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.
Are the SSB-Interacting Proteins RecO, RecG, PriA and the DnaB-Interacting Protein Rep Bound to Progressing Replication Forks in Escherichia coli? - Bentchikou, Esma and Chagneau, Carine and Long, Emilie and Matelot, Melody and Allemand, Jean-Francois and Michel, Benedicte
PLOS ONE 10 (2015) 

Abstract : In all organisms several enzymes that are needed upon replication impediment are targeted to replication forks by interaction with a replication protein. In most cases these proteins interact with the polymerase clamp or with single-stranded DNA binding proteins (SSB). In Escherichia coli an accessory replicative helicase was also shown to interact with the DnaB replicative helicase. Here we have used cytological observation of Venus fluorescent fusion proteins expressed from their endogenous loci in live E. coli cells to determine whether DNA repair and replication restart proteins that interact with a replication protein travel with replication forks. A custom-made microscope that detects active replisome molecules provided that they are present in at least three copies was used. Neither the recombination proteins RecO and RecG, nor the replication accessory helicase Rep are detected specifically in replicating cells in our assay, indicating that either they are not present at progressing replication forks or they are present in less than three copies. The Venus-PriA fusion protein formed foci even in the absence of replication forks, which prevented us from reaching a conclusion.
Polymerase exchange during Okazaki fragment synthesis observed in living cells (Retraction of vol 335, pg 328, 2012) - Lia, Giuseppe and Michel, Benedicte and Allemand, Jean-Francois
SCIENCE 3461466 (2014) 
RecA-Promoted, RecFOR-Independent Progressive Disassembly of Replisomes Stalled by Helicase Inactivation (Retracted article. See vol. 54, pg. 537, 2014) - Lia, Giuseppe and Rigato, Annafrancesca and Long, Emilie and Chagneau, Carine and Le Masson, Marie and Allemand, Jean-Francois and Michel, Benedicte
MOLECULAR CELL 49547-557 (2013) 

Abstract : In all organisms, replication impairment is a recognized source of genomic instability, raising an increasing interest in the fate of inactivated replication forks. We used Escherichia coli strains with a temperature-inactivated replicative helicase (DnaB) and in vivo single-molecule microscopy to quantify the detailed molecular processing of stalled replication forks. After helicase inactivation, RecA binds to blocked replication forks and is essential for the rapid release of hPol III. The entire holoenzyme is disrupted little by little, with some components lost in few minutes, while others are stable in 70\% of cells for at least 1 hr. Although replisome dissociation is delayed in a recA mutant, it is not affected by RecF or RecO inactivation. RecFOR are required for full RecA filaments formation, and we propose that polymerase clearance can be catalyzed by short, RecFOR-independent RecA filaments. Our results identify a function for the universally conserved, central recombination protein RecA.
Polymerase Exchange During Okazaki Fragment Synthesis Observed in Living Cells (Retracted, see vol 346, pg 1466, 2014) - Lia, Giuseppe and Michel, Benedicte and Allemand, Jean-Francois
SCIENCE 335328-331 (2012) 

Abstract : DNA replication machineries have been studied extensively, but the kinetics of action of their components remains largely unknown. We report a study of DNA synthesis during replication in living Escherichia coli cells. Using single-molecule microscopy, we observed repetitive fluorescence bursts of single polymerase IIIs (Pol IIIs), indicating polymerase exchange at the replication fork. Fluctuations in the amount of DNA-bound single-stranded DNA-binding protein (SSB) reflect different speeds for the leading-and lagging-strand DNA polymerases. Coincidence analyses of Pol III and SSB fluctuations show that they correspond to the lagging-strand synthesis and suggest the use of a new Pol III for each Okazaki fragment. Based on exchanges involving two Pol IIIs, we propose that the third polymerase in the replisome is involved in lagging-strand synthesis.
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.
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.
Molecular motors for DNA translocation in prokaryotes - Allemand, Jean-Francois and Maier, Berenike and Smith, Douglas E.

Abstract : DNA transport is an essential life process. From chromosome separation during cell division or sporulation, to DNA virus ejection or encapsidation, to horizontal gene transfer, it is ubiquitous in all living organisms. Directed DNA translocation is often energetically unfavorable and requires an active process that uses energy, namely the action of molecular motors. In this review we present recent advances in the understanding of three molecular motors involved in DNA transport in prokaryotes, paying special attention to recent studies using single-molecule techniques. We first discuss DNA transport during cell division, then packaging of DNA in phage capsids, and then DNA import during bacterial transformation.
Energy Propagation Through a Protometabolism Leading to the Local Emergence of Singular Stationary Concentration Profiles - Emond, Matthieu and Le Saux, Thomas and Allemand, Jean-Francois and Pelupessy, Philippe and Plasson, Raphael and Jullien, Ludovic
CHEMISTRY-A EUROPEAN JOURNAL 1814375-14383 (2012) 

Abstract : Living systems rely on chains of energy transfer from an energy source to maintain their metabolism. This task requires functionally identified components and organizations. However, propagation of a sustained energy flux through a cascade of reaction cycles has never been reproduced at a steady state in a simple chemical system. By using energy patterning and a diffusing hub reactant, we achieved the transfer of energy through an abiotic protometabolism. Patterned illumination was applied to a liquid solution of a reversible photoacid. It resulted in the local onset of a proton pump, which subsequently drove an extended reactiondiffusion cycle that involved pH-sensitive reactants. Thus, light has been used for locally setting out of chemical equilibrium a reaction involving blind reactants. The spontaneous onset of an energy-transfer chain notably drives the local generation of singular dissipative chemical structures; continuous matter fluxes are dynamically maintained at boundaries between spatially and chemically segregated zones, in the absence of any membrane or predetermined material structure.
Soft magnetic tweezers: A proof of principle - Mosconi, Francesco and Allemand, Jean Francois and Croquette, Vincent

Abstract : We present here the principle of soft magnetic tweezers which improve the traditional magnetic tweezers allowing the simultaneous application and measurement of an arbitrary torque to a deoxyribonucleic acid (DNA) molecule. They take advantage of a nonlinear coupling regime that appears when a fast rotating magnetic field is applied to a superparamagnetic bead immersed in a viscous fluid. In this work, we present the development of the technique and we compare it with other techniques capable of measuring the torque applied to the DNA molecule. In this proof of principle, we use standard electromagnets to achieve our experiments. Despite technical difficulties related to the present implementation of these electromagnets, the agreement of measurements with previous experiments is remarkable. Finally, we propose a simple way to modify the experimental design of electromagnets that should bring the performances of the device to a competitive level. (C) 2011 American Institute of Physics. [doi:10.1063/1.3531959]
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.
Separating speed and ability to displace roadblocks during DNA translocation by FtsK - Crozat, Estelle and Meglio, Adrien and Allemand, Jean-Francois and Chivers, Claire E. and Howarth, Mark and Venien-Bryan, Catherine and Grainge, Ian and Sherratt, David J.
EMBO JOURNAL 291423-1433 (2010) 

Abstract : FtsK translocates dsDNA directionally at >5 kb/s, even under strong forces. In vivo, the action of FtsK at the bacterial division septum is required to complete the final stages of chromosome unlinking and segregation. Despite the availability of translocase structures, the mechanism by which ATP hydrolysis is coupled to DNA translocation is not understood. Here, we use covalently linked translocase subunits to gain insight into the DNA translocation mechanism. Covalent trimers of wild-type subunits dimerized efficiently to form hexamers with high translocation activity and an ability to activate XerCD-dif chromosome unlinking. Covalent trimers with a catalytic mutation in the central subunit formed hexamers with two mutated subunits that had robust ATPase activity. They showed wild-type translocation velocity in single-molecule experiments, activated translocation-dependent chromosome unlinking, but had an impaired ability to displace either a triplex oligonucleotide, or streptavidin linked to biotin-DNA, during translocation along DNA. This separation of translocation velocity and ability to displace roadblocks is more consistent with a sequential escort mechanism than stochastic, hand-off, or concerted mechanisms. The EMBO Journal (2010) 29, 1423-1433. doi: 10.1038/emboj.2010.29; Published online 8 April 2010
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.
Bacterial translocation motors investigated by single molecule techniques - Allemand, Jean-Francois and Maier, Berenike

Abstract : Translocation of DNA and protein fibers through narrow constrictions is a ubiquitous and crucial activity of bacterial cells. Bacteria use specialized machines to support macromolecular movement. A very important step toward a mechanistic understanding of these translocation machines is the characterization of their physical properties at the single molecule level. Recently, four bacterial transport processes have been characterized by nanomanipulation at the single molecule level, DNA translocation by FtsK and SpoIIIE, DNA import during transformation, and the related process of a type IV pilus retraction. With all four processes, the translocation rates, processivity, and stalling forces were remarkably high as compared with single molecule experiments with other molecular motors. Although substrates of all four processes proceed along a preferential direction of translocation, directionality has been shown to be controlled by distinct mechanisms.
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
Some nonlinear challenges in biology - Mosconi, Francesco and Julou, Thomas and Desprat, Nicolas and Sinha, Deepak Kumar and Allemand, Jean-Francois and Croquette, Vincent and Bensimon, David
NONLINEARITY 21T131-T147 (2008) 

Abstract : Driven by a deluge of data, biology is undergoing a transition to a more quantitative science. Making sense of the data, building new models, asking the right questions and designing smart experiments to answer them are becoming ever more relevant. In this endeavour, nonlinear approaches can play a fundamental role. The biochemical reactions that underlie life are very often nonlinear. The functional features exhibited by biological systems at all levels (from the activity of an enzyme to the organization of a colony of ants, via the development of an organism or a functional module like the one responsible for chemotaxis in bacteria) are dynamically robust. They are often unaffected by order of magnitude variations in the dynamical parameters, in the number or concentrations of actors (molecules, cells, organisms) or external inputs (food, temperature, pH, etc). This type of structural robustness is also a common feature of nonlinear systems, exemplified by the fundamental role played by dynamical fixed points and attractors and by the use of generic equations (logistic map, Fisher-Kolmogorov equation, the Stefan problem, etc.) in the study of a plethora of nonlinear phenomena. However, biological systems differ from these examples in two important ways: the intrinsic stochasticity arising from the often very small number of actors and the role played by evolution. On an evolutionary time scale, nothing in biology is frozen. The systems observed today have evolved from solutions adopted in the past and they will have to adapt in response to future conditions. The evolvability of biological system uniquely characterizes them and is central to biology. As the great biologist T Dobzhansky once wrote: `nothing in biology makes sense except in the light of evolution'.
Single-molecule micromanipulation techniques - Neuman, K. C. and Lionnet, T. and Allemand, J.-F

Abstract : Single-molecule micrornanipulation techniques traditionally have been developed for biophysical applications, but they are being increasingly employed in materials science applications such as theology and polymer dynamics. Continuing developments and improvements in single-molecule manipulation techniques afford new opportunities in a broad range of fields. In this review we present an overview of current single-molecule manipulation techniques, with an emphasis on optical and magnetic tweezers, followed by a description of the elastic properties of single biopolymers. We then review die use of micrornanipulation techniques to locally probe material properties. To provide some insight into biophysical questions addressed by these techniques, we describe two applications, which further serve to illustrate the power and versatility of single-molecule micromanipulation techniques. We conclude with a brief discussion of emerging applications and techniques.