laboratoire de physique statistique
laboratoire de physique statistique


Revisiting Sequencing by Hybridization at the Single Molecule Level using the Unzipping Assay - Croquette, Vincent and Raj, Saurabh and Allemand, Jean-Francois and Bensimon, David and Boule, Jean-Baptiste
Single molecule studies of helicases with magnetic tweezers - Hodeib, Samar and Raj, Saurabh and Manosas, M. and Zhang, Weiting and Bagchi, Debjani and Ducos, Bertrand and Allemand, Jean-Francois and Bensimon, David and Croquette, Vincent
METHODS 1053-15 (2016) 

Abstract : Helicases are a broad family of enzymes that perform crucial functions in DNA replication and in the maintenance of DNA and RNA integrity. A detailed mechanical study of helicases on DNA and RNA is possible using single molecule manipulation methods. Among those, magnetic tweezers (or traps) present a convenient, moderate throughput assay (tens of enzymes can be monitored simultaneously) that allow for high resolution (single base-pair) studies of these enzymes in various conditions and on various substrates (double and single stranded DNA and RNA). Here we discuss various implementation of the basic assay relevant for these studies. (C) 2016 Elsevier Inc. All rights reserved.
Cell-cell contacts confine public goods diffusion inside Pseudomonas aeruginosa clonal microcolonies - Julou, Thomas and Mora, Thierry and Guillon, Laurent and Croquette, Vincent and Schalk, Isabelle J. and Bensimon, David and Desprat, Nicolas

Abstract : The maintenance of cooperation in populations where public goods are equally accessible to all but inflict a fitness cost on individual producers is a long-standing puzzle of evolutionary biology. An example of such a scenario is the secretion of siderophores by bacteria into their environment to fetch soluble iron. In a planktonic culture, these molecules diffuse rapidly, such that the same concentration is experienced by all bacteria. However, on solid substrates, bacteria form dense and packed colonies that may alter the diffusion dynamics through cell-cell contact interactions. In Pseudomonas aeruginosa microcolonies growing on solid substrate, we found that the concentration of pyoverdine, a secreted iron chelator, is heterogeneous, with a maximum at the center of the colony. We quantitatively explain the formation of this gradient by local exchange between contacting cells rather than by global diffusion of pyoverdine. In addition, we show that this local trafficking modulates the growth rate of individual cells. Taken together, these data provide a physical basis that explains the stability of public goods production in packed colonies.
Detection and Quantification through a Lipid Membrane Using the Molecularly Controlled Semiconductor Resistor - Bavli, Danny and Tkachev, Maria and Piwonski, Hubert and Capua, Eyal and de Albuquerque, Ian and Bensimon, David and Haran, Gilad and Naaman, Ron
LANGMUIR 281020-1028 (2012) 

Abstract : The detection of covalent and noncovalent binding events between molecules and biomembranes is a fundamental goal of contemporary biochemistry and analytical chemistry. Currently, such studies are performed routinely using fluorescence methods, surface-plasmon resonance spectroscopy, and electrochemical methods. However, there is still a need for novel sensitive miniaturizable detection methods where the sample does not have to be transferred to the sensor, but the sensor can be brought into contact with the sample studied. We present a novel approach for detection and quantification of processes occurring on the surface of a lipid bilayer membrane, by monitoring the current change through the n-type GaAs-based molecularly controlled semiconductor resistor (MOCSER), on which the membrane is adsorbed. Since GaAs is susceptible to etching in an aqueous environment, a protective thin film of methoxysilane was deposited on the device. The system was found to be sensitive enough to allow monitoring changes in pH and in the concentration of amino acids in aqueous solution on top of the membrane. When biotinylated lipids were incorporated into the membrane, it was possible to monitor the binding of streptavidin or avidin. The device modified with biotin-streptavidin complex was capable of detecting the binding of streptavidin antibodies to immobilized streptavidin with high sensitivity and selectivity. The response depends on the charge on the analyte. These results open the way to facile electrical detection of protein membrane interactions.
ATP-Independent Cooperative Binding of Yeast Isw1a to Bare and Nucleosomal DNA - De Cian, Anne and Praly, Elise and Ding, Fangyuan and Singh, Vijender and Lavelle, Christophe and Le Cam, Eric and Croquette, Vincent and Pietrement, Olivier and Bensimon, David
PLOS ONE 7 (2012) 

Abstract : Among chromatin remodeling factors, the ISWI family displays a nucleosome-enhanced ATPase activity coupled to DNA translocation. While these enzymes are known to bind to DNA, their activity has not been fully characterized. Here we use TEM imaging and single molecule manipulation to investigate the interaction between DNA and yeast Isw1a. We show that Isw1a displays a highly cooperative ATP-independent binding to and bridging between DNA segments. Under appropriate tension, rare single nucleation events can sometimes be observed and loop DNA with a regular step. These nucleation events are often followed by binding of successive complexes bridging between nearby DNA segments in a zipper-like fashion, as confirmed by TEM observations. On nucleosomal substrates, we show that the specific ATP-dependent remodeling activity occurs in the context of cooperative Isw1a complexes bridging extranucleosomal DNA. Our results are interpreted in the context of the recently published partial structure of Isw1a and support its acting as a ``protein ruler'' (with possibly more than one tick).
Single-molecule mechanical identification and sequencing - Ding, Fangyuan and Manosas, Maria and Spiering, Michelle M. and Benkovic, Stephen J. and Bensimon, David and Allemand, Jean-Francois and Croquette, Vincent
NATURE METHODS 9367-U74 (2012) 

Abstract : High-throughput, low-cost DNA sequencing has emerged as one of the challenges of the postgenomic era. Here we present the proof of concept for a single-molecule platform that allows DNA identification and sequencing. In contrast to most present methods, our scheme is not based on the detection of the fluorescent nucleotides but on DNA hairpin length. By pulling on magnetic beads tethered by a DNA hairpin to the surface, the molecule can be unzipped. In this open state it can hybridize with complementary oligonucleotides, which transiently block the hairpin rezipping when the pulling force is reduced. By measuring from the surface to the bead of a blocked hairpin, one can determine the position of the hybrid along the molecule with nearly single-base precision. Our approach can be used to identify a DNA fragment of known sequence in a mix of various fragments and to sequence an unknown DNA fragment by hybridization or ligation.
Allosteric inhibition of individual enzyme molecules trapped in lipid vesicles - Piwonski, Hubert M. and Goomanovsky, Mila and Bensimon, David and Horovitz, Amnon and Haran, Gilad

Abstract : Enzymatic inhibition by product molecules is an important and widespread phenomenon. We describe an approach to study product inhibition at the single-molecule level. Individual HRP molecules are trapped within surface-tethered lipid vesicles, and their reaction with a fluorogenic substrate is probed. While the substrate readily penetrates into the vesicles, the charged product (resorufin) gets trapped and accumulates inside the vesicles. Surprisingly, individual enzyme molecules are found to stall when a few tens of product molecules accumulate. Bulk enzymology experiments verify that the enzyme is noncompetitively inhibited by resorufin. The initial reaction velocity of individual enzyme molecules and the number of product molecules required for their complete inhibition are broadly distributed and dynamically disordered. The two seemingly unrelated parameters, however, are found to be substantially correlated with each other in each enzyme molecule and over long times. These results suggest that, as a way to counter disorder, enzymes have evolved the means to correlate fluctuations at structurally distinct functional sites.
Monitoring microbial population dynamics at low densities - Julou, Thomas and Desprat, Nicolas and Bensimon, David and Croquette, Vincent

Abstract : We propose a new and simple method for the measurement of microbial concentrations in highly diluted cultures. This method is based on an analysis of the intensity fluctuations of light scattered by microbial cells under laser illumination. Two possible measurement strategies are identified and compared using simulations and measurements of the concentration of gold nanoparticles. Based on this comparison, we show that the concentration of Escherichia coli and Saccharomyces cerevisiae cultures can be easily measured in situ across a concentration range that spans five orders of magnitude. The lowest measurable concentration is three orders of magnitude (1000x) smaller than in current optical density measurements. We show further that this method can also be used to measure the concentration of fluorescent microbial cells. In practice, this new method is well suited to monitor the dynamics of population growth at early colonization of a liquid culture medium. The dynamic data thus obtained are particularly relevant for microbial ecology studies. (C) 2012 American Institute of Physics. []
Mechanism of strand displacement synthesis by DNA replicative polymerases - Manosas, Maria and Spiering, Michelle M. and Ding, Fangyuan and Bensimon, David and Allemand, Jean-Francois and Benkovic, Stephen J. and Croquette, Vincent
NUCLEIC ACIDS RESEARCH 406174-6186 (2012) 

Abstract : 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.
Monogalactopyranosides of fluorescein and fluorescein methyl ester: synthesis, enzymatic hydrolysis by biotnylated beta-galactosidase, and determination of translational diffusion coefficient - Mandal, Prasun K. and Cattiaux, Laurent and Bensimon, David and Mallet, Jean-Maurice

Abstract : Fluorescein monoglycosides (D-galactopyranoside (FMG) and D-glucopyranoside) and their methyl ester (MFMG) have been prepared from acetobromoglucose/galactose and fluorescein methyl ester in good yields. Enzymatic hydrolysis experiments (using biotinylated beta-galactosidase) of the galacto derivatives have been performed and kinetic parameters were calculated. A 15-20 times increase of the fluorescence intensity has been observed during the hydrolysis. A linear increase of fluorescence has been noted at short time and low concentration of substrate, making these compounds useful and sensitive probes for galactosidases. The magnitude of the Michaelis-Menten constant (K-m) value for MFMG is higher than that of FMG suggesting a possible conformational change of the fluorogenic substrate. K-m value for biotinylated beta-Gal with FMG is lower than that for the native enzyme. This observation indicates higher substrate affinity of the biotinylated enzyme in comparison to the native enzyme. Translational diffusion coefficients have been measured, for both fluorogenic substrates and both the products, employing fluorescence correlation spectroscopy. Translational diffusion coefficients for fluorogenic substrates and the enzymatic hydrolysis products have been measured to be similar, in the range of 3.5-4.5 x 10(-10) m(2) s(-1). Thus an enhancement or retardation of the enzymatic kinetics due to difference in translational mobility of substrate and product is not that apparent. (C) 2012 Elsevier Ltd. All rights reserved.
Single molecule actuation and detection on a lab-on-a-chip magnetoresistive platform - Chaves, R. C. and Bensimon, D. and Freitas, P. P.

Abstract : On-chip magnetic tweezers based on current loops were integrated with magnetoresistive sensors. Magnetic forces up to 1.0 +/- 0.3 pN are produced to actuate on DNA anchored to the surface of a flow cell and labeled with micrometer-sized magnetic beads. The levitation of the beads stretches the immobilized DNA. The relative position of the magnetic beads is monitored using spin-valve sensors. A bead vertical displacement resolution of 60 nm is derived for DNA molecular motor activity in a tweezer steady current regime. (C) 2011 American Institute of Physics. [doi:10.1063/1.3560853]
Nucleosome-remodelling machines and other molecular motors observed at the single-molecule level - Lavelle, Christophe and Praly, Elise and Bensimon, David and Le Cam, Eric and Croquette, Vincent
FEBS JOURNAL 2783596-3607 (2011) 

Abstract : Through its capability to transiently pack and unpack our genome, chromatin is a key player in the regulation of gene expression. Single-molecule approaches have recently complemented conventional biochemical and biophysical techniques to decipher the complex mechanisms ruling chromatin dynamics. Micromanipulations with tweezers (magnetic or optical) and imaging with molecular microscopy (electron or atomic force) have indeed provided opportunities to handle and visualize single molecules, and to measure the forces and torques produced by molecular motors, along with their effects on DNA or nucleosomal templates. By giving access to dynamic events that tend to be blurred in traditional biochemical bulk experiments, these techniques provide critical information regarding the mechanisms underlying the regulation of gene activation and deactivation by nucleosome and chromatin structural changes. This minireview describes some single-molecule approaches to the study of ATP-consuming molecular motors acting on DNA, with applications to the case of nucleosome-remodelling machines.
MAGNETIC TWEEZERS FOR THE STUDY OF DNA TRACKING MOTORS - Manosas, Maria and Meglio, Adrien and Spiering, Michelle M. and Ding, Fangyuan and Benkovic, Stephen J. and Barre, Francois-Xavier and Saleh, Omar A. and Allemand, Jean Francois and Bensimon, David and Croquette, Vincent

Abstract : Single-molecule manipulation methods have opened a new vista on the study of molecular motors. Here we describe the use of magnetic traps for the investigation of the mechanism of DNA based motors, in particular helicases and translocases.
Photocontrol of protein activity in a single cell of a live organism - Sinha, Deepak K. and Neveu, Pierre and Gagey, Nathalie and Aujard, Isabelle and Benbrahim-Bouzidi, Chouaha and Le Saux, Thomas and Rampon, Christine and Gauron, Carole and Goetz, Bernard and Dubruille, Sylvie and Baaden, Marc and Leucht, Christof and Bally-Cuif, Laure and Volovitch, Michel and Bensimon, David and Vriz, Sophie and Jullien, Ludovic
Active and passive mechanisms of helicases - Manosas, Maria and Xi, Xu Guang and Bensimon, David and Croquette, Vincent
NUCLEIC ACIDS RESEARCH 385518-5526 (2010) 

Abstract : 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.
Measurement of the Torque on a Single Stretched and Twisted DNA Using Magnetic Tweezers - Mosconi, Francesco and Allemand, Jean Francois and Bensimon, David and Croquette, Vincent

Abstract : 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.
Single-molecule Visualization of Binding Modes of Helicase to DNA on PEGylated Surfaces - Yokota, Hiroaki and Han, Yong-Woon and Allemand, Jean-Francois and Xi, Xu Guang and Bensimon, David and Croquette, Vincent and Ito, Yoshihiro and Harada, Yoshie
CHEMISTRY LETTERS 38308-309 (2009) 

Abstract : Binding modes of helicase to various DNA substrates were visualized by single-molecule fluorescence imaging on silicate coverslips coated with poly(ethylene glycol) (PEG) which minimizes the background noise from nonspecific protein adsorption. The results clearly show that the helicase has higher affinity for double-stranded DNA (dsDNA) with a single-stranded DNA (ssDNA) tail than that without one and that multiple helicases can bind to 4.7-kilonucleotide (knt) ssDNA. The study reported here will enhance the capabilities of single-molecule fluorescence studies on DNA-protein interactions.
Mechanisms of chiral discrimination by topoisomerase IV - Neuman, K. C. and Charvin, G. and Bensimon, D. and Croquette, V.

Abstract : 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.
Single DNA/protein studies with magnetic traps - Meglio, Adrien and Praly, Elise and Ding, Fangyuan and Allemand, Jean-Francois and Bensimon, David and Croquette, Vincent

Abstract : Magnetic traps provide a simple technique to pull and twist a variety of biomolecules and monitor the resulting change in extension. They have been used with great success to investigate the interaction of stretched and supercoiled DNA and DNA fibers (e.g. chromatin) with a great variety of enzymes. In this small review we will address their recent use in the study of topoisomerases, gyrase, DNA translocases and various structural proteins.
A caged retinoic acid for one- and two-photon excitation in zebrafish embryos - Neveu, Pierre and Aujard, Isabelle and Benbrahim, Chouaha and Le Saux, Thomas and Allemand, Jean-Francois and Vriz, Sophie and Bensimon, David and Jullien, Ludovic