Displacement and dissociation of oligonucleotides during DNA hairpin closure under strain.

The hybridization kinetic of an oligonucleotide to its template is a fundamental step in many biological processes such as replication arrest, CRISPR recognition, DNA sequencing, DNA origami, etc. Although single kinetic descriptions exist for special cases of this problem, there are no simple general prediction schemes. In this work, we have measured experimentally, with no fluorescent labelling, the displacement of an oligonucleotide from its substrate in two situations: one corresponding to oligonucleotide binding/unbinding on ssDNA and one in which the oligonucleotide is displaced by the refolding of a dsDNA fork. In this second situation, the fork is expelling the oligonucleotide thus significantly reducing its residence time. To account for our data in these two situations, we have constructed a mathematical model, based on the known nearest neighbour dinucleotide free energies, and provided a good estimate of the residence times of different oligonucleotides (DNA, RNA, LNA) of various lengths in different experimental conditions (force, temperature, buffer conditions, presence of mismatches, etc.). This study provides a foundation for the dynamics of oligonucleotide displacement, a process of importance in numerous biological and bioengineering contexts.

Strand switching mechanism of Pif1 helicase induced by its collision with a G-quadruplex embedded in dsDNA.

G-rich sequences found at multiple sites throughout all genomes may form secondary structures called G-quadruplexes (G4), which act as roadblocks for molecular motors. Among the enzymes thought to process these structures, the Pif1 DNA helicase is considered as an archetypical G4-resolvase and its absence has been linked to G4-related genomic instabilities in yeast. Here we developed a single-molecule assay to observe Pif1 opening a DNA duplex and resolving the G4 in real time. In support of former enzymological studies, we show that the helicase reduces the lifetime of G4 from hours to seconds. However, we observe that in the presence of a G4, Pif1 exhibits a strong strand switching behavior, which can lead to Pif1 escaping G4 resolution, depending on the structural context surrounding the substrate. This behavior is also detected in the presence of other roadblocks (LNA or RNA). We propose that the efficiency of Pif1 to remove a roadblock (G4 or other) is affected by its strand switching behavior and depends on the context surrounding the obstacle. We discuss how this switching behavior may explain several aspects of Pif1 substrate preference and affect its activity as a G4 resolvase in vivo.

Folding and persistence times of intramolecular G-quadruplexes transiently embedded in a DNA duplex.

G-quadruplex (G4) DNA structures have emerged as important regulatory elements during DNA metabolic transactions. While many in vitro studies have focused on the kinetics of G4 formation within DNA single-strands, G4 are found in vivo in double-stranded DNA regions, where their formation is challenged by the complementary strand. Since the energy of hybridization of Watson-Crick structures dominates the energy of G4 folding, this competition should play a critical role on G4 persistence. To address this, we designed a single-molecule assay allowing to measure G4 folding and persistence times in the presence of the complementary strand. We quantified both folding and unfolding rates of biologically relevant G4 sequences, such as the cMYC and cKIT oncogene promoters, human telomeres and an avian replication origin. We confirmed that G4s are found much more stable in tested replication origin and promoters than in human telomere repeats. In addition, we characterized how G4 dynamics was affected by G4 ligands and showed that both folding rate and persistence time increased. Our assay opens new perspectives for the measurement of G4 dynamics in double-stranded DNA mimicking a replication fork, which is important to understand their role in DNA replication and gene regulation at a mechanistic level.

RecA-promoted, RecFOR-independent progressive disassembly of replisomes stalled by helicase inactivation.

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.

Parallelized DNA tethered bead measurements to scrutinize DNA mechanical structure.

Tethering beads to DNA offers a panel of single molecule techniques for the refined analysis of the conformational dynamics of DNA and the elucidation of the mechanisms of enzyme activity. Recent developments include the massive parallelization of these techniques achieved by the fabrication of dedicated nanoarrays by soft nanolithography. We focus here on two of these techniques: the Tethered Particle motion and Magnetic Tweezers allowing analysis of the behavior of individual DNA molecules in the absence of force and under the application of a force and/or a torque, respectively. We introduce the experimental protocols for the parallelization and discuss the benefits already gained, and to come, for these single molecule investigations.

Single molecule studies of helicases with magnetic tweezers.

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.

Single-molecule mechanical identification and sequencing.

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.

Separating speed and ability to displace roadblocks during DNA translocation by FtsK.

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.

Mechanism of strand displacement synthesis by DNA replicative polymerases.

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.

Magnetic tweezers principles and promises.

Magnetic tweezers have become popular with the outbreak of single molecule micromanipulation: catching a single molecule of DNA, RNA or a single protein and applying mechanical constrains using micron-size magnetic beads and magnets turn out to be easy. Various factors have made this possible: the fact that manufacturers have been preparing these beads to catch various biological entities-the ease of use provided by magnets which apply a force or a torque at a distance thus inside a flow cell-some chance: since the forces so generated are in the right range to stretch a single molecule. This is a little less true for torque. Finally, one feature which also appears very important is the simplicity of their calibration using Brownian motion. Here we start by describing magnetic tweezers used routinely in our laboratory where we have tried to develop a device as simple as possible so that the experimentalist can really focus on the biological aspect of the biomolecules that he/she is interested in. We discuss the implications of the various components and their important features. Next, we summarize what is easy to achieve and what is less easy. Then we refer to contributions by other groups who have brought valuable insights to improve magnetic tweezers.

Observing G4 formation and its resolution by Pif1 in real time by manipulation under magnetic tweezers.

G-quadruplexes (G4s) are nucleic acids secondary structures that may form in guanine-rich sequences, either intra or inter-molecularly. Ability of a primary sequence to form a G4 can be predicted computationally with an improving accuracy as well as tested in bulk using biophysical measurements. As a result, G4 density maps have been devised for a large number of genomes from all life kingdoms. Experimental validation of the formation of G4s in vivo however remains indirect and relies on their stabilization with small molecules, antibodies or proteins, or mutational studies, in order to measure downstream effects on gene expression or genome stability for example. Although numerous techniques exist to observe spontaneous formation of G4s in single-stranded DNA, observing G4 formation in double-stranded DNA (dsDNA) is more challenging. However, it is particularly relevant to understand if a given G4 sequence forms stably in a dsDNA context, if it is stable enough to dock proteins or pose a challenge to molecular motors such as helicases or polymerases. In essence, G4s can be a threat to genomic stability but carry as well as the potential to be elements of a structural language in the non-replicating genome. To study quantitatively the formation dynamics and stability of single intramolecular G4s embedded in dsDNA, we have adapted techniques of DNA manipulation under magnetic tweezers. This technique also allows to study encounters of molecular motors with G4 at a single molecule resolution, in order to gain insight into the specificity of G4 resolution by molecular motors, and its efficiency. The procedures described here include the design of the G4 substrate, the study of G4 formation probability and lifetime in dsDNA, as well as procedures to characterize the encounter between the Pif1 helicase and a G4 until G4 resolution. The procedures that we described here can easily be extended to the study of other G4s or molecular motors.

Energy propagation through a protometabolism leading to the local emergence of singular stationary concentration profiles.

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 reaction-diffusion 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.

Novel approaches to study helicases using magnetic tweezers.

Helicases form a universal family of molecular motors that bind and translocate onto nucleic acids. They are involved in essentially every aspect of nucleic acid metabolism: from DNA replication to RNA decay, and thus ensure a large spectrum of functions in the cell, making their study essential. The development of micromanipulation techniques such as magnetic tweezers for the mechanistic study of these enzymes has provided new insights into their behavior and their regulation that were previously unrevealed by bulk assays. These experiments allowed very precise measures of their translocation speed, processivity and polarity. Here, we detail our newest technological advances in magnetic tweezers protocols for high-quality measurements and we describe the new procedures we developed to get a more profound understanding of helicase dynamics, such as their translocation in a force independent manner, their nucleic acid binding kinetics and their interaction with roadblocks.

Oriented loading of FtsK on KOPS.

In Escherichia coli, the ATP-dependent DNA translocase FtsK transports DNA across the site of cell division and activates recombination by the XerCD recombinases at a specific site on the chromosome, dif, to ensure the last stages of chromosome segregation. DNA transport by FtsK is oriented by 8-base-pair asymmetric sequences ('KOPS'). Here we provide evidence that KOPS promote FtsK loading on DNA and that translocation is oriented at this step.

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.

Asymmetric DNA requirements in Xer recombination activation by FtsK.

In bacteria with circular chromosomes, homologous recombination events can lead to the formation of chromosome dimers. In Escherichia coli, chromosome dimers are resolved by the addition of a crossover by two tyrosine recombinases, XerC and XerD, at a specific site on the chromosome, dif. Recombination depends on a direct contact between XerD and a cell division protein, FtsK, which functions as a hexameric double stranded DNA translocase. Here, we have investigated how the structure and composition of DNA interferes with Xer recombination activation by FtsK. XerC and XerD each cleave a specific strand on dif, the top and bottom strand, respectively. We found that the integrity and nature of eight bottom-strand nucleotides and three top-strand nucleotides immediately adjacent to the XerD-binding site of dif are crucial for recombination. These nucleotides are probably not implicated in FtsK translocation since FtsK could translocate on single stranded DNA in both the 5'-3' and 3'-5' orientation along a few nucleotides. We propose that they are required to stabilize FtsK in the vicinity of dif for recombination to occur because the FtsK-XerD interaction is too transient or too weak in itself to allow for XerD catalysis.

Single-molecule kinetic locking allows fluorescence-free quantification of protein/nucleic-acid binding.

Fluorescence-free micro-manipulation of nucleic acids (NA) allows the functional characterization of DNA/RNA processing proteins, without the interference of labels, but currently fails to detect and quantify their binding. To overcome this limitation, we developed a method based on single-molecule force spectroscopy, called kinetic locking, that allows a direct in vitro visualization of protein binding while avoiding any kind of chemical disturbance of the protein's natural function. We validate kinetic locking by measuring accurately the hybridization energy of ultrashort nucleotides (5, 6, 7 bases) and use it to measure the dynamical interactions of Escherichia coli/E. coli RecQ helicase with its DNA substrate.

Parallel, linear, and subnanometric 3D tracking of microparticles with Stereo Darkfield Interferometry.

While crucial for force spectroscopists and microbiologists, three-dimensional (3D) particle tracking suffers from either poor precision, complex calibration, or the need of expensive hardware, preventing its massive adoption. We introduce a new technique, based on a simple piece of cardboard inserted in the objective focal plane, that enables simple 3D tracking of dilute microparticles while offering subnanometer frame-to-frame precision in all directions. Its linearity alleviates calibration procedures, while the interferometric pattern enhances precision. We illustrate its utility in single-molecule force spectroscopy and single-algae motility analysis. As with any technique based on back focal plane engineering, it may be directly embedded in a commercial objective, providing a means to convert any preexisting optical setup in a 3D tracking system. Thanks to its precision, its simplicity, and its versatility, we envision that the technique has the potential to enhance the spreading of high-precision and high-throughput 3D tracking.

Detection of genetic variation and base modifications at base-pair resolution on both DNA and RNA.

Accurate decoding of nucleic acid variation is critical to understand the complexity and regulation of genome function. Here we use a single-molecule magnetic tweezer (MT) platform to identify sequence variation and map a range of important epigenetic base modifications with high sensitivity, specificity, and precision in the same single molecules of DNA or RNA. We have also developed a highly specific amplification-free CRISPR-Cas enrichment strategy to isolate genomic regions from native DNA. We demonstrate enrichment of DNA from both E. coli and the FMR1 5'UTR coming from cells derived from a Fragile X carrier. From these kilobase-length enriched molecules we could characterize the differential levels of adenine and cytosine base modifications on E. coli, and the repeat expansion length and methylation status of FMR1. Together these results demonstrate that our platform can detect a variety of genetic, epigenetic, and base modification changes concomitantly within the same single molecules.

Analysis of DNA supercoil induction by FtsK indicates translocation without groove-tracking.

FtsK is a bacterial protein that translocates DNA in order to transport chromosomes within the cell. During translocation, DNA's double-helical structure might cause a relative rotation between FtsK and the DNA. We used a single-molecule technique to quantify this rotation by observing the supercoils induced into the DNA during translocation of an FtsK complex. We find that FtsK induces approximately 0.07 supercoils per DNA helical pitch traveled. This rate indicates that FtsK does not track along DNA's groove, but it is consistent with our previous estimate of FtsK's step size. We show that this rate of supercoil induction is markedly near to the ideal value that would minimize in vivo disturbance to the chromosomal supercoil density, suggesting an origin for the unusual rotational behavior of FtsK.