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PubMed2003

Monitoring the synthetic reaction of a polyamide/peptide conjugate using electrospray ionization mass spectrometry.

Recently, the chemical structures of a series of monoimidazole/polyamine conjugates were studied in this laboratory using electrospray ionization mass spectrometry (ESI-MS) combined with tandem mass spectrometry (ESI-MS/MS). The method was found to be a powerful tool for the identification of this class of compounds. During the synthesis of targeted polyamide/peptide conjugates as derivatives or analogues of netropsin and distamycin, the method was applied to analyze and track the coupling reaction for the formation of the polyamide, which was difficult to achieve using thin layer chromatography (TLC). Characteristic fragmentation pathways for a nitro-monoimidazole conjugate, an amino-monoimidazole conjugate, and the final product (a nitro-diimidazole conjugate) were explored. The fragmentations of these conjugates were strongly affected by the presence of an amino group instead of a nitro group in the molecule, and led to the identification of the three compounds in the reacting solution or in the final reaction mixture. Consequently, the reaction could be monitored successfully and the synthetic route optimized.

Models, Chemical↗

PubMed1999

No braiding of Holliday junctions in positively supercoiled DNA molecules.

The Holliday junction is a prominent intermediate in genetic recombination that consists of four double helical arms of DNA flanking a branch point. Under many conditions, the Holliday junction arranges its arms into two stacked domains that can be oriented so that genetic markers are parallel or antiparallel. In this arrangement, two strands retain a helical conformation, and the other two strands effect the crossover between helical domains. The products of recombination are altered by a crossover isomerization event, which switches the strands fulfilling these two roles. It appears that effecting this switch from the parallel conformation by the simplest mechanism results in braiding the crossover strands at the branch point. In previous work we showed by topological means that a short, parallel, DNA double crossover molecule with closed ends did not braid its branch point; however, that molecule was too short to adopt the necessary positively supercoiled topology. Here, we have addressed the same problem using a larger molecule of the same type. We have constructed a parallel DNA double crossover molecule with closed ends, containing 14 double helical turns in each helix between its crossover points. We have prepared this molecule in a relaxed form by simple ligation and in a positively supercoiled form by ligation in the presence of netropsin. The positively supercoiled molecule is of the right topology to accommodate braiding. We have compared the relaxed and supercoiled versions for their responses to probes that include hydroxyl radicals, KMnO4, the junction resolvases endonuclease VII and RuvC, and RuvC activation of KMNO4 sensitivity. In no case did we find evidence for a braid at the crossover point. We conclude that Holliday junctions do not braid at their branch points, and that the topological problem created by crossover isomerization in the parallel conformation is likely to be solved by distributing the stress over the helices that flank the branch point.

Bacterial Proteins↗

PubMed1996

Preferential inhibition of DNA polymerases alpha, delta, and epsilon from Novikoff hepatoma cells by inhibitors of cell proliferation.

DNA polymerases alpha, delta and epsilon from normal regenerating rat liver and Novikoff hepatoma cells were purified about 300-fold, characterized, and checked for sensitivity towards drugs known to inhibit cell proliferation. Characterization included (a) identification of associated proteins, (b) measurement of physiochemical constants (including sedimentation coefficients, diffusion coefficients, calculation of relative molecular masses), (c) quantification of catalytic activities using specific DNA primer templates (Km values) and specific inhibitors (Ki values), and (d) discrimination between DNA polymerases from normal cells and those from malignant cells using inhibitors of cell proliferation. (a) DNA primase associated with DNA polymerase alpha, and 3'-5' exonuclease accompanying DNA polymerases delta and epsilon had similar activities. (b) Comparison of physicochemical and catalytic properties of DNA polymerases from both sources revealed similarities but also some important differences. Sedimentation and diffusion coefficients of DNA polymerases alpha and epsilon from malignant cells differed significantly. (c) The DNA-binding domain of DNA polymerases alpha and epsilon from hepatoma cells was altered since Km values, determined with several specific DNA primer-templates, were higher. Furthermore, dNTP-binding sites of DNA polymerases from malignant cells, when probed with specific inhibitors (aphidicolin, butylphenyl-dGTP, carbonyldiphosphonate, and dideoxy-TTP) showed significantly lower Ki values, indicating lower affinity to deoxyribonucleoside 5'-triphosphates. (d) Sixteen drugs representative of various modes of interaction with DNA and protein were chosen. Dose/response experiments were performed and the concentration at which the polymerizing activity was reduced to 50% was calculated (K50 values). Preferential inhibition of DNA polymerases alpha, delta, and epsilon from Novikoff hepatoma cells was found for: the intercalating drugs doxorubicin, daunorubicin, amsacrine, mitoxantrone, quinacrine and ethidium bromide, the minor-groove binders distamycin and netropsin, the ATPase-blocking agents novobiocin and coumamycin, and the topoisomerase I inhibitors camptothecin and topotecan. When the sensitivity of polymerases delta and epsilon was measured using poly(dA.dT) as a primer-template, the preferential inhibition of the enzymes from malignant cells was even more pronounced. Drugs known to trap the DNA-topoisomerase-II complex, etoposide, nalidixic acid, teniposide, and merbarone did not affect DNA polymerases irrespective of the source. Since the majority of the inhibitors used, particularly intercalators and minor-groove binders, act by modification of the primer-template, inhibition of DNA synthesis must have occurred through weakening of non-covalent bonds between DNA and catalytic polypeptides. Consequently, preferential inhibition of DNA polymerases from malignant cells seems to be indicative of abnormally diminished binding of the enzymes to their primer-templates. This effect may be caused by conformational alterations in polymerases from malignant cells which affect the DNA binding domains. Similarly, changes in physicochemical and kinetic constants are indicative of alterations of dNTP-binding domains.

Animals↗

PubMed1976

Further characterization of a DNA polymerase activity in mouse sperm nuclei.

The presence of a nuclear DNA polymerase in mouse sperm from adult testes has been confirmed and the properties of this enzyme further investigated. This activity was shown to be greatly enhanced by treating the spermatozoa with methanol or ethanol before incubation in the reaction medium or by their addition in small amounts to this medium. It was protected against degradation by nuclear proteases by adding soybean trypsin inhibitor and was stimulated by ATP. It was found to be Mg2+ dependent (optimum concentration: 7.5 mM), DNA dependent, and all four deoxynucleoside triphosphates were needed for optimal reaction. The radioactive acid-precipitable product of polymerization was not eliminated by organic solvents, nor by pronase, ribonuclease or by nuclease S1; however, it was converted to a large extent to acid-soluble products by pancreatic deoxyribonuclease. Since it was only partially solubilized by Triton X-100, it therefore did not appear to be preferentially associated with the nuclear membranes. The activity recovered after incubation depended also on the pH (optimum at pH 8.3) and did not work well in a medium for DNA polymerase alpha. The temperature for maximum incorporation of nucleotides was found to be 32 degrees C and, under our conditions, the reaction was linear for 30 min. The DNA polymerase activity was inhibited by low and high concentrations of KCl. It was not lowered by N-ethylmaleimide or p-hydroxymercuribenzoate; urea slightly stimulated the reaction and this stimulation was reversed by subsequent treatment with N-ethylmaleimide. Actinomycin D (40 mug/ml), ethidium bromide (25--50 muM), netropsin (5--50 mug/ml), and spermidine (0.5--2.5 mM) lowered the polymerization of DNA precursors. The nuclear enzyme could shift from the endogenous template to activated exogenous calf thymus DNA, the resulting nuclear radioactivity being reduced. The endogenous DNP template ability was not increased by deoxyribonuclease activation according to the method of Aposhian and Kornberg (J. Biol. Chem. (1962) 237, 519--525) suggesting that the amount of DNA polymerase associated with chromatin was probably limiting the reaction. The DNA polymerase activity detected in mouse sperm nuclei has numerous properties of low molecular weight DNA polymerases (DNA polymerase beta) reported in several eukaryotic organisms.

Animals↗

PubMed1981

Differential reactivities at restriction enzyme sites.

A method has been developed to measure the rates of digestion by restriction enzymes at individual sites. This involves a simple arithmetical treatment of the integrated areas from a densitometer scan of an ethidium bromide stained gel. We have used this method to study the digestion by HpaI, HincII and SalI of pBR322 and phi X174 DNA, and the effect of various DNA binding ligands. One of the two HpaI sites in phi X174 DNA is much more sensitive to inhibition by ligands such as netropsin, which display a preference for AT base pairs, than is the other site. Inspection of the sequences flanking the restriction sites shows that the former contains a much higher proportion of AT base-pairs than dose the latter. The opposite phenomenon is observed with the two HincII sites in pBR322. This illustrates the importance of neighbouring sequences in the interaction between restriction enzymes and their cleavage sites in DNA.

Base Sequence↗

PubMed1987

Theoretical study on binding of Hoechst 33258 with oligonucleotides.

Computer modelling with an energy minimization procedure is used here to obtain stereochemical and energetic details for complexes of the dye Hoechst 33258 with different oligonucleotide sequences. An optimised model of the dye with d(A)5 X d(T)5 is in conformity with previous proposed models. It has bifurcated hydrogen bonds between N2H and N4H of benzimidazole rings with N3 of adenine and O2 of thymine. Relative binding energies with different oligonucleotides show preference for AT containing sequences, with an intermediate affinity between that for netropsin and distamycin-2. Reduced binding is observed at high ionic concentration. The benzimidazole rings are twisted with respect to the phenol ring in the optimal model. This gives desired curvature to the molecule which is stabilised by intermolecular forces.

Benzimidazoles↗

PubMed1983

Chain length-dependent association of distamycin-type oligopeptides with A X T and G X C pairs in polydeoxynucleotide duplexes.

Different binding affinities of various distamycin analogs including the deformylated derivative with poly(dA-dC) X poly(dG-dT) were investigated using CD measurements. The inhibitory effect of distamycins on the DNAase I cleavage activity of DNA duplexes strongly supports the binding data. The base specificity of the ligand interaction with duplex DNA depends on the chain length of distamycin analogs. Netropsin, distamycin-2 and the deformylated distamycin-3 show no binding to dG X dC containing sequences at moderate ionic strength and are classified as highly dA X dT specific. In contrast distamycin having three, four or five methylpyrrolecarboxamide groups also forms more or less stable complexes with dG X dC-containing duplexes. These ligands possess a lower basepair specificity. The correlation between binding behavior and oligopeptide structure shows that presence of the number of hydrogen acceptor and donor sites determines the basepair and sequence specificity. The additional interaction with dG X dC pairs becomes essential when the number of hydrogen acceptor sites exceeds n = 3.

Animals↗

PubMed1984

Effect of intercalating and groove-binding ligands on formation of covalent complexes between nitracrine (Ledakrin, C-283) or 8-methoxypsoralen and DNA.

The effect of ethidium bromide, actinomycin D, distamycin A and netropsin on covalent binding of nitracrine (1-nitro-9-(3,3-N,N-dimethylaminopropylamino)acridine, Ledakrin, C-283) and 8-methoxypsoralen to DNA was examined. The competition was assayed either directly with [3H]- and [14C]nitracrine or indirectly by estimation of transcriptional template activity of nitracrine-DNA and 8-methoxypsoralen-DNA complexes formed in the presence of the ligands. A higher protective effect of ethidium bromide and distamycin on the photo-binding of 8-methoxypsoralen than on the dithiothreitol-dependent attachement of nitracrine to DNA assayed at 0.15 M KCl or NaCl was observed. The non-intercalating antibiotics showed lower competitive effect on 8-methoxypsoralen binding than ethidium bromide. Actinomycin D showed relatively low competition for both drugs with DNA. In contrast to the reaction of 8-methoxypsoralen, the decrease of nitracrine binding in the presence of competing ligands considerably depends on ionic strength. Particularly high inhibition of the adduct formation in the presence of ethidium at 1 M KCl was shown, while the amount of nitracrine bound in the presence of distamycin increases at elevated ionic strength. The results may indicate steric demands of the reaction between nitracrine and DNA.

Aminoacridines↗

PubMed1988

Assignment of DNA binding sites for 4',6-diamidine-2-phenylindole and bisbenzimide (Hoechst 33258). A comparative footprinting study.

DNA binding sites for the minor groove-binding ligands DAPI (4',6-diamidine-2-phenylindole) and Hoechst 33258 (bisbenzimide) have been analysed using DNAase I and micrococcal nuclease footprinting techniques. Both drugs appear to bind to AT-rich regions containing at least four such basepairs. Hoechst 33258 seems to bind relatively poorly to nucleotide sequences containing the alternating step TpA. However, in contrast to DAPI, it can more readily accommodate the presence of guanosine residues at the end of the binding site. We compare the DNA binding sites for DAPI and Hoechst 33258 with those determined for the related minor groove-binding ligands, berenil, netropsin and distamycin A, under comparable conditions, and discuss the importance of using different footprinting probes when analysing drug-DNA interactions.

Benzimidazoles↗

PubMed1975

Basic polypeptides as histone models: circular dichroism of complexes of model polypeptides with DNA.

Circular dichroism (CD) was used to study the complexes of DNA (in 0.15M NaCl) with two polypeptides considered as models of the histone molecules. CD spectra in the region of DNA absorption were studied with respect to the concentration used for annealing and to the molecular weight and composition of the DNA used. The properties of supernatants after centrifugation of aggregated complexes were examined. The effect of selectively bound antibiotics (actinomycin D and netropsin) on CD sprectra of complexes was investigated. The induced CD of proflavine molecules bound to DNA in the various complexes was also studied. It was concluded that changes in the CD spectra of DNA in complexes with the polypeptides are due to the formation of chiral superstructures, even if some conformational changes of DNA molecules themselves may also be decisive in some cases. The superstructure is affected by the composition of DNA, the role of (G + C) rich segments being particularly important.

Binding Sites↗

PubMed1993

A thermodynamic and spectroscopic study on the binding of berenil to poly d(AT) and to poly (dA) x poly (dT).

The complete thermodynamic profile for the non-intercalative binding of berenil to the alternating copolymer poly d(AT) and to the homopolymer poly (dA) x poly (dT) was investigated. Differential Scanning Calorimetry (DSC) and UV absorbance spectroscopy have been used to characterize and to compare the binding of berenil to the different synthetic polymers. Both double stranded DNA's show two types of binding; one stronger binding mode at low berenil concentrations and a weaker, in the case of poly d(AT)-berenil complexes slightly cooperative binding mode at higher drug to base pair ratios. For the interaction of berenil with poly d(AT) the thermodynamic data delta G(bind)0 = -33 kJ/mol drug, delta H(bind)0 = -29 kJ/mol of drug and delta S(bind)0 = +13 J/Kmol of drug were calculated. For the minor groove binding of berenil to poly (dA) x poly (dT) the following values were obtained: delta G(bind)0 = -34 kJ/mol of drug, delta H(bind)0 = -25 kJ/mol of drug and delta S(bind)0 = +30 J/Kmol of drug. Temperature-dependent UV absorbance spectroscopy revealed for both duplexes a biphasic "melting" behavior. However, the saturated nucleic acids (drug to base pair ratio 0.33) "melted" monophasically and with a decreased length of the cooperative unit. The obtained apparent equilibrium constants K(app) for the complexation with the discharged drug molecule showed to be a sensitive function of the ionic environment. But in contradiction to the expected release of two counterions into the solvent only a value of 1.0 was observed for the alternating copolymer poly d(AT). The complexation of berenil with poly (dA) x poly (dT) is followed by a release of 1.4 ions indicating stronger electrostatic interactions. For both polynucleotides the predicted release of two ions is not achieved. This is due to the presence of a binding mode, which involves less electrostatic interactions. From the complete data set it is proposed that the mode of binding is closely related to that found for the analogue minor groove binders DAPI and netropsin.

Diminazene↗

PubMed1993

Berenil recognizes and changes the characteristics of adenine and thymine polynucleotide structures.

Using circular dichroism, differential scanning calorimetry and susceptibility to DNAse I cleavage assays, we show that the interaction of berenil, a minor-groove binding drug, with poly(dA-dT).poly(dA-dT) and poly(dA).poly(dT) involves important changes in the polynucleotide conformation. The effect of berenil on poly(dA-dT).poly(dA-dT) comprises a clear alteration in CD spectra even at drug/DNA ratios smaller than the stoichiometric value. Berenil recognizes and binds to the alternating-B conformation of DNA changing it to a new conformation which appears to show some of the peculiarities of poly(dA).poly(dT), possibly through a modification in the helical parameters at the TpA and ApT steps. Such alteration is accompanied by a small calorimetric enthalpy change. Moreover, the calorimetric enthalpy does not change significantly whatever the input ratio of drug to poly(dA-dT).poly(dA-dT), indicating that berenil binding does not substantially alters the enthalpy of transition. In addition to increasing the melting temperature of the polynucleotide, berenil reduces the cooperativity of the poly(dA-dT).poly(dA-dT) transition slightly more than either distamycin or netropsin.

Binding Sites↗

PubMed1996

Electric linear dichroism as a new tool to study sequence preference in drug binding to DNA.

An original approach using electric linear dichroism (ELD) and natural DNAs and polynucleotides of differing base composition has been developed with the aim to investigate the sequence-dependent recognition of DNA by drugs. Both intercalators and minor groove binders have been studied as well as certain hybrid molecules. The results indicate that the orientation of drugs upon binding to nucleic acids can change markedly according to the target sequence. Among the intercalators tested, only actinomycin D and hycanthone show a clear preference for GC- and AT-rich sequences, respectively. For minor groove binders, the linear dichroism showing a strong dependence on base composition of the DNA and polynucleotides is most pronounced. Netropsin and distamycin bind to DNA with a marked AT specificity. Hoechst 33258, berenil and DAPI exhibit positive and negative dichroism signals at AT and GC sites respectively, suggesting that at least two types of drug-DNA interaction are involved depending on the AT/GC content of the DNA. Further investigations using polynucleotides with inosine substituted for guanosine, and competition experiments with intercalative drugs suggest that Hoechst 33258, berenil and DAPI interact with GC sequences via a non-classical intercalation process.

Amino Acid Sequence↗

PubMed1989

A role of DNA topoisomerases in the active dissociation of DNA minor groove-ligand complexes. A flow cytometric study of inhibitor effects.

The biological effects of a number of DNA ligands which interact with the minor groove of B-form DNA (e.g. netropsin, distamycin and Hoechst 33258) are thought to arise from the direct disturbance of the processes of DNA replication and transcription. Although ligand binding appears to be an important factor in cytotoxicity, the pathways by which drug molecules can be actively dissociated from nuclear DNA are unknown. Recent evidence suggests that minor groove ligands can distort the manner in which DNA associates with nucleosomal core particles and we have hypothesized that in an intact cell such imposed torsional stress could be subject to the action of cellular topoisomerases. We have used flow cytometry to study the effects of various inhibitors (including topoisomerase-interactive drugs) on the responses of a mutant cell line (HoeR415) to Hoechst 33342, given that the mutant shows resistance to the cytotoxicity of this DNA-specific dye due to an enhanced capacity to dissociate nuclear DNA-dye complexes. Ligand-DNA dissociation in the mutant was found to be energy-dependent but not specifically, affected by the drug-efflux blocker verapamil or by inhibitors of DNA synthesis. The topoisomerase II inhibitors novobiocin, VP16, nalidixic acid and the topoisomerase I-interactive drug camptothecin inhibited ligand-DNA dissociation to various extents with novobiocin being the most effective (100% inhibition at 1 mM). Both novobiocin and camptothecin were without effect on the nuclear loss of a DNA intercalator, adriamycin. We conclude that efficient topoisomerase activity is required for the active dissociation of DNA minor groove-ligand complexes.

Animals↗

PubMed2004

Analysis of GAA/TTC DNA triplexes using nuclear magnetic resonance and electrospray ionization mass spectrometry.

The formation of a GAA/TTC DNA triplex has been implicated in Friedreich's ataxia. The destabilization of GAA/TTC DNA triplexes either by pH or by binding to appropriate ligands was analyzed by nuclear magnetic resonance (NMR) and positive-ion electrospray mass spectrometry. The triplexes and duplexes were identified by changes in the NMR chemical shifts of H8, H1, H4, 15N7, and 15N4. The lowest pH at which the duplex is detectable depends upon the overall stability and the relative number of Hoogsteen C composite function G to T composite function A basepairs. A melting pH (pHm) of 7.6 was observed for the destabilization of the (GAA)2T4(TTC)2T4(CTT)2 triplex to the corresponding Watson-Crick duplex and the T4(CTT)2 overhang. The mass spectrometric analyses of (TTC)6.(GAA)6 composite function(TTC)6 triplex detected ions due to both triplex and single-stranded oligonucleotides under acidic conditions. The triplex ions disappeared completely at alkaline pH. Duplex and single strands were detectable only at neutral and alkaline pH values. Mass spectrometric analyses also showed that minor groove-binding ligands berenil, netropsin, and distamycin and the intercalating ligand acridine orange destabilize the (TTC)6.(GAA)6 composite function (TTC)6 triplex. These NMR and mass spectrometric methods may function as screening assays for the discovery of agents that destabilize GAA/TTC triplexes and as general methods for the characterization of structure, dynamics, and stability of DNA and DNA-ligand complexes.

Base Pairing↗

PubMed2005

Cold stress-induced pea DNA helicase 47 is homologous to eIF4A and inhibited by DNA-interacting ligands.

Helicases are ubiquitous molecular motor proteins that play important role in maintaining the genome integrity and thus involved in plant growth and development. Here, we report the cloning of cDNA (1.64 kb) and genomic DNA (2.2 kb) of cold stress-induced pea DNA helicase 47 (PDH47) and characterization of its encoded protein. It belongs to DEAD-box protein family and shows striking identity (93%) with tobacco eIF4A. The transcript was induced under cold (4 degrees C) stress. The purified PDH47 protein (47 kDa) contains ATP-/Mg2+-dependent DNA unwinding as well as DNA-/Mg2+-dependent ATPase activities. The ATPase activity of PDH47 is stimulated more by ssDNA as compared to dsDNA and RNA. The activities of PDH47 are inhibited by various DNA-interacting ligands such as nogalamycin, daunorubicin, ethidium bromide, mitoxantrone, actinomycin, and cisplatin with apparent Ki values ranging from 0.5 to 8.0 microM. Interestingly, netropsin and distamycin inhibited the helicase but not the ATPase activity. The inhibition might be due to the intercalation of inhibitors into duplex DNA, which can impede the translocation of the PDH47. This study should help in our better understanding of cold stress signaling and mechanism of DNA unwinding in plants.

Adenosine Triphosphate↗

PubMed2004

Synthesis and cytotoxic effect of carbocyclic potential minor groove binders.

New carbocyclic potential minor groove binders were synthesised, using 3-nitrobenzoyl chloride and aliphatic alpha,omega-diamines with three, four and five methylene fragments. The half structures, compounds IV-VI can be compared to bis-amidines, compounds X-XII to bis-netropsin. All of the compounds were investigated antiproliferative and cytotoxic effects in the standard cell line of mammalian tumour MCF-7.

Acids, Carbocyclic↗

PubMed1996

The detection of B-form/A-form junction in a deoxyribonucleotide duplex.

The transition of the 14-meric deoxyoligonucleotide duplex d-(ACCCCCTTTTTTTG).d-(CAAAAAAAGGGGGT) from the B- to the A-conformation in water/trifluorethanol (TFE) solution was studied with the use of circular dichroism. An increase in the fraction of TFE induces a two-step B-A transition. In the first step, up to 73% TFE, the A-form is generated from the GC-rich part; in the second step, 73-82% TFE, the AT-rich part shifts to the A-form. By this we suggest the existence of a B/A junction near 73% TFE. Emergence of the B/A junction has been directly confirmed with the use of distamycin A and netropsin, ligands known to selectively bind to AT stretches of B-DNA. It can be shown that both ligands suppress formation of the A-form in the B-philic part. The free energy value for the B/A junction was estimated to be 2.1 kcal/mol, which agrees well with known data for polymeric DNAs. The obtained results may have biological relevance in connection with recently published x-ray data about the occurrence of the B/A junction in the complex of DNA with reverse transcriptase of HIV.

Base Composition↗