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Biomedical subjects

M Guéron

Publications and source records attributed to M Guéron.

At least 19 recordsLinked to original sources
PubMed2002

Design and implementation of a protection system for NMR spectrometers.

We have implemented a scheme, SPECMON, for monitoring various parameters of a spectrometer, such as nitrogen pressure and sample temperature, and taking corrective action. The scheme is based on considerations of protection management which are of general application. Evaluation of the spectrometer state is incorporated in macros of the application software (VNMR) and is therefore very flexible. In contrast, corrective action is limited to the single one which is deemed fully safe: complete shutdown of the spectrometer and logging. Shutdown is implemented by a minor hardware modification of the spectrometer: the introduction of a second input to a relay already present for protection of the spectrometer power supply. Monitoring is handled by the host computer, and the shutdown command is transmitted via control lines of its series port, independent of the standard connection between the host computer and the NMR system console. The monitoring system (software and hardware) is unobtrusive in normal conditions, and it can be tested without affecting the operation of the spectrometer.

Journal Article↗
PubMed2000

The solution structure and internal motions of a fragment of the cytidine-rich strand of the human telomere.

We present the solution structure of d(CCCTA2CCCTA2CCCTA2CCCT), a fragment of the vertebrate telomere which folds intramolecularly. The four cytidine stretches form an i-motif which includes six intercalated C.C+ pairs and terminates with the cytidines at the 5' extremity of each stretch. Above, the second TA2 linker loops across one of the narrow grooves, while at the bottom, the first and third linkers loop across the wide grooves. At 30 degrees C, the spectra of the first and third linkers are quasi-degenerate. Severe broadening at lower temperature indicates that this results from motional averaging between at least two structures of each bottom loop, and makes it impossible to solve the configuration of the bottom loops directly, in contrast to the rest of the structure. We therefore turned to the modified sequence d(CCCTA(2)5MCCCTA2CCCUA2CCCT) in which the two base substitutions (underlined) break the quasi-symmetry between linkers 1 and 3. The three loops follow approximately the hairpin "second pattern" of Hilbers. In the first loop, T4 is in the syn orientation, whereas its analog in the third loop, U16, oriented anti, is in a central location, where it interacts with bases of both loops, thus contributing to their tight association. The only motion is a syn/anti flip of A18 in the third loop. Returning to the telomere fragment, we show that each of the bottom loops switches between the structures identified in the first and third loops of the modified structure. The motions are concerted, and the resulting configurations of the bottom loop cluster present a bulge to either right (T4 syn) or left (T16 syn).

Base Pairing↗
PubMed2000

A unified theory of the B-Z transition of DNA in high and low concentrations of multivalent ions.

We showed recently that the high-salt transition of poly[d(G-C)]. poly[d(G-C)] between B-DNA and Z-DNA (at [NaCl] = 2.25 M or [MgCl(2)] = 0.7 M) can be ascribed to the lesser electrostatic free energy of the B form, due to better immersion of the phosphates in the solution. This property was incorporated in cylindrical DNA models that were analyzed by Poisson-Boltzmann theory. The results are insensitive to details of the models, and in fair agreement with experiment. In contrast, the Z form of the poly[d(G-m5C)] duplex is stabilized by very small concentrations of magnesium. We now show that this striking difference is accommodated quantitatively by the same electrostatic theory, without any adjustable parameter. The different responses to magnesium of the methylated and nonmethylated polymers do not come from stereospecific cation-DNA interactions: they stem from an experimentally derived, modest difference in the nonelectrostatic component of the free energy difference (or NFED) between the Z and B forms. The NFED is derived from circular DNA measurements. The differences between alkaline earth and transition metal ions are explained by weak coordination of the latter. The theory also explains the induction of the transition by micromolar concentrations of cobalt hexammine, again without specific binding or adjustable parameters. Hence, in the case of the B-Z transition as in others (e.g., the folding of tRNA and of ribozymes), the effect of multivalent cations on nucleic acid structure is mediated primarily by nonspecific ion-polyelectrolyte interactions. We propose this as a general rule for which convincing counter-examples are lacking.

Circular Dichroism↗
PubMed2000

The i-motif in nucleic acids.

Seven years after the discovery of the DNA i-motif, partial explanations for its occurrence have been uncovered, possibly involving CHellipsisO hydrogen bonds across the narrow grooves. Investigations of its biological significance have been encouraged by the demonstration and description of the intramolecular i-motif structure of human telomeric and centromeric sequences, by the recent observation of an intercalated RNA structure and by the discovery of proteins that associate with DNA sequences carrying cytosine repeats. The compatibility of the intercalation with peptide and phosphorothioate DNA analogs is favorable for possible pharmaceutical applications.

DNA↗
PubMed1999

Determination of the residence time of water molecules hydrating B'- DNA and B-DNA, by one-dimensional zero-enhancement nuclear Overhauser effect spectroscopy.

The residence time of water in the minor groove of the d(CGCGAATTCGCG) duplex has been determined by a recent measurement combining nuclear Overhauser enhancements (NOE, ROE) and 17O relaxation dispersion. The time is in the range of nanoseconds, so that it may be measured by a rather simple method proposed here, namely the choice of conditions such that the NOE between the observed DNA proton and a nearby water proton is zero. This condition is realized when the residence time of the water molecule is 0.178 times the nuclear magnetic resonance period (e.g. 0.297 ns at 600 MHz). It may be achieved by varying the magnetic field and/or the temperature. The zero-NOE measurement may be performed by one-dimensional NMR, and has therefore good sensitivity. We have developed excitation sequences which suppress two spurious contributions to the NOE: from neighboring exchangeable protons and from H3' protons whose chemical shift is close to that of water. The method is applied here to the comparison of residence times of water next to B-DNA and next to B'-DNA, the latter corresponding to better stacked, propeller-twisted base-pairs and a correspondingly narrower minor groove. In the minor groove of [d(CGCGAATTCGCG)]2, a B'-DNA duplex, the residence time of the water molecule next to H2 of adenine(6) (underlined), is 0.6 ns at 10 degreesC, in good agreement with the value obtained previously. The residence time is slightly but distinctly shorter for the water next to A5, suggesting non-cooperative departure of these two molecules which are presumed to be part of the hydration spine. Near A5 and A4 of [d(AAAAATTTTT)]2, another B'-DNA duplex, the residence times are approximately twice as long, but the activation enthalpies are about the same, ca. 38 kJ/mol. The residence time in the minor groove of the regular B-DNA sequence d(CGCGATCGCG) was 0.3 ns at 10 degreesC, shorter than in the case of the B'-DNA sequences by factors of 2 and 4, respectively. The temperature dependence is less, with an activation enthalpy of 27 kJ/mol. The major groove residence times are comparable for the three sequences, and a few times shorter than those of minor groove water. A value of 0.36 ns, or even more in case of rotation of water, is obtained around -8 degreesC. The most striking aspect of these results is the relatively small difference in the residence times of reputedly fast and slow-exchanging water molecules bound to DNA in biological conditions. This suggests that the spine of hydration is perhaps not a major stabilizer of the B'-DNA structure as compared with B-DNA.

DNA↗
PubMed1998

An intramolecular i-motif: the solution structure and base-pair opening kinetics of d(5mCCT3CCT3ACCT3CC).

We present a high-definition structure of d(5mCCT3CCT3ACCT3CC), a DNA sequence which resembles a four-times repeat of the C-rich strand of telomeres and centromeres. The structure is monomeric. The CC stretches form four hemi-protonated C.C base-pairs, belonging to two parallel-stranded duplexes which intercalate head-to-tail into an i-motif core. The four grooves of the core are similar to those observed previously in i-motif tetramers, with P-P distances around 0.9 nm and 1.4 nm for the narrow and wide grooves, respectively. At 0 degrees C, the structure is formed even at pH 7, despite the required protonation of cytidine pairs, suggesting that it may be biologically relevant.The intercalation topology of the i-motif core is read off the pattern of inter-residue cross-peaks along each groove: between H1' protons across the narrow grooves, and between amino and H2' protons across the wide grooves. In the hemi-protonated C.C pairs, the imino proton is shared equally between the two bases, as shown by the equal intensities of the NOESY cross-peaks between the imino proton and the two cis amino protons of the pair. Short inter-sugar distances and the direction of CH1' bonds are consistent with CH1'...O4' hydrogen bonds across the narrow grooves, as suggested by Berger et al. (1996). Proc. Natl. Acad. Sci. USA, 93, 12116-12121. At one extremity of the i-motif core, the T3A linker loops across one of the two wide grooves. It extends the core by stacking of A11, which also forms a strongly propeller-twisted reverse-Hoogsteen pair with T8. At the other extremity, the two T3 linkers loop side by side across the two narrow grooves, extending the core by stacking of a T5.T16 pair which connects the two linkers. In this T.T pair between parallel strands, the hydrogen bonds are from imino proton to O4, and the base-pair lifetime is 6 ms at 0 degrees C. The structures of segments 1 to 7 and 12 to 18, which form the i-motif core and the T3 loops, are related by a 2-fold pseudo-symmetry: the geometries and environment are so similar that the NOESY spectra are barely resolved. These various interactions illustrate how linker sequences may affect the stability, intercalation topology and folding pattern of the intramolecular i-motif.

Base Composition↗
PubMed1996

A comparison of AMP degradation in the perfused rat heart during 2-deoxy-D-glucose perfusion and anoxia. Part I: The release of adenosine and inosine.

AMP degradation is studied in two models of the Langendorff-perfused rat heart which generate a large release of purines: the 2-deoxy-D-glucose (2DG)-perfused heart and the anoxic heart. In the 2DG model, mitochondrial energy generation is quasi-normal, despite a very low ATP concentration. Furthermore, inorganic phosphate (Pi) concentration is low, an important difference with anoxia where Pi is very high, up to 82 mM. Coronary release of purines is measured by high performance liquid chromatography, and myocardial metabolite content by 31P nuclear magnetic resonance spectroscopy. In the 2DG-perfused hearts with glucose or acetate, the purine release consists nearly exclusively of inosine [up to 130 nmol/(min x gww)] while adenosine is less than 1 nmol/(min x gww). A possible interpretation is that AMP degradation proceeds mainly through deamination to inosine monophosphate by AMP deaminase (the IMP pathway). In contrast, the purine release in anoxia (100% N2) contains comparable quantities of adenosine and inosine [respectively 30 and 20 nmol/(min x gww)], indicating that part of AMP is dephosphorylated directly to adenosine. Comparison with the 2DG model suggests that the release of adenosine in the anoxic heart is a result of inhibition of AMP deaminase by Pi.

Adenosine↗
PubMed1996

AMP degradation in the perfused rat heart during 2-deoxy-D-glucose perfusion and anoxia. Part II: The determination of the degradation pathways using an adenosine deaminase inhibitor.

Using the adenosine deaminase inhibitor erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA), we determine the contribution of the adenosine pathway to the abundant purine release of two Langendroff-perfused rat heart models which differ particularly in inorganic phosphate (Pi) content: the 2-deoxy-D-glucose (2DG) perfused heart and the anoxic heart. We measure the release of coronary purines by high performance liquid chromatography, and the content of myocardial metabolites by 31P nuclear magnetic resonance spectroscopy. In the 2DG-perfused heart (2 mM for 45 min), the release of inosine [130 nmol/(min.gww)] is much larger than that of adenosine, and EHNA (50 microM) has little effect, showing that the pathway of inosine monophosphate (IMP) accounts for 97% of purine catabolism. In the anoxic heart (100% N2 for 45 min), where inosine and adenosine release are comparable in the absence of EHNA, the inhibitor reduces the release of inosine and catabolites from 50 to 20 nmol/(min.gww) and increases that of adenosine [from 30 to 55 nmol/(min.gww)], showing that the contributions of the IMP and adenosine pathways are 23 and 77%. The difference between the two models has been ascribed to the inhibition of AMP deaminase by Pi in the anoxic heart (Chen W, et al., 1996). We discuss the physiological significance of this heart-specific duality of degradation pathways.

Adenine↗
PubMed1995

Terminal base pairs of oligodeoxynucleotides: imino proton exchange and fraying.

We have estimated the dissociation constant of the terminal base pairs of the B-DNA duplexes formed by 5'-d(CGCGATCGCG) and 5'-d(TAGCGCTA) by two methods, one based on the change in imino proton chemical shift with temperature and the other on the apparent pK shift of the imino proton, as monitored by the change in chemical shift of aromatic protons. These methods do not rely on imino proton exchange, whose rate was also measured. (1) The effect of ammonia on the imino proton exchange rate of the terminal pair of the 5'-d(CGCGATCGCG) duplex is 67 times less than on the isolated nucleoside. This provides an upper limit on the exchange rate from the closed pair. In fact, the effect is just as predicted from the dissociation constant, assuming that there is no exchange at all from the closed pair and that, as has been argued previously, external catalysts act on the open state as they do on the isolated nucleoside. The inhibition of catalyzed proton exchange in the closed pair, despite exposure of one face of the pair to solvent, is a new feature of the exchange process. It will allow determination of the dissociation constant of terminal pairs from the exchange rate. (2) Intrinsic catalysis of proton exchange is less efficient for the terminal pair than for an internal one. A possible explanation is that proton transfer across the water bridge responsible for intrinsic catalysis is slower, as expected if the open-state separation of the bases is larger in a terminal pair. This observation may lead to a direct method for the study of fraying. (3) At 0 degrees C, the dissociation constant of the second pair of the 5'-d(CGCGATCGCG) duplex is close to the square of the constant for the terminal pair, as predicted from a simple model of fraying. The enthalpy and entropy of opening of the terminal pairs may be compared with those of nearest neighbor interactions derived from calorimetry [Breslauer, K. J., et al. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 3746-3750].

Base Composition↗
PubMed1995

Solution structures of the i-motif tetramers of d(TCC), d(5methylCCT) and d(T5methylCC): novel NOE connections between amino protons and sugar protons.

BACKGROUND: At slightly acid or even neutral pH, oligodeoxynucleotides that include a stretch of cytidines form a tetramer structure in which two parallel-stranded duplexes have their hemi-protonated C.C+ base pairs face-to-face and fully intercalated, in a so-called i-motif, first observed serendipitously in [d(TC5)]4. RESULTS: A high-definition structure of [d(TCC)]4 was computed on the basis of inter-residue distances corresponding to 21 NOESY cross-peaks measured at short mixing times. A similarly defined structure of [d(5mCCT)]4 was also obtained. A small number of very characteristic (amino proton)-(sugar proton) cross-peaks entails the intercalation topology. The structure is generally similar to that of [d(TC5)]4. The sequence d(T5mCC) forms two tetramers in comparable proportions. The intercalation topologies are read off the two patterns of (amino proton)-(sugar proton) cross-peaks: one is the same as in the d(TCC) tetramer, the other has the intercalated strands shifted by one base, which avoids the steric hindrance between the methyl groups of the 5mC pairs of the two duplexes. CONCLUSIONS: The structures obtained in this work and the procedures introduced to characterize them and to solve the problems linked to the symmetry of the structure provide tools for further exploring the conditions required for formation of the i-motif.

Base Sequence↗
PubMed1994

Intramolecular folding of a fragment of the cytosine-rich strand of telomeric DNA into an i-motif.

In the recently discovered i-motif, four stretches of cytosine form two parallel-stranded duplexes whose C.C+ base pairs are fully intercalated. The i-motif may be recognized by characteristic Overhauser cross-peaks of the proton NMR spectrum, reflecting short H1'-H1' distances across the minor groove, and short internucleotide amino-proton-H2'/H2" across the major groove. We report the observation of such cross-peaks in the spectra of a fragment of the C-rich telomeric strand of vertebrates, d[CCCTAA]3CCC. The spectra also demonstrate that the cytosines are base-paired and that proton exchange is very slow, as reported previously for the i-motif. From UV absorbance and gel chromatography measurements, we assign these properties to an i-motif which includes all or nearly all the cytosines, and which is formed by intramolecular folding at slightly acid or neutral pH. A fragment of telomeric DNA of Tetrahymena, d[CCCCAA]3CCCC, has the same properties. Hence four consecutive C stretches of a C-rich telomeric strand can fold into an i-motif. Hypothetically, this could occur in vivo.

Animals↗
PubMed1993

Acid multimers of oligodeoxycytidine strands: stoichiometry, base-pair characterization, and proton exchange properties.

The structure recently proposed for the acid form of the oligonucleotide 5'-d(TC5) is a four-strand "tetrad" in which two parallel-stranded, base-paired duplexes are intimately associated, with their hemiprotonated C-C+ base pairs face-to-face and fully intercalated, in a so-called "i-motif" (Gehring et al., 1993). We use the amino and imino proton spectra to establish the structure and symmetry of the base pairs, properties which are a primary element in the resolution of the acid form describe above. The amino proton spectrum gives the best lower limit (8 x 10(4) s-1) on the rate of the imino proton jumping process which is responsible for the base-pair symmetry. The stoichiometry of the acid form of other deoxycytidine sequences is studied by gel filtration chromatography and in one case by an NMR equilibrium titration. In all cases, i.e., d(C12), d(T2C8T2), d(C4TC4), d(TC5), d(C5), d(C4), d(TC4), d(TC3T), and d(TC3), the acid form elutes as a tetramer. A single-strand component is also present in some cases. But no dimer is observed, except for some samples prepared by quenching from high temperatures. The characteristic H1'-H1' interresidue NOESY cross-peaks of the d(TC5) structure (Gehring et al., 1993) are also found in all the tetramers where they have been searched for, i.e., those of d(T2C8T2), d(C4TC4), d(TC3T), and d(TC3) (not shown), suggesting that these tetramers also are built on the i-motif and that such structures may be formed generally by strands containing a stretch of as little as three deoxycytidines. From the NMR titration of d(TC3), we derive a free energy of -7.6 kJ/mol per cytidine base pair for the formation of the tetramer from single strands. The free energy released by packing a base pair into the i-motif is comparable to that released in forming the base pair itself. Imino proton exchange is limited by base-pair opening, thanks to efficient intrinsic exchange catalysis: this explains the lack of effect of added catalysts. The base-pair lifetime is hundreds of times longer than in any DNA duplex, presumably due to the base-pair intercalation geometry. The variation of the lifetime along the sequence of the d(TC5) tetramer provides support for the recently proposed structure. The internal amino proton exchanges from the open state of the C-C+ pair, at a rate compatible with a pK of 9 appropriate for C+. But the external proton exchanges from the closed state, as with a pK of 17!(ABSTRACT TRUNCATED AT 400 WORDS)

Base Composition↗
PubMed1993

A tetrameric DNA structure with protonated cytosine.cytosine base pairs.

Oligomers containing tracts of cytidine form hemiprotonated base pairs at acid pH and have been considered to be double-stranded. We have solved the structure of the DNA oligomer 5'-d(TCCCCC) at acid pH and find that it is a four-stranded complex in which two base-paired parallel-stranded duplexes are intimately associated, with their base pairs fully intercalated. The relative orientation of the duplexes is antiparallel, so that each base pair is face-to-face with its neighbours. The NMR spectrum displays only six spin systems, showing that the structure is highly symmetrical on the NMR timescale; the four strands are equivalent. A model derived by energy minimization and constrained molecular dynamics shows excellent compatibility with the observed nuclear Overhauser effects (NOEs) particularly for the very unusual inter-residue sugar-sugar NOEs H1'-H1', H1'-H2" and H1'-H4'. These NOEs are probably diagnostic for such tetrameric structures.

Base Composition↗
PubMed1993

Composite cylinder models of DNA: application to the electrostatics of the B-Z transition.

We develop and test a Poisson-Boltzmann model of the electrostatics of the B-Z transition of DNA. Starting from the detailed geometries of the two forms, we compute at each radius the fractions of DNA matter, of volume forbidden (for nonpoint-like ions), and of volume accessible to the center of ions. These radial distributions are incorporated in a composite cylinder model; availability to ions (porosity) and the dielectric constant at each radial distance are then obtained. The phosphate charge is distributed with cylindrical symmetry on two layers at the appropriate radial distances. The porous sheath, between the axis and the charge distribution, provides much more room for ions in B-DNA than in Z-DNA. By using previously developed methods, the Poisson-Boltzmann problem of such cylinders is easily solved. The computational load is small, so that results can be obtained for a large set of salt concentrations and for a number of ionic radii. The variation of the electrostatic free energy difference with salt concentration compares favorably with the experimental value (it is half as large). There is also qualitative agreement with experiments on supercoiled DNA, including a maximum of the free energy difference at submolar salt concentrations. The results for this cylinder with porous sheath are in line with those of the earlier simple planar model and of a plain cylinder with sheath, which is also presented here. They are thus insensitive to details of the model. They support the proposition that the main electrostatic feature of the B-Z transition is the better immersion of the B-DNA phosphates into the solution. They also give confidence in the validity of the Poisson-Boltzmann approach, despite the large salt concentrations involved. Prior studies using an approach based on the potential of mean force are discussed.

Biophysical Phenomena↗
PubMed1992

A simple explanation of the electrostatics of the B-to-Z transition of DNA.

Whereas the phosphates of B-DNA jut out into the solution, those of Z-DNA, being closer to DNA matter, are less subject to electrostatic screening by counterions. We present simple planar models of B- and Z-DNA that reflect these geometric features. The ionic strength dependence of the difference in the Poisson-Boltzmann electrostatic free energy of the models agrees with that measured by Pohl [Pohl, F. M. (1983) Cold Spring Harbor Symp. Quant. Biol. 47, 113-118]. This indicates that the electrostatics of the B-to-Z transition are primarily controlled by a qualitative geometrical difference and not by details of the DNA geometry or by complex electrostatic properties of the ionic solution.

DNA↗
PubMed1992

Proton exchange in DNA-luzopeptin and DNA-echinomycin bisintercalation complexes: rates and processes of base-pair opening.

Imino proton exchange studies are reported on the complexes formed by bisintercalation of luzopeptin around the two central A.T pairs of the d(CCCATGGG) and d(AGCATGCT) duplexes and of echinomycin around the two central C.G pairs of the d(AAACGTTT) and d(CCAAACGTTTGG) duplexes. The depsipeptide backbone of the drugs occupies the minor groove of the complexes at the bisintercalation site. The exchange time of the amide protons of the depsipeptide rings provides a lower estimate of the complex lifetime: 20 min at 15 degrees C for the echinomycin complexes and 4 days at 45 degrees C for the luzopeptin complexes. The exchange time of imino protons is always shorter than the complex lifetime. Hence, base pairs open even within the complexed oligomers. For the two base pairs sandwiched between the aromatic rings of the drug, the base-pair lifetime is strongly increased, and the dissociation constant is correspondingly reduced. Hence, the lifetime of the open state is unchanged. This suggests similar open states in the free duplex and in the complex. In contrast to the sandwiched base pairs, the base pairs flanking the intercalation site are not stabilized in the complex. Thus, the action of the bisintercalating drug may be compared to a vise clamping the inner base pairs. Analysis suggests that base-pair opening may require prior unwinding or bending of the DNA duplex.

Ammonia↗