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
Kinetics of Reactive Modules Adds Discriminative Dimensions for Selective Cell Imaging - Querard, Jerome and Le Saux, Thomas and Gautier, Arnaud and Alcor, Damien and Croquette, Vincent and Lemarchand, Annie and Gosse, Charlie and Jullien, Ludovic
CHEMPHYSCHEM 171396-1413 (2016) 

Abstract : Living cells are chemical mixtures of exceptional interest and significance, whose investigation requires the development of powerful analytical tools fulfilling the demanding constraints resulting from their singular features. In particular, multiplexed observation of a large number of molecular targets with high spatiotemporal resolution appears highly desirable. One attractive road to address this analytical challenge relies on engaging the targets in reactions and exploiting the rich kinetic signature of the resulting reactive module, which originates from its topology and its rate constants. This review explores the various facets of this promising strategy. We first emphasize the singularity of the content of a living cell as a chemical mixture and suggest that its multiplexed observation is significant and timely. Then, we show that exploiting the kinetics of analytical processes is relevant to selectively detect a given analyte: upon perturbing the system, the kinetic window associated to response read-out has to be matched with that of the targeted reactive module. Eventually, we introduce the state-of-the-art of cell imaging exploiting protocols based on reaction kinetics and draw some promising perspectives.
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.
DNA and RNA helicases singlemolecule assays - Raj, S. and Manosas, M. and Fiorini, F. and Bagchi, D. and Le Hir, H. and Banroques, J. and Taner, K. and Croquette, V.
Human Upf1 is a highly processive RNA helicase and translocase with RNP remodelling activities - Fiorini, Francesca and Bagchi, Debjani and Le Hir, Herve and Croquette, Vincent

Abstract : RNA helicases are implicated in most cellular RNA-dependent events. In eukaryotes however, only few have been functionally characterized. Upf1 is a RNA helicase essential for nonsense-mediated mRNA decay (NMD). Here, using magnetic tweezers and bulk assays, we observe that human Upf1 is able to translocate slowly over long single-stranded nucleic acids with a processivity >10 kb. Upf1 efficiently translocates through double-stranded structures and protein-bound sequences, demonstrating that Upf1 is an efficient ribonucleoprotein complex remodeler. Our observation of processive unwinding by an eukaryotic RNA helicase reveals that Upf1, once recruited onto NMD mRNA targets, can scan the entire transcript to irreversibly remodel the mRNP, facilitating its degradation by the NMD machinery.
Direct Observation of Stalled Fork Restart and Lesion Bypass via Fork Regression in the T4 Replication System - Croquette, Vincent and Manosas, Maria and Perumal, Senthil K. and Benkovic, Stephen J.
BIOPHYSICAL JOURNAL 104367A-368A (2013) 
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.
RecG and UvsW catalyse robust DNA rewinding critical for stalled DNA replication fork rescue - Manosas, Maria and Perumal, Senthil K. and Bianco, Piero and Ritort, Felix and Benkovic, Stephen J. and Croquette, Vincent

Abstract : Helicases that both unwind and rewind DNA have central roles in DNA repair and genetic recombination. In contrast to unwinding, DNA rewinding by helicases has proved difficult to characterize biochemically because of its thermodynamically downhill nature. Here we use single-molecule assays to mechanically destabilize a DNA molecule and follow, in real time, unwinding and rewinding by two DNA repair helicases, bacteriophage T4 UvsW and Escherichia coli RecG. We find that both enzymes are robust rewinding enzymes, which can work against opposing forces as large as 35 pN, revealing their active character. The generation of work during the rewinding reaction allows them to couple rewinding to DNA unwinding and/or protein displacement reactions central to the rescue of stalled DNA replication forks. The overall results support a general mechanism for monomeric rewinding enzymes.
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.
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. []
Collaborative coupling between polymerase and helicase for leading-strand synthesis - Manosas, Maria and Spiering, Michelle M. and Ding, Fangyuan and Croquette, Vincent and Benkovic, Stephen J.
NUCLEIC ACIDS RESEARCH 406187-6198 (2012) 

Abstract : Rapid and processive leading-strand DNA synthesis in the bacteriophage T4 system requires functional coupling between the helicase and the holoenzyme, consisting of the polymerase and trimeric clamp loaded by the clamp loader. We investigated the mechanism of this coupling on a DNA hairpin substrate manipulated by a magnetic trap. In stark contrast to the isolated enzymes, the coupled system synthesized DNA at the maximum rate without exhibiting fork regression or pauses. DNA synthesis and unwinding activities were coupled at low forces, but became uncoupled displaying separate activities at high forces or low dNTP concentration. We propose a collaborative model in which the helicase releases the fork regression pressure on the holoenzyme allowing it to adopt a processive polymerization conformation and the holoenzyme destabilizes the first few base pairs of the fork thereby increasing the efficiency of helicase unwinding. The model implies that both enzymes are localized at the fork, but does not require a specific interaction between them. The model quantitatively reproduces homologous and heterologous coupling results under various experimental conditions.
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.
Dda Helicase Tightly Couples Translocation on Single-Stranded DNA to Unwinding of Duplex DNA: Dda Is an Optimally Active Helicase - Byrd, Alicia K. and Matlock, Dennis L. and Bagchi, Debjani and Aarattuthodiyil, Suja and Harrison, David and Croquette, Vincent and Raney, Kevin D.

Abstract : Helicases utilize the energy of ATP hydrolysis to unwind double-stranded DNA while translocating on the DNA. Mechanisms for melting the duplex have been characterized as active or passive, depending on whether the enzyme actively separates the base pairs or simply sequesters single-stranded DNA (ssDNA) that forms due to thermal fraying. Here, we show that Dda translocates unidirectionally on ssDNA at the same rate at which it unwinds double-stranded DNA in both ensemble and single-molecule experiments. Further, the unwinding rate is largely insensitive to the duplex stability and to the applied force. Thus, Dda transduces all of its translocase activity into DNA unwinding activity so that the rate of unwinding is limited by the rate of translocation and that the enzyme actively separates the duplex. Active and passive helicases have been characterized by dividing the velocity of DNA unwinding in base pairs per second (V-un) by the velocity of translocation on ssDNA in nucleotides per second (V-trans). If the resulting fraction is 0.25, then a helicase is considered to be at the lower end of the ``active'' range. In the case of Dda, the average DNA unwinding velocity was 257 +/- 42 bp/s, and the average translocation velocity was 267 +/- 15 nt/s. The V-un/V-trans value of 0.96 places Dda in a unique category of being an essentially ``perfectly'' active helicase. (C) 2012 Elsevier Ltd. All rights reserved.
Direct Observation of Stalled Fork Restart via Fork Regression in the T4 Replication System - Manosas, Maria and Perumal, Senthil K. and Croquette, Vincent and Benkovic, Stephen J.
SCIENCE 3381217-1220 (2012) 

Abstract : The restart of a stalled replication fork is a major challenge for DNA replication. Depending on the nature of the damage, different repair processes might be triggered; one is template switching, which is a bypass of a leading-strand lesion via fork regression. Using magnetic tweezers to study the T4 bacteriophage enzymes, we have reproduced in vitro the complete process of template switching. We show that the UvsW DNA helicase in cooperation with the T4 holoenzyme can overcome leading-strand lesion damage by a pseudostochastic process, periodically forming and migrating a four-way Holliday junction. The initiation of the repair process requires partial replisome disassembly via the departure of the replicative helicase. The results support the role of fork regression pathways in DNA repair.
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]
Temperature Modulation and Quadrature Detection for Selective Titration of Two-State Exchanging Reactants - Zrelli, K. and Barilero, T. and Cavatore, E. and Berthoumieux, H. and Le Saux, T. and Croquette, V. and Lemarchand, A. and Gosse, C. and Jullien, L.
ANALYTICAL CHEMISTRY 832476-2484 (2011) 

Abstract : Biological samples exhibit huge molecular diversity over large concentration ranges. Titrating a given compound in such mixtures is often difficult, and innovative strategies emphasizing selectivity are thus demanded. To overcome limitations inherent to thermodynamics, we here present a generic technique where discrimination relies on the dynamics of interaction between the target of interest and a probe introduced in excess. Considering an ensemble of two-state exchanging reactants submitted to temperature modulation, we first demonstrate that the amplitude of the out-of-phase concentration oscillations is maximum for every compound involved in a reaction whose equilibrium constant is equal to unity and whose relaxation time is equal to the inverse of the excitation angular frequency. Taking advantage of this feature, we next devise a highly specific detection protocol and validate it using a microfabricated resistive heater and an epifluorescence microscope, as well as labeled oligonucleotides to model species displaying various dynamic properties. As expected, quantification of a sought for strand is obtained even if interfering reagents are present in similar amounts. Moreover, our approach does not require any separation and is compatible with imaging. It could then benefit some of the numerous binding assays performed every day in life sciences.
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.
Coupling DNA Unwinding Activity With Primer Synthesis in the Bacteriophage T4 Primosome - Croquette, Vincent and Manosas, Maria and Spiering, Michelle M. and Zhuang, Zhihao and Benkovic, Stephen J.