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Active and passive mechanisms of helicases
Laboratoire Physique des biomolécules - M. Manosas, X. G. Xi, D. Bensimon and V. Croquette
Nucleic Acids Res. - 38(16) :5518–26 - DOI:10.1093/nar/gkq273 - 2010
In this work, we discuss the active or passive character of helicases. In the past years, several studies have used the theoretical framework proposed by Betterton and Julicher [Betterton, M.D. and Julicher, F. (2005) Opening of nucleic-acid double strands by helicases: active versus passive opening. Phys. Rev. E, 71, 11904-11911.] to analyse the unwinding data and assess the mechanism of the helicase under study (active versus passive). However, this procedure has given rise to apparently contradictory interpretations: helicases exhibiting similar behaviour have been classified as both active and passive enzymes [Johnson, D.S., Bai, L. Smith, B.Y., Patel, S.S. and Wang, M.D. (2007) Single-molecule studies reveal dynamics of DNA unwinding by the ring-shaped T7 helicase. Cell, 129, 1299-1309; Lionnet, T., Spiering, M.M., Benkovic, S.J., Bensimon, D. and Croquette, V. (2007) Real-time observation of bacteriophage T4 gp41 helicase reveals an unwinding mechanism Proc. Natl Acid. Sci., 104, 19790-19795]. In this work, we show that when the helicase under study has not been previously well characterized (namely, if its step size and rate of slippage are unknown) a multi-parameter fit to the afore-mentioned model can indeed lead to contradictory interpretations. We thus propose to differentiate between active and passive helicases on the basis of the comparison between their observed translocation velocity on single-stranded nucleic acid and their unwinding rate of double-stranded nucleic acid (with various GC content and under different tensions). A threshold separating active from passive behaviour is proposed following an analysis of the reported activities of different helicases. We study and contrast the mechanism of two helicases that exemplify these two behaviours: active for the RecQ helicase and passive for the gp41 helicase.
Mechanism of strand displacement synthesis by DNA replicative polymerases
Laboratoire Physique des biomolécules - M. Manosas, M. M. Spiering, F. Ding, D. Bensimon, J.-F. Allemand, S. J. Benkovic and V. Croquette
Nucleic Acids Res. - 40(13) :6174-86 - PMID:22434889 - 2010
Replicative holoenzymes exhibit rapid and processive primer extension DNA synthesis, but inefficient strand displacement DNA synthesis. We investigated the bacteriophage T4 and T7 holoenzymes primer extension activity and strand displacement activity on a DNA hairpin substrate manipulated by a magnetic trap. Holoenzyme primer extension activity is moderately hindered by the applied force. In contrast, the strand displacement activity is strongly stimulated by the applied force; DNA polymerization is favoured at high force, while a processive exonuclease activity is triggered at low force. We propose that the DNA fork upstream of the holoenzyme generates a regression pressure which inhibits the polymerization-driven forward motion of the holoenzyme. The inhibition is generated by the distortion of the template strand within the polymerization active site thereby shifting the equilibrium to a DNA-protein exonuclease conformation. We conclude that stalling of the holoenzyme induced by the fork regression pressure is the basis for the inefficient strand displacement synthesis characteristic of replicative polymerases. The resulting processive exonuclease activity may be relevant in replisome disassembly to reset a stalled replication fork to a symmetrical situation. Our findings offer interesting applications for single-molecule DNA sequencing.
Micropillar array chips toward new immunodiagnosis
Laboratoire Sciences Analytiques Bioanalytiques et Miniaturisation - H.Y. Li, V. Dauriac, V. Thibert, H. Senechal, G. Peltre, X.X. Zhang, S. Descroix
Lab. Chip - 10(19) :2597-604 - DOI:10.1039/c005034b - 2010
In this paper, we demonstrate the possibility to use a micropillar array to perform molecular immunodiagnosis. A polydimethylsiloxane (PDMS) microdevice consisting of a rectangular array of micropillars (45 µm in height, 100 × 100 µm square cross section) was used to replace microchannels or gels (polyacrylamide or agarose) to perform electrokinetic separation. This microarray was used to mimic highly diluted gel and to maintain electrolyte within the pillar zone by capillary effect. The electrolyte composition (glycerol and agarose content) was investigated in order to improve protein separation by isoelectric focusing (IEF). The influence of glycerol on focusing time and on the different evaporative contributions was further evaluated. In order to perform an immunodiagnostic of milk allergy, different surface treatments were optimized to prevent milk allergen adsorption on PDMS surface. Poly(dimethylacrylamide)-co-allyl glycidyl ether (PDMA-AGE) as well as gelatin led to a satisfactory signal to noise ratio. Finally the possibility to perform protein mixture separation using this micropillar array chip followed by immunoblotting was demonstrated by using the serum from an allergic individual, confirming the great potential of this analytical platform in the field of immunodiagnosis.
Total on-line analysis of a target protein from plasma by immunoextraction, digestion and liquid chromatography-mass spectrometry
Laboratoire Sciences Analytiques Bioanalytiques et Miniaturisation - A. Cingöz, F. Hugon-Chapuis, V. Pichon
J. Chrom. B - 878(2) :213-21 - DOI:10.1016/j.jchromb.2009.07.032 - 2010
A total on-line analysis of a target protein from a plasma sample was made using a selective immunoextraction step coupled on-line to an immobilized enzymatic reactor (IMER) for the protein digestion followed by LC-MS/MS analysis. For the development of this device, cytochrome c was chosen as model protein due to its well-known sequence. An immunosorbent (IS) based on the covalent immobilization of anti-cytochrome c antibodies on a solid support was made and an immunoextraction procedure was carefully developed to assess a selective extraction of the target protein from plasma. For the first time, IS was easily coupled on-line with a laboratory-made IMER based on pepsin. The whole on-line device (IS-IMER-LC-MS/MS) allowed the quantification of cytochrome c from 8.5pmol to 1.7nmol in buffer medium. Finally, this device was applied to the analysis of only 85pmol of cytochrome c from plasma with a RSD value lower than 10% (n=3).
Supercritical fluid chromatography hyphenated with twin comprehensive two-dimensional gas chromatography for ultimate analysis of middle distillates
Laboratoire Sciences Analytiques Bioanalytiques et Miniaturisation - F. Adam, D. Thiébaut, F. Bertoncini, M. Courtiade, M.C. Hennion
J. Chrom. A - 1217(8) :1386-94 - DOI:10.1016/j.chroma.2009.11.092 - 2010
This paper reports the conditions of online hyphenation of supercritical fluid chromatography (SFC) with twin comprehensive two-dimensional gas chromatography (twin-GCxGC) for detailed characterization of middle distillates; this is essential for a better understanding of reactions involved in refining processes. In this configuration, saturated and unsaturated compounds that have been fractionated by SFC are transferred on two different GC x GC columns sets (twin-GCxGC) placed in the same GC oven. Cryogenic focusing is used for transfer of fractions into the first dimension columns before simultaneous GCxGC analysis of both saturated and unsaturated fractions. The benefits of SFC-twin-GC x GC are demonstrated for the extended alkane, iso-alkane, alkene, naphthenes and aromatics analysis (so-called PIONA analysis) of diesel samples which can be achieved in one single injection. For that purpose, saturated and unsaturated compounds have been separated by SFC using a silver loaded silica column prior to GC x GC analysis. Alkenes and naphthenes are quantitatively recovered in the unsaturated and saturated fractions, respectively, allowing their identification in various diesel samples. Thus, resolution between each class of compounds is significantly improved compared to a single GCxGC run, and for the first time, an extended PIONA analysis of diesel samples is presented.
Indirect Grafting of Acetonitrile-Derived Films on Metallic Substrates
Laboratoire Sciences Analytiques Bioanalytiques et Miniaturisation - A. Berisha, C. Combellas, F. Kanoufi, J. Pinson, S. Ustaze, F.I. Podvorica
Chem. Mater - 22(9) :2962-9 - DOI:10.1021/cm100295n - 2010
Strongly bonded organic films with amino groups are obtained on gold, copper, and silicon surfaces by reduction of 2,6-dimethyl benzenediazonium in acetonitrile (ACN). The sterically hindered 2,6-dimethylphenyl radical is unable to attach to the surface, but it abstracts an hydrogen atom from ACN to give the cyanomethyl radical (·CH2CN) that reacts with the surface. A spontaneous reaction is also possible on copper. The film is characterized by IR spectroscopy, scanning electron microscopy, ellipsometry, water contact angles, and cyclic voltammetry. A mechanism is elaborated that accounts for the formation, grafting of the cyanomethyl radical, and finally formation of amino multilayers.
Dansyl-peptides matrix-assisted laser desorption/ionization mass spectrometric (MALDI-MS) and tandem mass spectrometric (MS/MS) features improve the liquid chromatography/MALDI-MS/MS analysis of the proteome
Laboratoire Spectrométrie de masse biologique et protéomique - Chiappetta G, Ndiaye S, Demey E, Haddad I, Marino G, Amoresano A, Vinh J.
Rapid Commun Mass Spectrom - 24(20) :3021-32 - DOI:10.1002/rcm.4734 - 2010
Peptide tagging is a useful tool to improve matrix-assisted laser desorption/ionization tandem mass spectrometric (MALDI-MS/MS) analysis. We present a new application of the use of the dansyl chloride (DNS-Cl). DNS-Cl is a specific primary amine reagent widely used in protein biochemistry. It adds a fluorescent dimethylaminonaphthalene moiety to the molecule. The evaluation of MALDI-MS and MS/MS analyses of dansylated peptides shows that dansylation raises the ionization efficiency of the most hydrophilic species compared with the most hydrophobic ones. Consequently, higher Mascot scores and protein sequence coverage are obtained by combining MS and MS/MS data of native and tagged samples. The N-terminal DNS-Cl sulfonation improves the peptide fragmentation and promotes the generation of b-fragments allowing better peptide sequencing. In addition, we set up a labeling protocol based on the microwave chemistry. Peptide dansylation proved to be a rapid and cheap method to improve the performance of liquid chromatography (LC)/MALDI-MS/MS analysis at the proteomic scale in terms of peptide detection and sequence coverage.
Cell shape and cell division in fission yeast
Laboratoire Biologie cellulaire systémique de la polarité et de la division - Piel M, Tran PT
Curr Biol. - 17(19) :R823-7 - DOI:10.1016/j.cub.2009.08.012 - 2009
The fission yeast Schizosaccharomyces pombe has served as an important model organism for investigating cellular morphogenesis. This unicellular rod-shaped fission yeast grows by tip extension and divides by medial fission. In particular, microtubules appear to define sites of polarized cell growth by delivering cell polarity factors to the cell tips. Microtubules also position the cell nucleus at the cell middle, marking sites of cell division. Here, we review the microtubule-dependent mechanisms that regulate cell shape and cell division in fission yeast.
Simple and rapid process for single cell micro-patterning
Laboratoire Biologie cellulaire systémique de la polarité et de la division - Ammar Azioune, Marko Storch, Michel Bornens, Manuel Théry and Matthieu Piel
Lab. Chip - 9(11) :1640-2 - DOI:10.1039/b821581m - 2009
We present a simple and environmentally friendly process for cell patterning on glass covered with an ultrathin layer of poly-l-lysine-grafted-polyethylene glycol (PLL-g-PEG) by exposure to deep UV light. The patterned substrates are stable for months in the lab atmosphere before incubation with proteins. Incubation with proteins resulted in well defined patterns, with high feature resolution. RPE-1 cells seeded on fibronectin/fibrinogen-Alexa 488 patterns were constrained for days on the deep UV exposed regions. Finally, large glass plates were patterned with high homogeneity enabling the assembly of micro-patterned microplates in 96-well format.
Pushing off the walls: a mechanism of cell motility in confinement
Laboratoire Biologie cellulaire systémique de la polarité et de la division - Hawkins RJ, Piel M, Faure-Andre G, Lennon-Dumenil AM, Joanny JF, Prost J, Voituriez R
Phys. Rev. Lett. - 102(5) :58103 - DOI:10.1103/PhysRevLett.102.058103 - 2009
We propose a novel mechanism of cell motility, which relies on the coupling of actin polymerization at the cell membrane to geometric confinement. We consider a polymerizing viscoelastic cytoskeletal gel confined in a narrow channel, and show analytically that spontaneous motion occurs. Interestingly, this does not require specific adhesion with the channel walls, and yields velocities potentially larger than the polymerization velocity. The contractile activity of myosin motors is not necessary to trigger motility in this mechanism, but is shown quantitatively to increase the velocity. Our model qualitatively accounts for recent experiments which show that cells without specific adhesion proteins are motile only in confined environments while they are unable to move on a flat surface, and could help in understanding the mechanisms of cell migration in more complex confined geometries such as living tissues.
Diffusion through colloidal shells under stress
Laboratoire Colloïdes et Matériaux Divisés - J. Guery, J. Baudry, D. A. Weitz, P. M. Chaikin, J. Bibette
Phys. Rev. E - 79 :060402 - DOI:10.1103/PhysRevE.79.060402 - 2009
The permeability of solids has long been associated with a diffusive process involving activated mechanism as originally envisioned by Eyring. Tensile stress can affect the activation energy but definitive experiments of the diffusion rate of species through a stressed solid are lacking. Here we use core-shell (liquid core–solid shell) colloidal particles that are sensitive to osmotic pressure to follow the permeation of encapsulated probes at various stresses. We unambiguously show that the tensile stress applied on colloidal shells linearly reduces the local energy barrier for diffusion.
Breaking of an Emulsion under an ac Electric Field
Laboratoire Colloïdes et Matériaux Divisés - A. R. Thiam, N. Bremond, J. Bibette
Phys. Rev. Lett. - 102(18) :18304 - DOI:10.1103/PhysRevLett.102.188304 - 2009
By using microfluidic chips, we investigate the stability regarding coalescence of droplet pairs under an electric field as a function of drop separation and ac field intensity. Three different regimes are found: stable, coalescence, and partial merging. From this, we identify the two breaking scenarios of a one dimensional train of droplets: in one case the coalescence front propagates; in the other case, in which pairs belong to the partial merging regime, the coalescence front can become heterogeneous. From these findings, we can propose a destruction mechanism for a macroscopic emulsion, which includes the packing condition for which total and immediate destruction is effective.
Direct observation of twisting steps during Rad51 polymerization on DNA
Laboratoire Macromolécules et Microsystèmes en Biologie et Médecine - Arata H, Dupont A, Miné-Hattab J, Disseau L, Renodon-Cornière A, Takahashi M, Viovy JL, Cappello G
Proc. Nat. Acad. Sci. USA - 106(46) :19239-44 - DOI:10.1073/pnas.0902234106 - 2009
The human recombinase hRad51 is a key protein for the maintenance of genome integrity and for cancer development. Polymerization and depolymerization of hRad51 on duplex DNA were studied here using a new generation of magnetic tweezers, measuring DNA twist in real time with a resolution of 5°. Our results combined with earlier structural information suggest that DNA is somewhat less extended by hRad51 than by RecA (4.5 vs. 5.1 Å per base pair) and untwisted by 18.2° per base pair. They also confirm a stoichiometry of 3–4 bp per protein in the hRad51-dsDNA nucleoprotein filament. At odds with earlier claims, we show that after initial deposition of a multimeric nucleus, nucleoprotein filament growth occurs by addition/release of single proteins, involving DNA twisting steps of 65° ± 5°. Simple numeric simulations show that this mechanism is an efficient way to minimize nucleoprotein filament defects. Nucleoprotein filament growth from a preformed nucleus was observed at hRad51 concentrations down to 10 nM, whereas nucleation was never observed below 100 nM in the same buffer. This behavior can be associated with the different stoichiometries of nucleation and growth. It may be instrumental in vivo to permit efficient continuation of strand exchange by hRad51 alone while requiring additional proteins such as Rad52 for its initiation, thus keeping the latter under the strict control of regulatory pathways.
Droplet breakup in microfluidic T-junctions at small capillary numbers
Laboratoire Microfluidique MEMS et nanostructures - M. C Jullien, M. J Tsang Mui Ching, C. Cohen, L. Ménétrier, P. Tabeling
Phys. Fluids - 21( 7) :7200-61 - DOI:10.1063/1.3170983 - 2009
We perform experimental studies of droplet breakup in microfluidic T-junctions in a range of capillary numbers lying between 4×10-4 and 2×10-1 and for two viscosity ratios of the fluids forming the dispersed and continuous phases. The present paper extends the range of capillary numbers explored by previous investigators by two orders of magnitude. We single out two different regimes of breakup. In a first regime, a gap exists between the droplet and the wall before breakup occurs. In this case, the breakup process agrees well with the analytical theory of Leshansky and Pismen [Phys. Fluids 21, 023303 (2009) ]. In a second regime, droplets keep obstructing the T-junction before breakup. Using physical arguments, we introduce a critical droplet extension for describing the breakup process in this case.
T7 RNA polymerase studied by force measurements varying cofactor concentration
Laboratoire Nanobiophysiques - P. Thomen, P. J. Lopez, U. Bockelmann, J. Guillerez, M. Dreyfus, and F. Heslot
Biophys. J. - 95(5) :2423–33 - DOI:10.1529/biophysj.107.125096 - 2009
RNA polymerases carry out the synthesis of an RNA copy from a DNA template. They move along DNA, incorporate nucleotide triphosphate (NTP) at the end of the growing RNA chain, and consume chemical energy. In a single-molecule assay using the T7 RNA polymerase, we study how a mechanical force opposing the forward motion of the enzyme along DNA affects the translocation rate. We also study the influence of nucleotide and magnesium concentration on this process. The experiment shows that the opposing mechanical force is a competitive inhibitor of nucleotide binding. Also, the single-molecule data suggest that magnesium ions are involved in a step that does not depend on the external load force. These kinetic results associated with known biochemical and mutagenic data, along with the static information obtained from crystallographic structures, shape a very coherent view of the catalytic cycle of the enzyme: translocation does not take place upon NTP binding nor upon NTP cleavage, but rather occurs after PPi release and before the next nucleotide binding event. Furthermore, the energetic bias associated with the forward motion of the enzyme is close to kT and represents only a small fraction of the free energy of nucleotide incorporation and pyrophosphate hydrolysis.
Probing DNA base pairing energy profiles using a nanopore
Laboratoire Nanobiophysiques - Virgile Viasnoff, Nicolas Chiaruttini, and Ulrich Bockelmann
European Biophysics Journal - 38(2) :263–9 - PMID:18836709 - 2009
We experimentally show that the voltage driven unzipping of long DNA duplexes by an a-hemolysin pore is sensitive to the shape of the base pairing energy landscape. Two sequences of equal global stability were investigated. The sequence with an homogeneous base pairing profile translocates faster than the one with alternative weak and strong regions. We could qualitatively account for theses observations by theoretically describing the voltage driven translocation as a biased random walk of the unzipping fork in the sequence dependent energy landscape.
Photomanipulation of a Droplet by the Chromocapillary Effect
Laboratoire Nanobioscience et Microsystèmes group - A. Diguet, R. M. Guillermic, N. Magome, A. Saint-Jalmes, Y. Chen, K. Yoshikawa and D. Baigl
Angew Chem Int Ed Engl. - 48(49) :9281-4 - DOI:10.1002/anie.200904868 - 2009
Ace of hearts: Liquid droplets can be manipulated in a controlled fashion along trajectories of any desired shape (such as a heart, see picture) by using light to create a wavelength-dependent interfacial tension gradient at a liquid/liquid interface. In this new phenomenon, the “chromocapillary effect”, an interfacial flow generates droplet motion in the direction opposite to the gradient.
Sequence-Independent and Reversible Photocontrol of Transcription/Expression Systems Using a Photosensitive Nucleic Acid Binder
Laboratoire Nanobioscience et Microsystèmes group - A. Estevez-Torres, C. Crozatier, A. Diguet, T. Hara, H. Saito, K. Yoshikawa and D. Baigl
Proc. Nat. Acad. Sci. USA - 106(30) :12219-23 - DOI:10.1073/pnas.0904382106 - 2009
To understand non-trivial biological functions, it is crucial to develop minimal synthetic models that capture their basic features. Here, we demonstrate a sequence-independent, reversible control of transcription and gene expression using a photosensitive nucleic acid binder (pNAB). By introducing a pNAB whose affinity for nucleic acids is tuned by light, in vitro RNA production, EGFP translation, and GFP expression (a set of reactions including both transcription and translation) were successfully inhibited in the dark and recovered after a short illumination at 365 nm. Our results indicate that the accessibility of the protein machinery to one or several nucleic acid binding sites can be efficiently regulated by changing the conformational/condensation state of the nucleic acid (DNA conformation or mRNA aggregation), thus regulating gene activity in an efficient, reversible, and sequence-independent manner. The possibility offered by our approach to use light to trigger various gene expression systems in a system-independent way opens interesting perspectives to study gene expression dynamics as well as to develop photocontrolled biotechnological procedures.
Mechanisms of chiral discrimination by topoisomerase IV
Laboratoire Physique des biomolécules - K. C. Neuman, G. Charvin, D. Bensimon and V. Croquette
Proc. Nat. Acad. Sci. USA - 106(17) :6986–91 - DOI:10.1073/pnas.0900574106 - 2009
Topoisomerase IV (Topo IV), an essential ATP-dependent bacterial type II topoisomerase, transports one segment of DNA through a transient double-strand break in a second segment of DNA. In vivo, Topo IV unlinks catenated chromosomes before cell division and relaxes positive supercoils generated during DNA replication. In vitro, Topo IV relaxes positive supercoils at least 20-fold faster than negative supercoils. The mechanisms underlying this chiral discrimination by Topo IV and other type II topoisomerases remain speculative. We used magnetic tweezers to measure the relaxation rates of single and multiple DNA crossings by Topo IV. These measurements allowed us to determine unambiguously the relative importance of DNA crossing geometry and enzymatic processivity in chiral discrimination by Topo IV. Our results indicate that Topo IV binds and passes DNA strands juxtaposed in a nearly perpendicular orientation and that relaxation of negative supercoiled DNA is perfectly distributive. Together, these results suggest that chiral discrimination arises primarily from dramatic differences in the processivity of relaxing positive and negative supercoiled DNA: Topo IV is highly processive on positively supercoiled DNA, whereas it is perfectly distributive on negatively supercoiled DNA. These results provide fresh insight into topoisomerase mechanisms and lead to a model that reconciles contradictory aspects of previous findings while providing a framework to interpret future results.
Measurement of the Torque on a Single Stretched and Twisted DNA Using Magnetic Tweezers
Laboratoire Physique des biomolécules - F. Mosconi, J.-F. Allemand, D. Bensimon and V. Croquette
Phys. Rev. Lett. - 102(7) :78301 - PMID:19257716 - 2009
We deduced the torque applied on a single stretched and twisted DNA by integrating the change in the molecule's extension with respect to force as it is coiled. While consistent with previous direct measurements of the torque at high forces (F > 1 pN), this method, which is simple and does not require a sophisticated setup, allows for lower force estimates. We used this approach to deduce the effective torsional modulus of DNA, which decreases with force, and to estimate the buckling torque of DNA as a function of force in various salt conditions.

638 publications.