Biomedical subjects

J C Maurizot

Publications and source records attributed to J C Maurizot.

At least 19 recordsLinked to original sources

PubMed2001

Fluorescence resonance energy transfer as a probe for G-quartet formation by a telomeric repeat.

The secondary structure of guanine-rich oligodeoxynucleotides has been investigated with fluorescent probes. Intramolecular folding of a telomeric oligonucleotide into a quadruplex led to fluorescence resonance energy transfer (FRET) between a donor (fluorescein) and an acceptor (tetramethylrhodamine) covalently attached to the 5' and 3' ends of the DNA, respectively. Depending on oligonucleotide length, quenching efficiency varied between 0.45 and 0.72 at 20 degrees C. The conjugation of the dyes to the oligonucleotide had a limited, but significant, influence on the thermodynamics of G-quartet formation. Intramolecular folding was demonstrated from the concentration independence of fluorescence resonance energy transfer over a wide concentration range. Folding of the oligonucleotide was confirmed by UV absorption, UV melting, and circular dichroism experiments. The folding of the G-quartet could be followed at concentrations as low as 100 pM. Fluorescence resonance energy transfer can thus be used to reveal the formation of multistranded DNA structures.

Chromosomes↗

PubMed2000

Spectroscopic studies on ethidium bromide binding to intramolecular parallel and antiparallel triple helices containing TA:T and GG:C triplets.

The interaction of ethidium bromide (EB), a DNA intercalator, with two intramolecular triplexes 5'd(G4A4G4-[T4]-C4T4C4-[T4]-G4T4G4), 5'd(G4T4G4-[T4]-G4A4G4-[T4]-C4T4C4) ([T4] represents a stretch of 4 thymine residues) and their precursor duplexes has been investigated by circular dichroism, fluorescence and UV absorption spectroscopy. Binding of EB induces a circular dichroism band in the region around 310 nm which is positive for the duplex forms but negative for the triplex forms. We observed that the binding of EB to the duplex form does not induce the formation of the triplex structures. Thermal denaturation experiments demonstrate that EB stabilizes more the parallel triple helix than the antiparallel one. Analysis of the binding process from fluorescence measurements shows that binding constants to the triple helical forms and to the hairpin reference duplex [T4]-G4A4G4-[T4]-C4T4C4) are close. However the binding site size is larger for the triplexes (4-6 base triplets) than for the duplex (2 base pairs).

Base Sequence↗

PubMed1998

Triple helix formation by (G,A)-containing oligonucleotides: asymmetric sequence effect.

Sequence effects on the stability of purine-motif (also called (G, A)-motif) triple helix have been investigated through two symmetry-related systems: one of them had a 5'(GGA)43' core sequence of triplex-forming oligonucleotides (TFOs), whereas the other one had a reversed 5'(AGG)43' core sequence. These (G,A)-containing TFOs were prone to self-associate into intermolecular complexes at room temperature. The competition of TFOs' self-association with triple helix formation was assessed, and minimized. By varying the lengths and the terminal base sequences of TFOs, the following were found that (1) The stability of two triple helices with identical length and base composition but reverse strand orientation may be significantly different (up to a factor of 6). (2) When the 5'(GGA)43' core sequence was extended at the 3'-end by a G, the 13-nt TFO exhibited 3- and 5-fold higher affinity toward the target double-stranded DNA (dsDNA) than the longer 14-nt and 15-nt TFOs in which one and two A(s) were added at the 3'-end of the 13-nt TFO, respectively. In contrast, when the similar extensions occurred at the 5'-end of the 5'(AGG)43' core sequence, the length increase provided a higher binding affinity of TFOs toward the target duplex. (3) The nature of the base triplets involved at the ends of triple helices may have great influence on triplex stability. The observed asymmetric sequence effect of the (G,A)-motif triple helix formation is discussed in terms of the binding strength of the first base triplet(s) at the 3' end which seems to be deeply involved in the nucleation step of triple helix formation and therefore to be a determining factor for triplex stability.

Adenine↗

PubMed1998

Circular dichroism and UV melting studies on formation of an intramolecular triplex containing parallel TA:T and GG:C triplets: netropsin complexation with the triplex.

We have used circular dichroism and UV absorption spectroscopy to characterize the formation and melting behaviour of an intramolecular DNA triple helix containing parallel T*A:T and G*G:C triplets. Our approach to induce and to stabilize a parallel triplex involves the oligonucleotide 5'-d(G4A4G4[T4]C4T4C4-[T4]G4T4G4) ([T4] represents a stretch of four thymine residues). In a 10 mM sodium cacodylate, 0.2 mM disodium EDTA (pH 7) buffer, we have shown the following significant results. (i) While in the absence of MgCl2 this oligonucleotide adopts an intramolecular hairpin duplex structure prolonged by the single strand extremity 5'-d([T4]G4T4G4), the presence of millimolar concentrations of MgCl2generates an intramolecular triplex (via double hairpin formation). (ii) In contrast to the antiparallel triplex formed by the oligonucleotide 5'-d(G4T4G4[T4]G4A4G4[T4]C4T4C4), the parallel triplex melts in a biphasic manner (a triplex to duplex transition followed by a duplex to coil transition) and is less stable than the antiparallel one. The enthalpy change associated with triplex formation (-37 kcal/mol) is approximately half that of duplex formation (-81 kcal/mol). (iii) The parallel triple helix is disrupted by increasing the concentration of KCl(>10 mM), whereas, under the same conditions, the antiparallel triplex remains stable. (iv) Netropsin, a natural DNA minor groove-binding ligand, binds to the central site A4/T4of the duplex or triplex in an equimolar stoichiometry. Its association constant K is smaller for the parallel triplex ( approximately 1 x 10(7) M-1) than for the antiparallel one ( approximately 1 x 10(8) M-1). In contrast to the antiparallel structure, netropsin binding has no apparent effect on thermal stability of the parallel triple helix.

Anti-Bacterial Agents↗

PubMed1998

Structure-specific binding recognition of a methanogen chromosomal protein.

The archaeon Methanosarcina thermophila expresses large amounts of a small basic protein, called MC1 (methanogen chromosomal protein), which was previously identified as a DNA-binding protein possibly involved in DNA compaction in some methanogenic species. We have investigated the binding of MC1 to various kinds of branched DNA molecules whose double helix axis is severely kinked. We show that MC1 is able to distinguish and to bind preferentially to four-way junctions. This preferential binding is observed in the absence and presence of divalent cations. However, we find that MC1 has a low affinity for bulged DNA structures. These results show how MC1 is able to discriminate between different deformations of the DNA double helix.

Archaeal Proteins↗

PubMed1998

The stability of duplexes involving AT and/or G4EtC base pairs is not dependent on their AT/G4EtC ratio content. Implication for DNA sequencing by hybridization.

Sequencing by the recently reported hybridization technique requires the formation of DNA duplexes with similar stabilities. In this paper we describe a new strategy to obtain DNA duplexes with a thermal stability independent of their AT/GC ratio content. Melting data were acquired on 35 natural and 27 modified duplexes of a given length and of varying base compositions. Duplexes built with AT and/or G4EtC base pairs exhibit a thermal stability restrained to a lower range of temperature than that of the corresponding natural compounds (16 instead of 51 degrees C). The 16 degrees C difference in thermal stability observed between the least stable and the most stable duplex built with AT and/or G4EtC base pairs is mainly due to the sequence effect and not to their AT/G4EtC ratio content. Thus N -4-ethyl-2'-deoxycytidine (d4EtC) hybridizes specifically with natural deoxyguanosine leading to a G4EtC base pair whose stability is very close to that of the natural AT base pair. Oligonucleotide probes involving d4EtC can be easily prepared by chemical synthesis with phosphoramidite chemistry. Modified DNA targets were successfully amplified by random priming or PCR techniques using d4EtCTP, dATP, dGTP and dTTP in the presence of DNA polymerase. This new system might be very useful for DNA sequencing by hybridization.

Adenine↗

PubMed1998

Interaction of methyl green with an oligonucleotide in intramolecular duplex and triplex conformations. Circular dichroism studies.

Interaction of methyl green with the oligonucleotide 5-dGGAAAAGG-[T4]-GGAAAAGG-[T4]-CCTTTTCC (where [T4] is a nucleotide sequence of four thymines) in hairpin duplex and in intramolecular triplex structures has been studied by circular dichroism. We found that methyl green binding to the duplex form shows a complex pattern, exhibiting an exciton contribution when the number of bound molecules increases. Differences between this pattern and previously published results on other DNAs reveals the presence of different types of complexes. In contrast to previous findings with the triple helix poly(dA).2poly(dT) we show that the methyl green is not toallly excluded from this triplex structure made of Pur:Pur:Pyr triplets.

Base Sequence↗

PubMed1998

Spectroscopic investigation of an intramolecular DNA triplex containing both G.G:C and T.A:T triads and its complex with netropsin.

The triple helix formation by the oligonucleotide 5'd(G4T4G4-[T4]-G4A4G4-[T4]-C4T4C4) ([T4] represents a stretch of 4 thymine residues) has been investigated by UV absorption spectroscopy and circular dichroism. In a 10 mM sodium cacodylate, 0.2 mM disodium EDTA (pH 7) buffer, we show the following significant results: i) In the absence of MgCl2, the oligonucleotide adopts a hairpin duplex structure with the dangling tail 5'd(G4T4G4-[T4]). This 5' extremity, which contains separated runs of four guanine residues, does not assume the expected tetraplex conformation observed when this sequence is free. ii) In the presence of MgCl2, the oligonucleotide folds back on itself twice to give a triple helix via a double hairpin formation, with [T4] single-strand loops. iii) The addition of high concentration of KCl to the preformed triplex does not disrupt the structure. Nevertheless, if the oligonucleotide is allowed to fold back in the presence of K+, triplex formation is inhibited. Circular dichroism studies demonstrate that the oligonucleotide adopts a dimeric conformation, resulting from the association of two hairpin duplexes, via the formation of an antiparallel G-quadruplex by the telomeric 5'd(G4T4G4-[T4]) extremities. iv) Under the experimental conditions used in this report, the triplex melts in a monophasic manner. v) Netropsin, a DNA minor groove ligand, binds to the central site A4/T4 of the duplex and to that of the triplex in an equimolar stoichiometry. In contrast with previous studies concerning pyr.pur:pyr triplexes, thermal denaturation experiments demonstrate that the netropsin binding stabilizes the intramolecular triplex.

Base Composition↗

PubMed1996

Distamycin A complexation with a nucleic acid triple helix.

The interaction of the minor groove binding drug distamycin with the T-A-T triple helix and the A-T double helix was studied using circular dichroism spectroscopy and thermal denaturation. The triple helix was made by the oligonucleotide (dA)12-x-(dT)12-x-(dT)12, where x is a hexaethylene glycol chain bridged between the 3'-phosphate of one strand and the 5'-phosphate of the following strand. This oligonucleotide is able to fold back on itself to form a very stable triplex. Changing the conditions allows the same oligonucleotide to be in a duplex form with a dangling arm. Circular dichroism spectroscopy demonstrates that the distamycin A molecule can bind to the triple-stranded form of this oligonucleotide. Spectral analysis shows that the bound distamycin exhibits a conformation and an environment slightly different from those which are observed when the drug is bound to the corresponding double-stranded structure. Furthermore, a second type of complex which is observed in the double-strand binding (two stacked distamycins in the minor groove) is not observed with the triple-stranded host. When distamycin is added to the triplex made of unbridged chains (dA)12 + 2(dT)12, the triplex dissociates to give a double-stranded structure. Thermal denaturation experiments demonstrate that distamycin binding destabilizes the triplex whereas it stabilizes the duplex. These results are compared with those obtained by the same experimental approaches on other minor groove binding drugs.

Antiviral Agents↗

PubMed1996

Preferential binding of the archaebacterial histone-like MC1 protein to negatively supercoiled DNA minicircles.

The interaction of the archaebacterial MC1 protein with 207 bp negatively supercoiled DNA minicircles has been examined by gel retardation assays and compared to that observed with the relaxed DNA minicircle. MC1 binding induces a drastic DNA conformational change of each minicircle, leading to an increase of the electrophoretic mobility of the DNA. A slight increase in salt concentration enhances the amount of bound MC1, and high NaCl concentrations are required to dissociate the complexes. Furthermore, the salt effect on binding depends on the supercoiling state of the DNA. The dissociation rates decrease with increasing linking difference of the minicircles relative to their relaxed configuration to reach a maximum at -2 turns. In addition, differences between the topoisomers are also observed in terms of stoichiometry of the strongest complexes. So with the -2 topoisomer the complex with two MC1 molecules is the most stable, while with the -1 and -3 topoisomers, the strongest ones are those with one MC1 molecule per DNA ring.

Archaeal Proteins↗

PubMed1996

Molecular modelling study of the netropsin complexation with a nucleic acid triple helix.

A detailed molecular mechanical study has been made on the complexes of netropsin with the double stranded oligonucleotide (dA)12.(dT)12 and with the triple helix (dA)12.(dT)12.(dT)12. The complexes were built using computer graphics and energy refined using JUMNA program. In agreement with circular dichroism experiments we have shown that 3 netropsins can bind the minor grooves of the triple helix and of the double helix. The groove geometry in the duplex and in the triplex is very similar. However a detailed analysis of the energetic terms shows, in agreement with thermal denaturation studies, that the affinity of netropsin toward the double helices is larger than towards triple helices.

DNA↗

PubMed1995

Conformational changes of DNA minicircles upon the binding of the archaebacterial histone-like protein MC1.

Binding of the archaebacterial histone-like protein MC1 to DNA minicircles has been examined by gel retardation and electron microscopy. MC1 preferentially binds to a 207-base pair relaxed DNA minicircle as compared with the linear fragment. Random binding is observed at very low ionic strength, and a slight increase in salt concentration highly favors the formation of a complex that corresponds to the binding of two MC1 molecules per DNA ring. Measurements of dissociation rates show that this complex is remarkably stable, and electron microscopy reveals that it is characterized by two diametrically opposed kinks. These results are discussed in regard to the mechanisms by which MC1 affects DNA structure.

Archaeal Proteins↗

PubMed1995

Interaction between cisplatin-modified DNA and the HMG boxes of HMG 1: DNase I footprinting and circular dichroism.

The interactions between the two boxes A and B of HMG 1 and cis-diamminedichloroplatinum(II)-modified DNA containing a single intrastrand cross-link at the d(GpG) site were studied by DNase I footprinting and circular dichroism. The DNAase I cleavage patterns of the HMG box-platinated DNA complexes are identical, the two boxes inhibiting the DNase I cutting over at least 15 and 12 nucleotide residues in the platinated strand and the complementary strand, respectively. As judged by circular dichroism, the two boxes have the same alpha-helical content (56%) and they induce the same conformational changes in the platinated DNA.

Amino Acid Sequence↗

PubMed1995

The secondary structure of the insect defensin A depends on its environment. A circular dichroism study.

Defensin A is an inducible antibacterial protein isolated from the larvae of Phormia terranovae. The conformation of defensin A has been previously determined by two-dimensional 1H-NMR for concentrations in the range of 4-8 mM in water (Bonmatin JM et al (1992) J Biomol NMR 2, 235-256). CD spectroscopic data of defensin A at lower concentrations (10(-5) to 10(-3) M) are reported herein. The ellipticity in the 200-240 nm wavelength range for various solvents varies as follows: acetonitrile < water < methanol < HFIP. The magnitude of theta 222 is strongly dependent on defensin concentration in a buffer solution, suggesting an aggregation process. The helical content of defensin A is maximum at a pH value range (7.5-8) for which the optimum antibacterial activity was observed (Cociancich S et al (1993) J Biol Chem 268, 19239-19245).

Acetonitriles↗

PubMed1994

Archaebacterial histone-like protein MC1 can exhibit a sequence-specific binding to DNA.

The binding of MC1 protein, the major chromosomal protein of the archaebacterium Methanosarcina sp. CHTI 55, to the region preceding the strongly expressed genes encoding methyl coenzyme reductase in a closely related micro-organism has been investigated. By gel retardation and DNAase I footprinting assays, we identified a preferential binding sequence in an open reading frame of unknown function. The large area of DNA protected against DNAase I is interrupted by a strong cleavage enhancement site on each strand. By circular permutation assays, we showed that the DNA bends upon MC1 binding. Furthermore we observed that the presence of a sequence outside the binding site can induce an unusual electrophoretic behaviour in some complexes.

Archaeal Proteins↗

PubMed1994

Fluorescence study on the non-specific binding of cyclic-AMP receptor protein to DNA: effect of pH.

The binding of the cyclic-AMP receptor protein (CRP) of Escherichia coli to a non-specific DNA fragment of 46 base pairs has been studied using fluorescence spectroscopy. The equilibrium binding constant was found to be several orders of magnitude lower than in the specific binding to a DNA fragment of the same size. The salt dependence of the equilibrium binding constant indicates that the CRP makes an identical number (8) of ion pairs to this non-specific DNA fragment in the presence and absence of cAMP. This number is larger than that previously found in the specific binding process. The effect of pH on the non-specific binding was investigated. The number of ion pairs does not vary between pH 6 and 8. From the variation of the binding constant with pH it was deduced that two histidines are involved in the binding in the absence of cAMP. These are most probably the histidines 199 of each subunit. In the presence of cAMP, only one histidine participates in the binding process, indicating an asymmetric interaction between the two subunits of the CRP and the DNA.

Cyclic AMP↗

PubMed1994

Comparison of solution structure of free and complexed lac operator by molecular modelling with NMR constraints.

The structure difference between the free operator of the lac system d(GCTCACAAT).d(ATTGTGAGC) and the same operator complexed to the headpiece of the lac repressor has been investigated by 2-D-1H NMR spectroscopy in conjunction with molecular modelling in internal coordinates (JUMNA). The free and complexed operator adopt both a right-handed B helical conformation, but a more detailed analysis of the conformational parameters using the Curves program shows striking differences in the groove geometries, the rises, the twists and the total bending.

Base Sequence↗

PubMed1994

Interaction of Hoechst 33258 with a DNA triple helix.

The interaction of Hoechst 33258 molecule, a minor groove binding drug, with T-A-T triple helix and A-T double helix was studied using circular dichroism spectroscopy and thermal denaturation. The triple helix consisted of an oligonucleotide (dA)12-x-(dT)12-x-(dT)12, where x is a hexa-ethylene glycol chain bridged between the 3' phosphate of one strand and the 5' phosphate of the following strand. This oligonucleotide is able to fold back on itself to form a very stable triplex. Circular dichroism spectroscopy demonstrates that Hoechst 33258 can bind to the triple helical structure. Spectral analysis shows that the bound drug exhibits a conformation and an environment slightly different in double-stranded and in triple-stranded structure. The affinity to the triple stranded structure is found smaller than to the double stranded one. Thermal denaturation experiments demonstrate that Hoechst 33258 destabilizes the triplex whereas it stabilizes the duplex.

Bisbenzimidazole↗