Yves Boulard

Publications of Yves Boulard

• Hyperpolarized 129Xe NMR signature of living biological cells.

  Authors: Céline Boutin, Hervé Desvaux, Marie Carrière, François Leteurtre, Nadège Jamin, Yves Boulard, Patrick Berthault

  NMR in biomedicine. 12/2011; 24(10):1264-9.

  We show that the differentiation between internal and external compartments of various biological cells in suspension can be made via simple NMR spectra of hyperpolarized (129) Xe. The spectralWe show that the differentiation between internal and external compartments of various biological cells in suspension can be made via simple NMR spectra of hyperpolarized (129) Xe. The spectral separation between the signals of (129) Xe in these two compartments is already known for red blood cells, because of the strong interaction of the noble gas with hemoglobin. The observation of two separate peaks in the 200-ppm region can be seen with both eukaryotic and prokaryotic cells, some of which are not known to contain paramagnetic proteins in large quantities. Using different experiments in which the cells are lysed, swell or are blocked in G2 phase, we demonstrate that the low-field-shifted peak observed corresponds to xenon in the aqueous pool inside the cells and not in the membranes. The presence of this additional peak is a clear indication of cell integrity, and its integration allows the quantification of the total cell volume. The relaxation time of intracellular xenon is sufficiently long to open up promising perspectives for cell characterization. The exchange time between the inner and outer cell compartments (on the order of 30 ms) renders possible the targeting of intracellular receptors, whereas the observation of chemical shift variations represents a method of revealing the presence of toxic species in the cells.

• A triple spin-labeling strategy coupled with DEER analysis to detect DNA modifications and enzymatic repair.

  Authors: Mélanie Flaender, Giuseppe Sicoli, Samia Aci-Seche, Thomas Reignier, Vincent Maurel, Christine Saint-Pierre, Yves Boulard, Serge Gambarelli, Didier Gasparutto

  Chembiochem : a European journal of chemical biology. 11/2011; 12(17):2560-3.

  In a spin: Spin-labeled oligonucleotides produced by click chemistry can be studied by EPR, by using a DEER sequence. This was used to test a complex triple-labeling strategy with damaged DNA.In a spin: Spin-labeled oligonucleotides produced by click chemistry can be studied by EPR, by using a DEER sequence. This was used to test a complex triple-labeling strategy with damaged DNA. Extensive and accurate analysis of DNA structure and enzymatic repair processes were performed after digestion by EndoIV. Modified DNA structures and DNA-protein interactions can now be readily studied.

• Crucial role of a dicarboxylic motif in the catalytic center of yeast RNA polymerases.

  Authors: Gwenaël Ruprich-Robert, Maxime Wery, Daphné Després, Yves Boulard, Pierre Thuriaux

  Current genetics. 07/2011; 57(5):327-34.

  The catalytic center of yeast RNA polymerase II and III contains an acidic loop borne by their second largest subunit (Rpb2-(832)GYNQED(837), Rpc128-(764)GYDIED(769)) and highly conserved in allThe catalytic center of yeast RNA polymerase II and III contains an acidic loop borne by their second largest subunit (Rpb2-(832)GYNQED(837), Rpc128-(764)GYDIED(769)) and highly conserved in all cellular and viral DNA-dependent RNA polymerases. A site-directed mutagenesis of this dicarboxylic motif reveals its strictly essential character in RNA polymerase III, with a slightly less stringent pattern in RNA polymerase II, where rpb2-E836Q and other substitutions completely prevent growth, whereas rpb2-E836A combines a dominant growth defect with severe lethal sectoring. A mild but systematic increase in RNA polymerase occupancy and a strict dependency on the transcript cleavage factor TFIIS (Dst1) also suggest a slower rate of translocation or higher probability of transcriptional stalling in this mutation. A conserved nucleotide triphosphate funnel domain binds the Rpb2-(832)GYNQED(837) loop by an Rpb2-R(1020)/Rpb2-D(837) salt-bridge. Molecular dynamic simulations reveal a second bridge (Rpb1-K(752)/Rpb2-E(836)), which may account for the critical role of the invariant Rpb2-E(836). Rpb2-E(836) and the funnel domain are not found among the RNA-dependent eukaryotic RNA polymerases and may thus represent a specific adaptation to double-stranded DNA templates.

• Cell uptake of a biosensor detected by hyperpolarized 129Xe NMR: the transferrin case.

  Authors: Céline Boutin, Antoine Stopin, Fatimazohra Lenda, Thierry Brotin, Jean-Pierre Dutasta, Nadège Jamin, Alain Sanson, Yves Boulard, François Leteurtre, Gaspard Huber, Aurore Bogaert-Buchmann, Nawal Tassali, Hervé Desvaux, Marie Carrière, Patrick Berthault

  Bioorganic & medicinal chemistry. 07/2011; 19(13):4135-43.

  For detection of biological events in vitro, sensors using hyperpolarized (129)Xe NMR can become a powerful tool, provided the approach can bridge the gap in sensitivity. Here we propose constructsFor detection of biological events in vitro, sensors using hyperpolarized (129)Xe NMR can become a powerful tool, provided the approach can bridge the gap in sensitivity. Here we propose constructs based on the non-selective grafting of cryptophane precursors on holo-transferrin. This biological system was chosen because there are many receptors on the cell surface, and endocytosis further increases this density. The study of these biosensors with K562 cell suspensions via fluorescence microscopy and (129)Xe NMR indicates a strong interaction, as well as interesting features such as the capacity of xenon to enter the cryptophane even when the biosensor is endocytosed, while keeping a high level of polarization. Despite a lack of specificity for transferrin receptors, undoubtedly due to the hydrophobic character of the cryptophane moiety that attracts the biosensor into the cell membrane, these biosensors allow the first in-cell probing of biological events using hyperpolarized xenon.

• Cadmium-glutathione solution structures provide new insights into heavy metal detoxification.

  Authors: Olivier Delalande, Hervé Desvaux, Emmanuel Godat, Alain Valleix, Christophe Junot, Jean Labarre, Yves Boulard

  The FEBS journal. 10/2010; 277(24):5086-96.

  Cadmium is a heavy metal and a pollutant that can be found in large quantities in the environment from industrial waste. Its toxicity for living organisms could arise from its ability to alterCadmium is a heavy metal and a pollutant that can be found in large quantities in the environment from industrial waste. Its toxicity for living organisms could arise from its ability to alter thiol-containing cellular components. Glutathione is an abundant tripeptide (γ-Glu-Cys-Gly) that is described as the first line of defence against cadmium in many cell types. NMR experiments for structure and dynamics determination, molecular simulations, competition reactions for metal chelation by different metabolites (γ-Glu-Cys-Gly, α-Glu-Cys-Gly and γ-Glu-Cys) combined with biochemical and genetics experiments have been performed to propose a full description of bio-inorganic reactions occurring in the early steps of cadmium detoxification processes. Our results give unambiguous information about the spontaneous formation, under physiological conditions, of the Cd(GS)(2) complex, about the nature of ligands involved in cadmium chelation by glutathione, and provide insights on the structures of Cd(GS)(2) complexes in solution at different pH. We also show that γ-Glu-Cys, the precursor of glutathione, forms a stable complex with cadmium, but biological studies of the first steps of cadmium detoxification reveal that this complex does not seem to be relevant for this purpose.

• Effect of pH and counterions on the encapsulation properties of xenon in water-soluble cryptophanes.

  Authors: Patrick Berthault, Hervé Desvaux, Thierry Wendlinger, Marina Gyejacquot, Antoine Stopin, Thierry Brotin, Jean-Pierre Dutasta, Yves Boulard

  Chemistry (Weinheim an der Bergstrasse, Germany). 09/2010; 16(43):12941-6.

  In the (129)Xe NMR-based biosensing approach in which the hyperpolarized noble gas is transported to biological receptors for a sensitive molecular imaging, cryptophanes are excellent xenon hostIn the (129)Xe NMR-based biosensing approach in which the hyperpolarized noble gas is transported to biological receptors for a sensitive molecular imaging, cryptophanes are excellent xenon host systems. However to avoid formation of self-organized systems, these hydrophobic cage molecules can be rendered water soluble by introduction of ionic groups. We show that the sensitivity of xenon to its local environment and the presence of these ionic functions can lead to interesting properties. For a first water-soluble cryptophane derivative, we show that a precise monitoring of the local pH can be performed. For a second cryptophane, the presence of ionic groups close to the cryptophane cavity modifies the xenon binding constant and in-out exchange rate. The latter allows the tuning of physical properties of xenon-cryptophane interactions without resorting to a change of the cavity size. These results open new perspectives on the influence of chemical modifications of cryptophanes for optimizing the biosensor properties.

• Lesion-induced DNA weak structural changes detected by pulsed EPR spectroscopy combined with site-directed spin labelling.

  Authors: Giuseppe Sicoli, Gérald Mathis, Samia Aci-Sèche, Christine Saint-Pierre, Yves Boulard, Didier Gasparutto, Serge Gambarelli

  Nucleic acids research. 04/2009;

  Double electron-electron resonance (DEER) was applied to determine nanometre spin-spin distances on DNA duplexes that contain selected structural alterations. The present approach to evaluate theDouble electron-electron resonance (DEER) was applied to determine nanometre spin-spin distances on DNA duplexes that contain selected structural alterations. The present approach to evaluate the structural features of DNA damages is thus related to the interspin distance changes, as well as to the flexibility of the overall structure deduced from the distance distribution. A set of site-directed nitroxide-labelled double-stranded DNA fragments containing defined lesions, namely an 8-oxoguanine, an abasic site or abasic site analogues, a nick, a gap and a bulge structure were prepared and then analysed by the DEER spectroscopic technique. New insights into the application of 4-pulse DEER sequence are also provided, in particular with respect to the spin probes' positions and the rigidity of selected systems. The lesion-induced conformational changes observed, which were supported by molecular dynamics studies, confirm the results obtained by other, more conventional, spectroscopic techniques. Thus, the experimental approaches described herein provide an efficient method for probing lesion-induced structural changes of nucleic acids.

• Sensitivity and multiplexing capabilities of MRI based on polarized 129Xe biosensors.

  Authors: Patrick Berthault, Aurore Bogaert-Buchmann, Hervé Desvaux, Gaspard Huber, Yves Boulard

  Journal of the American Chemical Society. 01/2009; 130(49):16456-7.

• Double electron-electron resonance (DEER): a convenient method to probe DNA conformational changes.

  Authors: Giuseppe Sicoli, Gérald Mathis, Olivier Delalande, Yves Boulard, Didier Gasparutto, Serge Gambarelli

  Angewandte Chemie (International ed. in English). 02/2008; 47(4):735-7.

• A cryptophane biosensor for the detection of specific nucleotide targets through xenon NMR spectroscopy.

  Authors: Vincent Roy, Thierry Brotin, Jean-Pierre Dutasta, Marie-Hélène Charles, Thierry Delair, François Mallet, Gaspard Huber, Hervé Desvaux, Yves Boulard, Patrick Berthault

  Chemphyschem : a European journal of chemical physics and physical chemistry. 11/2007; 8(14):2082-5.

• Functional organization of the Rpb5 subunit shared by the three yeast RNA polymerases.

  Authors: Cécile Zaros, Jean-François Briand, Yves Boulard, Sylvie Labarre-Mariotte, M Carmen Garcia-Lopez, Pierre Thuriaux, Francisco Navarro

  Nucleic acids research. 02/2007; 35(2):634-47.

  Rpb5, a subunit shared by the three yeast RNA polymerases, combines a eukaryotic N-terminal module with a globular C-end conserved in all non-bacterial enzymes. Conditional and lethal mutants of theRpb5, a subunit shared by the three yeast RNA polymerases, combines a eukaryotic N-terminal module with a globular C-end conserved in all non-bacterial enzymes. Conditional and lethal mutants of the moderately conserved eukaryotic module showed that its large N-terminal helix and a short motif at the end of the module are critical in vivo. Lethal or conditional mutants of the C-terminal globe altered the binding of Rpb5 to Rpb1-beta25/26 (prolonging the Bridge helix) and Rpb1-alpha44/47 (ahead of the Switch 1 loop and binding Rpb5 in a two-hybrid assay). The large intervening segment of Rpb1 is held across the DNA Cleft by Rpb9, consistent with the synergy observed for rpb5 mutants and rpb9Delta or its RNA polymerase I rpa12Delta counterpart. Rpb1-beta25/26, Rpb1-alpha44/45 and the Switch 1 loop were only found in Rpb5-containing polymerases, but the Bridge and Rpb1-alpha46/47 helix bundle were universally conserved. We conclude that the main function of the dual Rpb5-Rpb1 binding and the Rpb9-Rpb1 interaction is to hold the Bridge helix, the Rpb1-alpha44/47 helix bundle and the Switch 1 loop into a closely packed DNA-binding fold around the transcription bubble, in an organization shared by the two other nuclear RNA polymerases and by the archaeal and viral enzymes.

• NMR assessment of the global shape of a non-labelled DNA dodecamer containing a tandem of G-T mismatches.

  Authors: Francisco Alvarez Salgado, Hervé Desvaux, Yves Boulard

  Magnetic resonance in chemistry : MRC. 01/2007; 44(12):1081-9.

  We have carried out a solution study of two non-labelled self-complementary DNA dodecamers d(GACTGTACAGTC)2 and d(GACTGTGCAGTC)2 by NMR, the second sequence composed of two G-T mismatches. StructuresWe have carried out a solution study of two non-labelled self-complementary DNA dodecamers d(GACTGTACAGTC)2 and d(GACTGTGCAGTC)2 by NMR, the second sequence composed of two G-T mismatches. Structures were determined using distances extracted from NOE effects alone or using both NOE and RDC constraints, measured in three different liquid crystalline media. We ensured that our data on the influence of the mesogen on the DNA structures, and the way in which the RDCs were incorporated as constraints in the protocol refinement, were consistent. We also tested the influence of different sets of RDCs and the best means of optimizing the calculation of D(a) and R. Resolution and accuracy of the ten best energy final structures were compared. The addition of a small set of RDC constraints significantly improves the final determined structures. We took advantage of the specificity of the RDC, i.e. it contains orientational information, and explored the global shape of the DNA duplexes; it was found that the duplexes do not have a large curvature. For the G-T base pair, we observed, in this particular sequence (tandem of G-T mismatches), a new pattern of base pairing, which involved the formation of a bifurcated hydrogen bond.

• Yaf9, a novel NuA4 histone acetyltransferase subunit, is required for the cellular response to spindle stress in yeast.

  Authors: Ivan Le Masson, David Y Yu, Kurt Jensen, Anne Chevalier, Régis Courbeyrette, Yves Boulard, M Mitchell Smith, Carl Mann

  Molecular and cellular biology. 10/2003; 23(17):6086-102.

  Yaf9 is one of three proteins in budding yeast containing a YEATS domain. We show that Yaf9 is part of a large complex and that it coprecipitates with three known subunits of the NuA4 histoneYaf9 is one of three proteins in budding yeast containing a YEATS domain. We show that Yaf9 is part of a large complex and that it coprecipitates with three known subunits of the NuA4 histone acetyltransferase. Although Esa1, the catalytic subunit of NuA4, is essential for viability, we found that yaf9 Delta mutants are viable but hypersensitive to microtubule depolymerizing agents and synthetically lethal with two different mutants of the mitotic apparatus. Microtubules depolymerized more readily in the yaf9Delta mutant compared to the wild type in the presence of nocodazole, and recovery of microtubule polymerization and cell division from limiting concentrations of nocodazole was inhibited. Two other NuA4 mutants (esa1-1851 and yng2 Delta) and nonacetylatable histone H4 mutants were also sensitive to benomyl. Furthermore, wild-type budding yeast were more resistant to benomyl when grown in the presence of trichostatin A, a histone deacetylase inhibitor. These results strongly suggest that acetylation of histone H4 by NuA4 is required for the cellular resistance to spindle stress.

• Dilute liquid crystals used to enhance residual dipolar couplings may alter conformational equilibrium in oligosaccharides.

  Authors: Patrick Berthault, Damien Jeannerat, Franck Camerel, Francisco Alvarez Salgado, Yves Boulard, Jean-Christophe P Gabriel, Hervé Desvaux

  Carbohydrate research. 09/2003; 338(17):1771-85.

  The solution structures of a trisaccharide and a pentasaccharide containing the Lewis(x) motif were determined by two independent approaches using either dipolar cross-relaxation (NOE) or residualThe solution structures of a trisaccharide and a pentasaccharide containing the Lewis(x) motif were determined by two independent approaches using either dipolar cross-relaxation (NOE) or residual dipolar coupling (RDC) data. For the latter, one-bond 13C[bond](1)H RDC enhanced by two different mineral liquid crystals were used alone. Home-written programs were employed firstly for measuring accurately the coupling constants in the direct dimension of non-decoupled HSQC experiments, secondly for transforming each RDC data set into geometrical restraints. In this second program, the complete molecular structure was expressed in a unique frame where the alignment tensor is diagonal. Assuming that the pyranose rings are rigid, their relative orientation is defined by optimizing the glycosidic torsion angles. For the trisaccharide, a good agreement was observed between the results of both approaches (NOE and RDC). In contrast, for the pentasaccharide, strong discrepancies appeared, which seem to result from interactions between the pentasaccharide and the mesogens, affecting conformational equilibrium. This observation is of importance, as it reveals that using simultaneously NOE and RDC can be hazardous as the former represent 99% of the molecules free in solution, whereas the latter correspond to less than 1% of the structure bound to the mesogen.

• Solution structure of a DNA duplex containing a formamide-adenine base pair.

  Authors: Corinne Maufrais, Yves Boulard

  Canadian journal of physiology and pharmacology. 08/2002; 80(7):609-17.

  The N-(2-deoxy-beta3-D-erythro-pentofuranosyl) formamide residue results from a ring fragmentation product of thymine or cytosine. The presence of a formamide-adenine base pair in the sequenceThe N-(2-deoxy-beta3-D-erythro-pentofuranosyl) formamide residue results from a ring fragmentation product of thymine or cytosine. The presence of a formamide-adenine base pair in the sequence 5'd(AGGAACCACG).d(CGTGGFTCCT) has been studied by 1H and 31P nuclear magnetic resonance (NMR) and molecular dynamics. There are two possible isomers for the formamide side chain, either cis or trans. For each isomer, we observed an equilibrium in solution between two forms. First, a species where the formamide is intrahelical and paired with the facing adenine. For the cis isomer, the formamide is in a syn conformation and two hydrogen bonds with adenine are formed. The trans isomer is in an anti conformation and a single hydrogen bond is observed. In the second form, whatever the isomer, the formamide is rejected outside the helix, whereas the adenine remains inside.

• The solution structure of an oligonucleotide duplex containing a 2'-deoxyadenosine-3-(2-hydroxyethyl)- 2'-deoxyuridine base pair determined by NMR and molecular dynamics studies.

  Authors: Yves Boulard, G Victor Fazakerley, Lawrence C Sowers

  Nucleic acids research. 04/2002; 30(6):1371-8.

  Determination of the solution structure of the duplex d(GCAAGTC(HE)AAAACG)*d(CGTTTTAGACTTGC) containing a 3-(2-hydroxyethyl)-2'-deoxyuridine*deoxyadenine (HE*A) base pair is reported. TheDetermination of the solution structure of the duplex d(GCAAGTC(HE)AAAACG)*d(CGTTTTAGACTTGC) containing a 3-(2-hydroxyethyl)-2'-deoxyuridine*deoxyadenine (HE*A) base pair is reported. The three-dimensional solution structure, determined starting from 512 models via restrained molecular mechanics using inter-proton distances and torsion angles, converged to two final families of structures. For both families the HE and the opposite A residues are intrahelical and in the anti conformation. The hydroxyethyl chain lies close to the helix axis and for one family the hydroxyl group is above the HE*A plane and in the other case it is below. These two models were used to start molecular dynamic calculations with explicit solvent to explore the hydrogen bonding possibilities of the HE*A base pair. The dynamics calculations converge finally to one model structure in which two hydrogen bonds are formed. The first is formed all the time and is between HEO4 and the amino group of A, and the second, an intermittent one, is between the hydroxyl group and the N1 of A. When this second hydrogen bond is not formed a weak interaction CH...N is possible between HEC7H2 and N1A21. All the best structures show an increase in the C1'-C1' distance relative to a Watson-Crick base pair.