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J Portugal

Publications and source records attributed to J Portugal.

At least 19 recordsLinked to original sources
PubMed2001

Heterogeneous DNA binding modes of berenil.

Isothermal titration calorimetry (ITC) profiles of berenil bound to different DNAs show that, despite the strong preference of berenil for AT-rich regions in DNA, it can bind to other DNA sequences significantly. The ITC results were used to quantify the binding of berenil, and the thermodynamic profiles were obtained using natural DNAs as well as synthetic polynucleotides. ITC binding isotherms cannot be simply described when a single set of identical binding sites is considered, except for poly[d(A-T)2]. Ultraviolet melting of DNA and differential scanning calorimetry were also used to quantify several aspects of the binding of berenil to salmon testes DNA. We present evidence for secondary binding sites for berenil in DNA, corresponding to G+C rich sites. Berenil binding to poly[d(G-C)2] is also observed. Circular dichroism experiments showed that binding to GC-rich sites involves drug intercalation. Using a molecular modeling approach we demonstrate that intercalation of berenil into CpG steps is sterically feasible.

Animals↗
PubMed2001

Analysis of the effects of daunorubicin and WP631 on transcription.

The proficiency with which anthracyclines and other DNA-binding drugs target certain sequences in eukaryotic promoters offers a potential approach to interfere with the mechanisms that regulate gene expression in tumor cells. An in vitro transcription assay has been used to compare the ability of the bisintercalating anthracycline WP631 and the monointercalating anthracycline daunorubicin in terms of their ability to inhibit initiation of transcription of the adenovirus major late promoter linked to a G-less transcribed DNA template. Both drugs inhibit basal transcription by RNA polymerase II. However, WP631 is approximately 15 times more efficient at inhibiting transcription initiation from an adenovirus promoter containing an upstream Sp1-protein binding site. The differences in the ability of each drug to inhibit transcription initiation appear to be related to the competition between Sp1 and the anthracyclines for binding to the same site. To see whether WP631's strong effect on transcription can also be observed in cells, we compared the effects of WP631 and other anthracyclines on the transcription of the c-myc gene, which promoter contains Sp1 binding sites. The resulting data suggest that WP631 might circumvent some kinds of tumor resistance at rather low drug concentrations, inhibit c-myc expression in some cell lines, and exert its antitumoral effect by inducing apoptosis.

Animals↗
PubMed1999

Bisanthracycline WP631 inhibits basal and Sp1-activated transcription initiation in vitro.

An in vitro transcription assay was used to compare the capacity of the bisintercalating anthracycline WP631 (which displays a remarkably high DNA-binding affinity) and the monointercalating anthracycline daunomycin to inhibit transcription initiation of the adenovirus major late promoter linked to a G-less transcribed DNA template. Both drugs inhibit basal RNA synthesis in a concentration-dependent way, and the drug concentrations required to inhibit transcription initiation are similar. However, in this study WP631 was around 15 times more efficient at inhibiting transcription initiation when used with an adenovirus promoter containing an upstream Sp1-protein binding site under experimental conditions in which the Sp1 protein acted as a transactivator in vitro. The differences in the ability of each drug to inhibit transcription initiation were related to the competition between Sp1 and the drugs for the same binding site. Concentrations of WP631 as low as 60 nM could inhibit the Sp1-activated transcription initiation in vitro. In contrast, the concentration of daunomycin required to inhibit Sp1-activated transcription by 50% was almost the same as the concentration required to inhibit basal transcription. The efficiency of WP631 at displacing Sp1 from its putative binding site was confirmed using gel retardation and footprinting assays. These results are the first unequivocal example of a direct effect of an intercalator on activated transcription initiation.

Base Sequence↗
PubMed1998

Modulation of DNA-protein interactions in the P1 and P2 c-myc promoters by two intercalating drugs.

Regulation of transcription from the oncogene c-myc has an important role in the genesis of various tumors. Therefore, c-myc is a potential target for chemotherapy by drugs which are able to modify its activity directly. In this article, we identify the binding sites in the P1 and P2 promoter regions of c-myc for the intercalating antibiotics actinomycin D and elsamicin A. Gel retardation experiments indicate that actinomycin D or elsamicin A binding can inhibit the formation of several DNA-protein complexes. However, relatively low concentrations of elsamicin A, but not actinomycin D, appear to increase the level of binding to the P1 promoter of a protein factor. Using pure Sp1 transcription factor and an oligonucleotide containing the Sp1 putative binding site, we determined that the binding enhancement induced by small amounts of elsamicin was on the Sp1-DNA complex. Run-off transcription experiments in vitro showed that the effect of elsamicin A on Sp1 binding is followed by the maintenance or a relative rise in transcription levels from the P1 promoter of c-myc, while actinomycin D always inhibited the transcription from the P1 c-myc promoter in a concentration-dependent manner. Higher concentrations of elsamicin acted as an inhibitor of the transcription from the P1 start site but not from the P2.

Aminoglycosides↗
PubMed1998

A theoretical perusal of the satellite DNA curvature in tenebrionid beetles.

The curvature patterns of seven satellite DNAs taken from beetles belonging to the family Tenebrionidae (Coleoptera) were modelled utilising a number of computer programs that describe and plot the curvature profiles of DNA. The theoretical analysis agreed with the experimentally observed curvature of most of these satellite DNAs, and its absence in Tribolium freemani and Tenebrio obscurus satellite I. In many cases, the tenebrionid satellite DNAs lack periodically repeated runs of phased-A-tracts, yet they represent a clear example of curved DNA. The macroscopic curvature of satellites from these closely related organisms confirmed that other sequence elements must be participating in the bending of these DNAs. Our modelling approaches are discussed, together with previous experimental results, in terms of the role played by DNA curvature in the organisation of satellite DNA and the tight compacting of heterochromatin.

Animals↗
PubMed1997

Small ligands that neither bind to nor alter the structure of d(GA x TC)n sequences in DNA.

Three minor-groove binding ligands have been used to study the characteristics of two d(GA x CT)n DNAs embedded in longer DNA fragments. The binding of mithramycin, netropsin or Thia-Net to these sequences has been studied using DNAse I footprinting. None of these ligands appeared to bind to d(GA x CT)5 nor to d(GA x CT)22 extensively, although with mithramycin some protected bonds were detected at the very edge of these sequences. In general, these small ligands did not enhance the DNAse I cleavage patterns at the alternating d(GA x CT)n flanking sequences located near DNA regions where the drug was bound. The d(GA x CT)n sequences could act as a rigid block in which it is not easy to propagate structural changes, whereas other sequences flanking the binding sites showed cleavage enhancements.

Base Sequence↗
PubMed1997

Polymorphic curvature of satellite DNA in three subspecies of the beetle Pimelia sparsa.

The curvature of the monomeric repeats of satellite DNAs from three subspecies of the beetle Pimelia sparsa (Coleoptera, Tenebrionidae) has been analysed. Evidence of curvature was inferred from their retarded migration in native polyacrylamide gels, which was confirmed by direct electron microscopy visualisation. Sequence-comparison analysis, which included sequence alignments and modelling studies, was used to reveal the patterns of local bending and curvature. The effects of the minor-groove-binding drugs distamycin and berenil on the curvature were assayed, and analysed with respect to their (A+T)-rich sequence preferences. Since our study deals with satellite DNAs from closely related organisms (three subspecies), we correlated the differences in sequence, and also the high similarity conserved in the (A+T)-rich regions, with the changes in the patterns of curvature.

Animals↗
PubMed1996

T7 RNA polymerase cannot transcribe through a highly knotted DNA template.

The ability of T7 RNA polymerase to transcribe a plasmid DNA in vitro in its linear, supercoiled, relaxed and knotted forms was analysed. Similar levels of transcription were found on each template with the exception of plasmids showing varying degrees of knotting (obtained using stoichiometric amounts of yeast topoisomerase II). A purified fraction of knotted DNA with a high number of nodes (crosses) was found to be refractory to transcription. The unknotting of the knotted plasmids, using catalytic amounts of topoisomerase II, restored their capacity as templates for transcription to levels similar to those obtained for the other topological forms. These results demonstrate that highly knotted DNA is the only topological form of DNA that is not a template for transcription. We suggest that the regulation of transcription, which depends on the topological state of the template, might be related to the presence of knotted DNA with different number of nodes.

Bacteriophage T7↗
PubMed1996

Influence of elsamicin A on the activity of mammalian topoisomerase I.

The strong effect of elsamicin A on the mobility of DNA in agarose gels has been characterized. This antibiotic forms tight complexes that are resistant to an electrophoretic field, though they are not covalent and can be removed by phenol or 1-butanol extraction. In the presence of mammalian topoisomerase I, elsamicin A behaves as an intercalating agent in unwinding experiments performed with either phi X174 rf I (double-stranded, covalently closed DNA) or relaxed pUC19. The unwinding assay was used to calculate the apparent unwinding angle per bound antibiotic molecule, phi = 19 +/- 2.7 degrees. Moreover, an apparent binding constant for elsamicin was derived, under the experimental conditions of the topoisomerase I assays, using the Scatchard equation. The effects of elsamicin A on the mammalian topoisomerase I catalytic cycle do not seem to involve inhibition of the enzyme. Neither symptoms of trapping of covalent DNA-topoisomerase I cleavable complexes nor "nonspecific" inhibition, based solely on DNA binding, was apparent. Utilizing an experimental approach based on the use of relaxed plasmid DNA, we suggest that elsamicin might not be a topoisomerase I inhibitor.

Aminoglycosides↗
PubMed1995

Molecular dynamics study of the binding of elsamicin A to DNA.

Molecular mechanics (MM) and dynamics (MD) were used to examine the conformational flexibility of elsamicin A, an antitumour antibiotic, in aqueous solution. Furthermore, MM and MD simulations were performed to obtain the first three-dimensional model of the elsamicin-A-DNA complex. Comparison of MD simulations of free and DNA-bound elsamicin A provided insight into the mechanism of DNA cleavage. Moreover, the drug-DNA complexes were analyzed to investigate specific drug-DNA interactions, as well as the role of the solvent water molecules in the drug-DNA binding. On the basis of the results derived from these simulations, several issues related to the sequence-selective binding of elsamicin A to DNA are discussed.

Aminoglycosides↗
PubMed1995

Mitomycin C binding to poly[d(G-m5C)].

Poly[d(G-m5C)] was modified by reductively activated mitomycin C, an anti-tumour drug, under buffer conditions which are known to favour either the B or the Z conformations of DNA. C.d. and 31P-n.m.r. were used to characterize the poly[d(G-m5C)]-mitomycin cross-linked complexes, as well as the effects on the equilibrium between the B and Z forms of the polynucleotide. Mitomycin C appears to inhibit the B-->Z transition, even in the presence of 3 mM MgCl2, while the Z-form of poly[d(G-m5C)] does not interact significantly with the drug under bifunctionally activating conditions; thus no reversion from the Z-form to the B-form of the polynucleotide can be observed under the salt conditions which are required for the Z-form to exist.

Circular Dichroism↗
PubMed1995

Abortive transcription of the T7 promoter induced by elsamicin A.

The capacity of two intercalating drugs, actinomycin D and elsamicin A, to act as obstacles to transcript elongation by T7 RNA polymerase was analysed using a transcription assay in vitro. Whereas actinomycin D only delayed the production of full-length ('run-off') transcripts from the T7 promoter, elsamicin A arrested the transcription after a short transcript was made, and full-length transcripts were not obtained. The halt of transcription at the drug binding sites close to the promoter causes transcript arrest and results in the formation of a stable RNA hairpin. Actinomycin D appears to be able to detach from the template, allowing the phage polymerase to read through the pause site. In this way, full-length transcripts were obtained in a time-dependent way. With elsamicin A, a blockage of the transcription was observed which could be followed by the release of the phage RNA polymerase. The abortive transcription induced by elsamicin A might be explained considering that this DNA-binding drug drastically reduces the elongation rate and indirectly interferes with the polymerase during transcription, thus favouring its loose association with the nascent RNA and the inability of the enzyme to translocate along the DNA template. A direct interaction of the drug with the polymerase cannot be ruled out as an alternative explanation for the abortive transcription induced by elsamicin A.

Aminoglycosides↗
PubMed1994

Berenil acts as a poison of eukaryotic topoisomerase II.

The ability of the anti-trypanosomal drug berenil to interfere with the activities of eukaryotic type II topoisomerases is evaluated using two different types of reactions catalyzed by the enzyme: the relaxation of naturally supercoiled DNA and the decatenation of kinetoplast DNA (kDNA). The results indicate that berenil acts as an inhibitor of the catalytic activity, but the inhibiting ability appears to be smaller than for other minor-groove binding ligands.

Animals↗
PubMed1994

Calorimetric and spectroscopic studies on the poly[d(GA).d(CT)] structural polymorphism induced by zinc.

The interaction of zinc (II) with poly[d(GA).d(CT)] and salmon testes DNA has been investigated by Differential Scanning Calorimetry (DSC) and Circular Dichroism (CD). We have detected and energetically characterized the existence of two different structural forms in poly[d(GA).d(CT)] which behave differently during a DSC experiment. The overall melting of DNA shows two calorimetric transitions at different temperatures. Moreover, the presence of zinc, at an input ratio of ion to nucleotide (r) above two, renders a complex DSC profile which is characterized by a negative enthalpy transition. Besides, the low-temperature transition observed in the presence of zinc is practically reversible after re-cooling/re-heating cycles. Nevertheless, the high-temperature transition characterized by a negative delta H degree cal does not appear in re-heating experiments, and remains stable below 100 degrees C. A calorimetric negative enthalpy transition is also found using salmon DNA in the presence of zinc ions. It seems that the combination of a temperature effect and zinc binding might induce the production of a stable metal-DNA complex, which can also be detected by changes in some bands in the CD profiles. The experimental results show that the presence of DNA structures and binding processes involving a negative calorimetric enthalpy contribution might be more widespread than previously reckoned.

Animals↗
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↗
PubMed1993

PERFILS: a program for the quantitative treatment of footprinting data.

PERFILS, a computer program written in Borland TurboPascal, performs quantitative analysis of footprinting experiments using any IBM PC or compatible microcomputer. The program uses the height of the bands obtained from densitometric scanning of footprinting autoradiographs to calculate a differential cleavage plot. Such a plot displays, on a logarithmic scale, the difference of susceptibility of a DNA fragment to DNase I, or any other cleaving agent, in the presence of any ligand versus the sequence. PERFILS calculates the fractional cleavage values for control and ligand, giving a table of values for each internucleotidic bond and rendering the differential cleavage plot in only a few seconds.

Algorithms↗