Université PSL



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Coupling DNA unwinding activity with primer synthesis in the bacteriophage T4 primosome
M. Manosas, M. M. Spiering, Z. Zhuang, S. J. Benkovic and V. Croquette
Nature Chemical Biology - 5(12) :904–12 - DOI:10.1038/nchembio.236 - 2009
The unwinding and priming activities of the bacteriophage T4 primosome, which consists of a hexameric helicase (gp41) translocating 5' to 3' and an oligomeric primase (gp61) synthesizing primers 5' to 3', have been investigated on DNA hairpins manipulated by a magnetic trap. We find that the T4 primosome continuously unwinds the DNA duplex while allowing for primer synthesis through a primosome disassembly mechanism or a new DNA looping mechanism. A fused gp61-gp41 primosome unwinds and primes DNA exclusively via the DNA looping mechanism. Other proteins within the replisome control the partitioning of these two mechanisms by disfavoring primosome disassembly, thereby increasing primase processivity. In contrast to T4, priming in bacteriophage T7 and Escherichia coli involves discrete pausing of the primosome and dissociation of the primase from the helicase, respectively. Thus nature appears to use several strategies to couple the disparate helicase and primase activities within primosomes.
A nanostructure made of a small non-coding RNA
Cayrol B., Nogues C., Dawid A., Sagi I., Silberzan P., Isambert H.
JACS - 131(47) :17270-6 - DOI:10.1021/ja906076e - 2009
Natural RNAs, unlike many proteins, have never been reported to form extended nanostructures, despite their wide variety of cellular functions. This is all the more striking, as synthetic DNA and RNA forming large nanostructures have long been successfully designed. Here, we show that DsrA, a 87-nt noncoding RNA of Escherishia coli, self-assembles into a hierarchy of nanostructures through antisense interactions of three contiguous self-complementary regions. Yet, the extended nanostructures, observed using atomic force microscopy (AFM) and fluorescence microscopy, are easily disrupted into >100 nm long helical bundles of DsrA filaments, including hundreds of DsrA monomers, and are surprisingly resistant to heat and urea denaturation. Molecular modeling demonstrates that this structural switch of DsrA nanostructures into filament bundles results from the relaxation of stored torsional constraints and suggests possible implications for DsrA regulatory function.
Diffusion through colloidal shells under stress
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
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.
Determination of Cocaine in Human Plasma by Selective Solid-Phase Extraction Using an Aptamer-Based Sorbent
B. Madru, F. Chapuis-Hugon, E. Peyrin, V. Pichon
Anal. Chem. - 81(16) :7081-6 - DOI:10.1021/ac9006667 - 2009
A complete characterization is presented of a highly selective solid-phase extraction (SPE) sorbent which exploits the properties of aptamers. An oligosorbent based on aptamers immobilized on a solid support was synthesized and tested for the selective extraction of cocaine from human plasma. Anticocaine aptamers were immobilized to CNBr-activated Sepharose, and an extraction procedure was developed in pure media. Specific retention of cocaine on the oligosorbent was demonstrated, and the capacity of the support was determined. This oligosorbent was then applied to the selective extraction of cocaine from plasma at a concentration of 0.4 mg L(-1), i.e., corresponding to the plasma concentration reached after an intake of a single dose of cocaine. Extraction recovery close to 90% was obtained. Moreover, interfering compounds that perturbed cocaine quantification when using a standard SPE sorbent were not retained on the oligosorbent, thus allowing fast and reliable analyses of plasma samples with an estimated limit of detection of 0.1 microg mL(-1).
Direct observation of twisting steps during Rad51 polymerization on DNA
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
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
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
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.
Wetting and spreading
Bonn D., Eggers J., Iindekeu J., Meunier J., Rolley E.
REVIEWS OF MODERN PHYSICS - 81(2) :739-805 - DOI:10.1103/RevModPhys.81.739 - 2009
Wetting phenomena are ubiquitous in nature and technology. A solid substrate exposed to the environment is almost invariably covered by a layer of fluid material. In this review, the surface forces that lead to wetting are considered, and the equilibrium surface coverage of a substrate in contact with a drop of liquid. Depending on the nature of the surface forces involved, different scenarios for wetting phase transitions are possible; recent progress allows us to relate the critical exponents directly to the nature of the surface forces which lead to the different wetting scenarios. Thermal fluctuation effects, which can be greatly enhanced for wetting of geometrically or chemically structured substrates, and are much stronger in colloidal suspensions, modify the adsorption singularities. Macroscopic descriptions and microscopic theories have been developed to understand and predict wetting behavior relevant to microfluidics and nanofluidics applications. Then the dynamics of wetting is examined. A drop, placed on a substrate which it wets, spreads out to form a film. Conversely, a nonwetted substrate previously covered by a film dewets upon an appropriate change of system parameters. The hydrodynamics of both wetting and dewetting is influenced by the presence of the three-phase contact line separating ``wet'' regions from those that are either dry or covered by a microscopic film only. Recent theoretical, experimental, and numerical progress in the description of moving contact line dynamics are reviewed, and its relation to the thermodynamics of wetting is explored. In addition, recent progress on rough surfaces is surveyed. The anchoring of contact lines and contact angle hysteresis are explored resulting from surface inhomogeneities. Further, new ways to mold wetting characteristics according to technological constraints are discussed, for example, the use of patterned surfaces, surfactants, or complex fluids.

396 publications.