A with DNA.

The magnetic circular dichroism (MCD) of netropsin and distamycin-A is reported. New data for the interaction with dA ; dT base pairs in DNA were obtained from the MCD of their complexes with DNA duplex polymers. The MCD results allow an interpretation of the induced Cotton effects in the natural CD spectra of netropsin and distamycin-A complexes with DNA. While large distortions of the bases in DNA by the oligopeptide interaction is excluded, some subtle conformational variations of the DNA might explain the inhibition of the enzyme function of netropsin and distamycin-A on DNA.

Circular Dichroism↗

PubMed1990

Netropsin, distamycin and berenil interact differentially with a high-affinity binding site for the high mobility group protein HMG-I.

Netropsin, distamycin, berenil and the chromosomal protein HMG-I share the ability to bind preferentially to AT-rich regions of DNA. We studied the binding behaviour of the chemical agents towards a high-affinity binding site for HMG-I by DNase I and MPE footprinting and analyzed their ability to challenge HMG-I-DNA complexes by competition experiments. Significant differences in the binding affinities and in the efficiencies to abolish HMG-I-DNA complexes were observed for the three drugs. Netropsin proved to be the most avidly binding compound and the most efficient competitor raising the interesting possibility that netropsin affects cell growth by interfering with HMG-I-DNA interaction.

Amidines↗

PubMed1989

Subcellular distribution of a nitroxide spin-labeled netropsin in living KB cells. Electron paramagnetic resonance and sequence specificity studies.

A nitroxide spin-labeled netropsin was studied by EPR spectroscopy with respect to its uptake and localization in living KB cells. Whereas the drug was taken up readily, there was relatively little drug in the cytoplasm, but a significant concentration of the drug in the cell nucleus. The EPR signal in the latter site corresponded to a relatively freely rotating radical. The drug exhibited good intracellular stability up to 25 hr. While a delta Tm of 24 degrees between the spin-labeled netropsin and calf thymus DNA confirmed strong binding, the absence of any DNA elongation by viscometry was consistent with nonintercalative exterior binding which was confirmed to be minor groove specific by binding of the agent to T4 DNA with a delta Tm of 17.5 degrees. The sequence specificity of the DNA binding of the spin-labeled drug was confirmed by methidiumpropyl-EDTA (MPE) footprinting on a fragment of pBR322 DNA to be very similar to that of the parent netropsin, i.e. selective for AT-rich sites, with minor differences of protection afforded by introduction of the nitroxide label.

Base Sequence↗

PubMed1994

Inhibition of the in vitro integration of Moloney murine leukemia virus DNA by the DNA minor groove binder netropsin.

In search of potential inhibitors of integration of retroviral DNA into host cells genome, we have investigated the effect of the external DNA binder netropsin on the in vitro insertion of long terminal repeat (LTR) ends of Moloney murine leukemia virus (M.MuLV) as catalysed by integrase purified from baculovirus strain expression vector. In agreement with the preferential binding of netropsin to A+T rich sequences, footprinting experiments have shown that this drug selectively binds to the 5'-TTTCAT LTR end sequence which is included in the DNA binding site of integrase. This feature results in the potent inhibition of both reactions involved in the insertion process, namely, nucleolytic cleavage and strand transfer. The relation between netropsin binding to A+T rich region of M.MuLV LTR end and inhibition of insertion is strongly suggested from the inability of the drug to inhibit the insertion of HIV U3 LTR end which displays a G+C rich sequence. Selective inhibition of integration of viral DNA appears to be feasible using drugs recognizing LTR end sequences.

Amino Acid Sequence↗

PubMed1995

Mono-, di- and trimeric binding of a bis-netropsin to DNA.

An unusual 3:1 stoichiometry for complex formation between an elongated bis-netropsin compound and its binding site on DNA has been observed. Circular dichroism measurements distinguish two types of complexes formed between this bis-netropsin and poly[d(A-T)].poly[d(A-T)]. The first type is characterized by a 1:1 saturating ratio of bound molecules per ten base pairs. Formation of the second type results from the cooperative binding of two additional bis-netropsin molecules to the first type of complex. In contrast to these results observed for binding to the alternating polynucleotide, only the 1:1 type of complex is formed when this ligand binds to the homopolymer poly(dA).poly(dT).

Circular Dichroism↗

PubMed1995

Sequence-directed single strand cleavage of DNA by a netropsin-flavin hybrid molecule.

In an attempt to obtain sequence specific DNA-cleaving molecules, we have synthesized a series of hybrid minor groove binders composed of a photoactiveable isoalloxazine (flavin) chromophore linked through a polymethylenic chain to a bis-pyrrolecarboxamide moiety related to netropsin. Like netropsin, the hybrid derivatives preferentially bind to A+T-rich sequences. Activation of the flavin chromophore by visible light results in the appearance of single strand breaks in the vicinity of the DNA binding site. We have further investigated the cleavage affinity properties of one of these compounds referred to as netropsin-flavin (Net-Fla) and considered as representative of the series. Net-Fla cleaves only one strand at a specific locus downstream of 5'-AAAT-3', upstream of 5'-TAAA-3' and on either side of a 5'-AAAA-3' sequence. Net-Fla cleaves both strands downstream to 5'-AATT-3'. This makes the properties of Net-Fla similar to that of a restriction endonuclease and provides additional insight into establishing the rules for the readout of B-DNA helix by non-nucleotidic compounds. Using molecular modeling, we show that Net-Fla binds to an asymmetric site in one orientation. The values of the energetic minima lie in the same order as expected from the cleavage patterns, which suggests that the oriented cleavage is a consequence of a sequence-oriented binding of Net-Fla in the DNA minor groove.

Base Sequence↗