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Vincent Croquette

Publications and source records attributed to Vincent Croquette.

16 recordsLinked to original sources
PubMed2006

DNA mechanics as a tool to probe helicase and translocase activity.

Helicases and translocases are proteins that use the energy derived from ATP hydrolysis to move along or pump nucleic acid substrates. Single molecule manipulation has proved to be a powerful tool to investigate the mechanochemistry of these motors. Here we first describe the basic mechanical properties of DNA unraveled by single molecule manipulation techniques. Then we demonstrate how the knowledge of these properties has been used to design single molecule assays to address the enzymatic mechanisms of different translocases. We report on four single molecule manipulation systems addressing the mechanism of different helicases using specifically designed DNA substrates: UvrD enzyme activity detection on a stretched nicked DNA molecule, HCV NS3 helicase unwinding of a RNA hairpin under tension, the observation of RecBCD helicase/nuclease forward and backward motion, and T7 gp4 helicase mediated opening of a synthetic DNA replication fork. We then discuss experiments on two dsDNA translocases: the RuvAB motor studied on its natural substrate, the Holliday junction, and the chromosome-segregation motor FtsK, showing its unusual coupling to DNA supercoiling.

DNA↗
PubMed2006

Mechanically controlled DNA extrusion from a palindromic sequence by single molecule micromanipulation.

A magnetic tweezers setup is used to control both the stretching force and the relative linking number DeltaLk of a palindromic DNA molecule. We show here, in absence of divalent ions, that twisting negatively the molecule while stretching it at approximately 1 pN induces the formation of a cruciform DNA structure. Furthermore, once the cruciform DNA structure is formed, the extrusion of several kilo-base pairs of palindromic DNA sequence is directly and reversibly controlled by varying DeltaLk. Indeed the branch point behaves as a nanomechanical gear that links rotation with translation, a feature related to the helicity of DNA. We obtain experimentally a very good linear relationship between the extension of the molecule and DeltaLk. We use then this experiment to obtain a precise measurement of the pitch of B-DNA in solution: 3.61 +/- 0.03 nm/turn.

Biomechanical Phenomena↗
PubMed2006

Wringing out DNA.

The chiral nature of DNA plays a crucial role in cellular processes. Here we use magnetic tweezers to explore one of the signatures of this chirality, the coupling between stretch and twist deformations. We show that the extension of a stretched DNA molecule increases linearly by 0.42 nm per excess turn applied to the double helix. This result contradicts the intuition that DNA should lengthen as it is unwound and get shorter with overwinding. We then present numerical results of energy minimizations of torsionally restrained DNA that display a behavior similar to the experimental data and shed light on the molecular details of this surprising effect.

Base Pairing↗
PubMed2006

Structural plasticity of single chromatin fibers revealed by torsional manipulation.

Magnetic tweezers were used to study the mechanical response under torsion of single nucleosome arrays reconstituted on tandem repeats of 5S positioning sequences. Regular arrays are extremely resilient and can reversibly accommodate a large amount of supercoiling without much change in length. This behavior is quantitatively described by a molecular model of the chromatin three-dimensional architecture. In this model, we assume the existence of a dynamic equilibrium between three conformations of the nucleosome, corresponding to different crossing statuses of the entry/exit DNAs (positive, null or negative, respectively). Torsional strain displaces that equilibrium, leading to an extensive reorganization of the fiber's architecture. The model explains a number of long-standing topological questions regarding DNA in chromatin and may provide the basis to better understand the dynamic binding of chromatin-associated proteins.Note: In the supplementary information initially published online to accompany this article, Supplementary Figure 2 was mistakenly replaced by Supplementary Equation 2. The error has been corrected online.

Biomimetic Materials↗
PubMed2006

Direct observation of DNA distortion by the RSC complex.

The Snf2 family represents a functionally diverse class of ATPase sharing the ability to modify DNA structure. Here, we use a magnetic trap and an atomic force microscope to monitor the activity of a member of this class: the RSC complex. This enzyme caused transient shortenings in DNA length involving translocation of typically 400 bp within 2 s, resulting in the formation of a loop whose size depended on both the force applied to the DNA and the ATP concentration. The majority of loops then decrease in size within a time similar to that with which they are formed, suggesting that the motor has the ability to reverse its direction. Loop formation was also associated with the generation of negative DNA supercoils. These observations support the idea that the ATPase motors of the Snf2 family of proteins act as DNA translocases specialized to generate transient distortions in DNA structure.

Adenosine Triphosphatases↗
PubMed2005

Friction and torque govern the relaxation of DNA supercoils by eukaryotic topoisomerase IB.

Topoisomerases relieve the torsional strain in DNA that is built up during replication and transcription. They are vital for cell proliferation and are a target for poisoning by anti-cancer drugs. Type IB topoisomerase (TopIB) forms a protein clamp around the DNA duplex and creates a transient nick that permits removal of supercoils. Using real-time single-molecule observation, we show that TopIB releases supercoils by a swivel mechanism that involves friction between the rotating DNA and the enzyme cavity: that is, the DNA does not freely rotate. Unlike a nicking enzyme, TopIB does not release all the supercoils at once, but it typically does so in multiple steps. The number of supercoils removed per step follows an exponential distribution. The enzyme is found to be torque-sensitive, as the mean number of supercoils per step increases with the torque stored in the DNA. We propose a model for topoisomerization in which the torque drives the DNA rotation over a rugged periodic energy landscape in which the topoisomerase has a small but quantifiable probability to religate the DNA once per turn.

DNA Topoisomerases, Type I↗
PubMed2005

Stochastic resonance to control diffusive motion in chemistry.

This paper reports on a novel procedure to tune the effective diffusion coefficient of a field-sensitive reactant in the presence of a periodic external field. We investigate the motion of two negatively charged azo dyes interacting with alpha-cyclodextrin (alpha-CD) upon action of a periodic square wave electrical field. We show that the dyes exhibit an effective diffusion coefficient D(eff) that depends on the rate constants for dye complexation within alpha-CD, the period and the amplitude of the field. UV-vis absorption, gradient field (1)H NMR, and fluorescence correlation spectroscopy (FCS) after two photon excitation are used to evidence that D(eff) may be increased far beyond its intrinsic value when specific relations interpreted as a stochastic resonance are fulfilled. The present results may find useful applications in chemical kinetics as well as for molecular sorting.

Journal Article↗
PubMed2005

Single-molecule manipulation measurements of DNA transport proteins.

Single-molecule measurements of the manipulation of three different DNA motor proteins are reviewed. Despite some differences in the structure and mechanisms of the proteins, there are consistent phenomenological themes that relate them. Each of the experiments described represents a significant advance in the understanding of the mechanisms of DNA transport.

Bacillus Phages↗
PubMed2004

Molecular sorting by stochastic resonance.

To sort a targeted species from a mixture, we introduce a procedure that relies on the enhancement of its effective diffusion coefficient. We use the formation of a host-guest complex between alpha-cyclodextrin and a dye to evidence the dye dispersion when the medium is submitted to an oscillating field. In particular, we demonstrate that the effective diffusion coefficient of the dye may be increased far beyond its intrinsic value by tuning the driving field frequency in the stochastic resonance regime. We use this effect to selectively sort from a mixture a dye that is addressed by its rate constants for association with alpha-cyclodextrin.

Journal Article↗
PubMed2004

Single-molecule assay reveals strand switching and enhanced processivity of UvrD.

DNA helicases are enzymes capable of unwinding double-stranded DNA (dsDNA) to provide the single-stranded DNA template required in many biological processes. Among these, UvrD, an essential DNA repair enzyme, has been shown to unwind dsDNA while moving 3'-5' on one strand. Here, we use a single-molecule manipulation technique to monitor real-time changes in extension of a single, stretched, nicked dsDNA substrate as it is unwound by a single enzyme. This technique offers a means for measuring the rate, lifetime, and processivity of the enzymatic complex as a function of ATP, and for estimating the helicase step size. Strikingly, we observe a feature not seen in bulk assays: unwinding is preferentially followed by a slow, enzyme-translocation-limited rezipping of the separated strands rather than by dissociation of the enzymatic complex followed by quick rehybridization of the DNA strands. We address the mechanism underlying this phenomenon and propose a fully characterized model in which UvrD switches strands and translocates backwards on the other strand, allowing the DNA to reanneal in its wake.

Adenosine Triphosphate↗
PubMed2004

Diaroyl(methanato)boron difluoride compounds as medium-sensitive two-photon fluorescent probes.

This paper evaluates the use of diaroyl(methanato)boron difluoride compounds for designing efficient fluorescent probes through two-photon absorption. Three different pathways allowing for the syntheses of symmetrical and dissymmetrical molecules are reported. The stable diaroyl(methanato)boron difluoride derivatives can be easily obtained in good yields. They exhibit a large one-photon absorption that is easily tuned in the near-UV range. Their strong fluorescence emission covers the whole visible domain. In addition to these attractive linear properties, several diaroyl(methanato)boron difluoride derivatives possess significant cross sections for two-photon absorption. The derived structure-property relationships are promising for designing new generations of molecules relying on the diaroyl(methanato)boron difluoride backbone.

Boron Compounds↗
PubMed2004

Twisting and untwisting a single DNA molecule covered by RecA protein.

We study dsDNA-RecA interactions by exerting forces in the pN range on single DNA molecules while the interstrand topological state is controlled owing to a magnetic tweezers setup. We show that unwinding a duplex DNA molecule induces RecA polymerization even at moderate force. Once initial polymerization has nucleated, the extent of RecA coverage still depends on the degree of supercoiling: exerting a positive or negative torsional constraint on the fiber forces partial depolymerization, with a strikingly greater stability when ATPgammaS is used as a cofactor instead of ATP. This nucleofilament's sensitivity to topology might be a way for the bacterial cell to limit consumption of precious RecA monomers when DNA damage is addressed through homologous recombination repair.

Adenosine Triphosphate↗
PubMed2003

Supercoiling and denaturation in Gal repressor/heat unstable nucleoid protein (HU)-mediated DNA looping.

The overall topology of DNA profoundly influences the regulation of transcription and is determined by DNA flexibility as well as the binding of proteins that induce DNA torsion, distortion, and/or looping. Gal repressor (GalR) is thought to repress transcription from the two promoters of the gal operon of Escherichia coli by forming a DNA loop of approximately 40 nm of DNA that encompasses the promoters. Associated evidence of a topological regulatory mechanism of the transcription repression is the requirement for a supercoiled DNA template and the histone-like heat unstable nucleoid protein (HU). By using single-molecule manipulations to generate and finely tune tension in DNA molecules, we directly detected GalR/HU-mediated DNA looping and characterized its kinetics, thermodynamics, and supercoiling dependence. The factors required for gal DNA looping in single-molecule experiments (HU, GalR and DNA supercoiling) correspond exactly to those necessary for gal repression observed both in vitro and in vivo. Our single-molecule experiments revealed that negatively supercoiled DNA, under slight tension, denatured to facilitate GalR/HU-mediated DNA loop formation. Such topological intermediates may operate similarly in other multiprotein complexes of transcription, replication, and recombination.

DNA, Superhelical↗
PubMed2003

Stretching DNA and RNA to probe their interactions with proteins.

When interacting with a single stretched DNA, many proteins modify its end-to-end distance. This distance can be monitored in real time using various micromanipulation techniques that were initially used to determine the elastic properties of bare nucleic acids and their mechanically induced structural transitions. These methods are currently being applied to the study of DNA enzymes such as DNA and RNA polymerases, topoisomerases and structural proteins such as RecA. They permit the measurement of the probability distributions of the rate, processivity, on-time, affinity and efficiency for a large variety of DNA-based molecular motors.

DNA↗
PubMed2002

Magnetic tweezers: micromanipulation and force measurement at the molecular level.

Cantilevers and optical tweezers are widely used for micromanipulating cells or biomolecules for measuring their mechanical properties. However, they do not allow easy rotary motion and can sometimes damage the handled material. We present here a system of magnetic tweezers that overcomes those drawbacks while retaining most of the previous dynamometers properties. Electromagnets are coupled to a microscope-based particle tracking system through a digital feedback loop. Magnetic beads are first trapped in a potential well of stiffness approximately 10(-7) N/m. Thus, they can be manipulated in three dimensions at a speed of approximately 10 microm/s and rotated along the optical axis at a frequency of 10 Hz. In addition, our apparatus can work as a dynamometer relying on either usual calibration against the viscous drag or complete calibration using Brownian fluctuations. By stretching a DNA molecule between a magnetic particle and a glass surface, we applied and measured vertical forces ranging from 50 fN to 20 pN. Similarly, nearly horizontal forces up to 5 pN were obtained. From those experiments, we conclude that magnetic tweezers represent a low-cost and biocompatible setup that could become a suitable alternative to the other available micromanipulators.

Biophysical Phenomena↗