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M Le Bret

Publications and source records attributed to M Le Bret.

At least 37 records · Page 2Linked to original sources
PubMed1991

Sequence requirements for mammalian topoisomerase II mediated DNA cleavage stimulated by an ellipticine derivative.

Various antitumor drugs stabilize DNA topoisomerase II-DNA transient covalent complexes. The complexes distribution along pBR322 DNA was shown previously to depend upon the nature of the drug (Tewey et al. (1984) Science 226, 466-468). The position in pBR322 of DNA cleavage by calf DNA topoisomerase II for 115 such sites stabilized by an ellipticine derivative and the relative frequency of cleavage at most of these sites were determined. The nucleotide sequence surrounding the 25 strongest sites was analyzed and the following ellipticine specific consensus sequence was deduced: 5'-ANCNT(A/G)T.NN(G/C)N(A/G)-3' where cleavage occurs at the indicated mark. A thymine is always present at the 3' end of at least one strand of the strong cleavage sites, and the dinucleotide AT or GT at the 3' end of the break plays a major role in the complex stabilisation. The predictive value of cleavage of the consensus was tested for two regions of SV40 DNA and cleavage was indeed detected at the majority of the sites matching the consensus. Some complexes stabilized by ellipticine are resistant to salt dissociation and this property seems to be correlated with the presence of symmetrical sequences in the cleavage site with a center of symmetry staggered relatively to the center of symmetry of cleavage.

Animals↗
PubMed1991

Comparison of the bis-intercalating complexes formed between either ditercalinium or a flexible analogue and d(CpGpCpG)2 or d(TpTpCpGpCpGpApA)2 minihelices: 1H- and 31P-NMR analyses.

The 400-MHz 1H- and 162-MHz 31P-nmr have been used to study complexes constituted by (a) the d(TpTpCpGpCpGpApA)2 or the d(CpGpCpG)2 self-complementary oligonucleotides and (b) two bifunctional 7H-pyrido [4,3-c] carbazole dimer drugs, the antitumoral ditercalinium (NSC 366241), a dimer with a rigid bis-piperidine linking chain and its pharmacologically inactive analogue, a dimer with a flexible spermine-like linking chain. Nearly all proton and phosphorus signals have been assigned by two-dimensional (2D) nmr (correlated spectroscopy, homonuclear Hartmann-Hahn, nuclear Overhauser enhancement spectroscopy, 2D 31P (1H) heteronuclear correlated spectroscopy and 31P-31P chemical exchange experiments). Both drugs bis-intercalate into the two CpG sites. The complexes show small differences in the position of the 7H-pyrido [4,3-c] carbazole ring into the intercalation site and possibly in the ribose-phosphate backbone deformation. However, the inactive analogue exhibits a longer residence lifetime in octanucleotide than the ditercalinium does. All these results are discussed in terms of differences in dimer activities.

Base Sequence↗
PubMed1990

Molecular mechanics and dynamics of an abasic frameshift in DNA and comparison to NMR data.

In a previous publication (Ph. Cuniasse, L.C. Sowers, R. Eritja, B. Kaplan, M.F. Goodman, J.A.H. Cognet, M. Le Bret, W. Guschlbauer and G.V. Fazakerley, Biochemistry 28, 2018 (1989), we determined by two dimensional NMR studies and molecular mechanics calculations the three-dimensional structure of a non-selfcomplementary oligonucleotide: [sequence; see text] where dr, at the center of the first strand, is a model abasic site. In order to explain all the results arising from NMR measurements, we found that an equilibrium between two conformations was necessary. These conformations differ mainly by the sugar pucker of G5 which is C2' endo or C3' endo. The latter is stabilized by addition of counterions between phosphate residues P3 and P4. In this paper, we have constructed systematically, all possible structures as a function of torsion angles delta of dr4 and of G5 by molecular mechanics in the presence or absence of counterions. Since these conformations were not forced with NMR distance measurements, this method allows detailed comparisons between all possible conformations and NMR data. Maps of contour lines of the potential energy, of fits to NMR distance measurements, and of helical twist as a function of torsion angles delta of dr4 and of G5 unravel the difficulties associated with the study of the G5 sugar pucker conformation equilibrium. Sugar puckers and proton distances are very sensitive criteria to monitor molecular dynamics. Relying on these experimental criteria, we have tested many molecular dynamics preparation phases and we propose a new warm-up and equilibration procedure for molecular dynamics. Thus we show with a 290 ps molecular dynamic run that G5 is in conformational equilibrium and that all NMR data are well reproduced.

Base Composition↗
PubMed1989

Abasic frameshift in DNA. Solution conformation determined by proton NMR and molecular mechanics calculations.

We have determined the three-dimensional structure of a non-self-complementary oligodeoxynucleotide duplex that contains a model abasic site. The duplex contains six GC base pairs plus the abasic site at the center of one strand and corresponds to an abasic frameshift. Two-dimensional NMR studies on the nonexchangeable protons show that the guanine bases on either side of the abasic site are stacked over each other and that the abasic site is rotated out of the helix. Close proton-proton interactions are observed between the H4' proton of the abasic site and sugar protons of the guanosine in the 5' direction, which allows the position of the free sugar to be well-defined. NOE buildup curves from NOESY spectra recorded at very short mixing times were used to calculate a set of interproton distances. This data set was incorporated into the refinement of the oligonucleotide structure by molecular mechanics calculations. Two conformations that differ in the sugar conformation of the guanosine next to the abasic site in the 3' direction were necessary to fit all the NMR data. One of these two conformations could only be stabilized by addition of counterions at specific sites.

Chemical Phenomena↗
PubMed1989

Reassessment of structural characteristics of the d(CGCG)2:actinomycin D complex from complete 1H and 31P NMR.

Complexes formed between Actinomycin D (ActD) and the tetranucleotides d(AGCT)2 and d(CGCG)2 were studied in detail by one and two-dimensional 1H and 31P NMR. The 31P two dimensional chemical exchange experiment, at room temperature on saturated complexes (1:1), showed unambiguously that the asymmetrical phenoxazone ring binds to the unique GC site under the two possible orientations in the d(AGCT)2 tetranucleotide but adopts a single orientation in the d(CGCG)2 tetranucleotide. For the d(CGCG)2:Act D saturated complex, complete assignments of all protons and phosphorus signals of the two-nucleotide strands, as well as of the two cyclic pentapeptide chains has allowed us to study in details the conformational features of the complex from NOE and coupling constants analysis. The tetranucleotide remains in a right-handed duplex, but the sugar puckers are modified for residues at the intercalation site. A uniform C2' endo pucker is observed for residues on the strand facing the quinoid side of the phenoxazone ring while a C2' endo-C3-endo equilibrium about 60% of C2' endo is proposed for the two residues on the strand facing the benzenoid side of the phenoxazone ring. In contrast to previous studies on ActD-DNA interactions, we have been able to measure the 3J phosphorus-proton coupling constants at the intercalation site but also adjacent to it, showing that 31P chemical shifts are not simply related to the backbone conformation. Molecular mechanics calculations, using empirical distances deduced from NOE effects as restrained distances during minimizations, led to a model differing mainly from those previously published by orientation of the N methyl groups of both N-Methyl-Valines.

Base Sequence↗
PubMed1988

Chromatin reconstitution on small DNA rings. II. DNA supercoiling on the nucleosome.

DNA supercoiling on the nucleosome was investigated by relaxing with topoisomerase I mono- and dinucleosomes reconstituted on small DNA rings. Besides 359 base-pair (bp) rings whose linking differences were integers, two additional series of rings with fractional differences, 341 and 354 bp in size, were used. Mononucleosomes reconstituted on 359 bp rings were found to relax into a single mononucleosome form. In contrast, 341 and 354 bp mononucleosomes relaxed into a mixture of two forms, corresponding to two adjacent topoisomers. The observation that the ratio between these two forms was, within each ring series, virtually independent of the initial linking number of the topoisomer used for the reconstitution suggested that each partition reflected an equilibrium. Comparison with the equilibria observed for the same rings in the absence of histones showed that the formation of a single nucleosome is associated with a linking number change of -1.1(+/-0.1) turn. Dinucleosomes, in contrast, were not relaxed to completion and do not reach equilibria. The corresponding linking number change per nucleosome was, however, estimated to be similar to the above figure, in agreement with previous data from the literature obtained with circular chromatins containing larger numbers of nucleosomes. DNA structure in mononucleosomes was subsequently investigated by means of high-resolution electron microscopy and gel electrophoresis. It was found that the above linking number reduction could be ascribed to a particle with a large open extranucleosomal DNA loop and with no more than 1.5 turns of a superhelix around the histone core. A theoretical model of a nucleosome on a small ring was constructed in which one part of the DNA was wrapped around a cylinder and the other part was free to vary both in torsion and flexion. The linking number reduction predicted was found to be most consistent with experimental data when the twist of the DNA in the superhelix was between 10.5 and 10.65 pb per turn, suggesting that wrapping on the nucleosome does not alter the twist of the DNA significantly. A lower estimate of the linking number reduction associated with a two-turn nucleosome was also derived, based on an analysis of recent data obtained upon treatment of reconstituted minichromosomes with gyrase. The value, 1.6 turns, set a lower limit of 10.44 bp per turn for the twist of nucleosomal DNA, in agreement with the above estimate.(ABSTRACT TRUNCATED AT 400 WORDS)

Chromatin↗
PubMed1986

Identification of form III conformers in tRNAPhe from Escherichia coli by intramolecular photo-cross-linking.

In the absence of divalent cations, at neutral pH, low ionic strength, and low to moderate temperature, tRNAs are known to be in a denatured form, designated form III in the tRNA phase diagram by Cole et al. [Cole, P. E., Yang, S. R., & Crothers, D. M. (1972) Biochemistry 11, 4358-4368]. Form III tRNAPhe from Escherichia coli has been studied at pH 7, 5 mM Na+, and 10 degrees C. As judged from ethidium bromide intercalation, it exhibits extensive secondary structure. 4-Thiouridine in position 8 of the tRNAPhe sequence was used as a built-in photoaffinity probe. Spectroscopic and spectrofluorometric analysis in the near-UV range of form III tRNAPhe irradiated with broad-band near-UV light to completion of the reaction before or after reduction with NaBH4 revealed that the Pdo(4-5)Cyt (8-C) and Pdo(4-5)Urd (8-U) adducts form in equimolar yield. In different experiments, the overall yield of s4U conversion to these adducts varies between 20 and 40%. The remaining s4U is photolyzed to weakly absorbing product(s) in the near-UV range. The disappearance of s4U follows biexponential kinetics while the 8-C adduct formation follows monoexponential kinetics, indicating the presence of at least two tRNA classes of conformers, not in equilibrium on the time scale of the reaction. Migration on a denaturing polyacrylamide gel of irradiated form III tRNAPhe revealed three main bands, D1, D2, and D3, and no slowly migrating tRNA dimers. D1 migrates at the control position and presumably contains the photolysis product(s) P. The fast-migrating D2 and D3 bands contain 8-Pyr cross-links which were identified by sequence analysis as 8-(66-68) in D2 and 8-(40-43) and 8-(59-62) in D3. On the basis of these data, it is proposed that the minor poorly photoreactive class II conformers are the cloverleaf and close variants, whereas the major class I cross-linkable conformers are essentially long-extended secondary structures. Clearly, our data demonstrate the polymorphism of form III tRNAPhe.

Base Sequence↗
PubMed1985

Accessibility to bacterial nucleic acids of the intercalating drug aliphatic amino acid ellipticinium derivatives in Escherichia coli and Salmonella typhimurium.

The fluorescence of the aliphatic (amino acido)-N2-methyl-9-hydroxyellipticinium (AA-NMHE) derivatives [Auclair, C., Voisin, E., Banoun, H., Bernardou, J., Meunier, B., & Paoletti, C. (1984) J. Med. Chem. 27, 1161-1166], namely, dehydroglycino-NMHE, dehydroalanino-NMHE, dehydrovalino-NMHE, and dehydroleucino-NMHE, has been characterized. The changes in the fluorescence properties of the drugs, including increase in quantum yields, increase in fluorescence lifetimes, and occurrence of energy transfer upon binding to DNA in vitro, have been further investigated. The measurement of the fluorescence increment of AA-NMHE when bound to fluorescent sites inside intact bacteria has been found to be suitable for the determination of the accessibility of the drugs to bacterial nucleic acids according to the method of Lambert and Le Pecq [Lambert, B., & Le Pecq, J.B. (1984) Biochemistry 23, 166-176]. With this methodology, the kinetics of drug uptake, the ability of the drug to reach the bacterial nucleic acids at equilibrium, and the nature of the ligand binding model have been determined in two AA-NMHE-sensitive strains, Escherichia coli BL 101 (Lambert & Le Pecq, 1984) and Salmonella typhimurium TA 98 [Ames, B.N., Lee, F.D., & Durston, W.E. (1973) Proc. Natl. Acad. Sci. U.S.A. 70, 782-786]. The main results obtained are the following: At nonsaturating concentrations, each AA-NMHE exhibits a marked difference in its ability to reach the bacterial nucleic acids. This parameter seems to be correlated with the antibacterial efficiency of the drugs.(ABSTRACT TRUNCATED AT 250 WORDS)

Alkaloids↗
PubMed1984

Electron microscopic measurement of chain flexibility of poly(dG-dC).poly(dG-dC) modified by cis-diamminedichloroplatinum(II).

The antitumor drug cis-diamminedichloroplatinum (II) (cis-Pt) forms bidentate adducts with guanine residues of poly(dG-dC).poly(dG-dC). The secondary structure of the polymer is altered. In this work, high resolution pictures of naked molecules, obtained by dark field electron microscopy reveal DNA chain distortions with radii as small as 30 A. The extent of distortion increases with the drug/nucleotide ratio (rb). These alterations of the secondary structure are responsible for the apparent shortening of the molecules. Measurements of the persistence lengths of the polymer as well as the end-to-end distances of elementary segments of various lengths, are obtained from digitized electron micrographs. The measurements are used to monitor and quantify the observed modifications of polymer structure upon cis-Pt binding at various rb or incubation times. Poly(dG-m5dC).poly(dG-m5dC) in the B and Z forms have different persistence lengths. In the B form, this polymer is more altered by cis-Pt than in the Z one.

Cisplatin↗
PubMed1984

Quenching of DNA-ethidium fluorescence by amsacrine and other antitumor agents: a possible electron-transfer effect.

The antitumor agent amsacrine, 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA), when bound to double-stranded DNA, particularly poly(deoxyadenylicthymidylic acid), reduced the fluorescence of bound ethidium without physically displacing it from DNA. Fluorescence lifetime measurements showed that the reduction of fluorescence was not due to reduction of the lifetime of the excited state of ethidium. Rather, a proportion of the DNA-bound ethidium changed to a state where the fluorescence was highly quenched. Several other 9-anilinoacridine derivatives, and also 9-hydroxyellipticine, caused quenching of ethidium-DNA fluorescence, whereas 9-aminoacridine, proflavin, and ellipticine had no effect. Resonance energy transfer (Förster transfer) is not responsible for the effect since there is no spectral overlap between the absorption spectrum of any of the agents and the fluorescence emission spectrum of ethidium. It is suggested that quenching may occur as a result of reversible formation of electron-transfer complexes between the intercalating drug and the excited state of ethidium.

Aminoacridines↗
PubMed1983

Counter-ion condensation and system dimensionality.

Condensation of the counter-ions around a highly charged infinitely long cylindrical molecule, such as DNA, can be described in detail in terms of the solutions of the Poisson-Boltzmann (Gouy-Chapman) equation. By using the Alfrey-Berg-Morawetz (1951) solution of this equation one can show that a certain fraction of the counter-ions remain within finite distances of the poly-ion even when the volume of the system is expanded indefinitely; these ions can be appropriately called "condensed". The fraction of the macromolecule's charge represented by these ions is just 1-1/xi, where xi is the linear charge-density parameter of the macromolecule; this is also the value given by Manning's theory. The question arises: Is this property unique to the infinite cylinder? Using the same PB equation, we can consider the infinite charged plane and a large finite charged sphere for comparison. In the case of the plane all of the counter-ions are condensed in the above sense, not just a fraction, for any surface charge density of the plane. These ions form the classical Gouy double layer. On the other hand, none of the counter-ions of the charged sphere are condensed in the above sense, no matter how high the surface charge density. Thus the cylinder is a unique intermediate case in which a fraction of the counter-ions are condensed if the linear charge density is higher than the critical value of unity.

Chemical Phenomena↗
PubMed1979

Yeast mitochondrial deoxyribonuclease stimulated by ethidium bromide. 2. Mechanism of enzyme activation.

An endonuclease (EtdBr DNase), which is more active in the presence of EtdBr, has been purified from yeast mitochondrial membrane (Jacquemin-Sablon, H., et al. (1979) Biochemistry 18 (preceding paper in this issue)). This paper deals with the analysis of the mechanism of this activation. Determination of the enzyme activity in the presence of intercalating and nonintercalating agents showed that the enzyme does not recognize the DNA structure modifiction provoked by drug intercalation. Studies carried out with a series of phenanthridinium derivatives led to the following model. The EtdBr DNase activation would result from the formation of a ternary complex, DNA--drug--Triton X-100. The activation capacity of a drug depends on its ability to bind simultaneoulsy to the DNA (not necessarily by intercalation) and the detergent. When this complex is formed, the DNA molecule is surrounded with Triton X-100 molecules which constitute an hydrophobic environment and make the substrate more prone to interaction with the enzyme. The implications of this model are discussed.

Deoxyribonucleases↗
PubMed1977

A reexamination of the problem of resonance energy transfer between DNA intercalated chromophores using bisintercalating compounds.

The rate of energy transfer between DNA intercalated ethidium cations calculated by Paoletti and Le Pecq1 using the Forster theory differs from the measured one by a factor of twenty two, if the proper geometrical factors are taken into account. By changing some of the parameters used in the calculation, the discrepancy can be reduced but not eliminated. This led us to the study of other systems where experimental and calculated results can be more directly compared. The apparent rate of energy transfer between ethidium and one of its non fluorescent analogues and between various pairs of intercalated chromophores has been studied. The fluorescence anisotropy decay of acridine dimers in glycerol or bisintercalated in DNA has been measured. These studies show that the Forster theory of energy transfer does not apply to the case of identical chromophores when they are relatively close to each other.

Acridines↗