Stalled replication forks are sources of genetic instability. Multiple fork-remodeling enzymes recruited to stalled forks, but how they work promote fork restart is poorly understood. By combining ensemble biochemical assays and single-molecule studies with magnetic tweezers, we show that SMARCAL1 branch migration DNA-annealing activities directed by the single-stranded DNA-binding protein RPA selectively regress caused blockage leading-strand polymerase restore normal a lagging-strand gap....

In this work, we discuss the active or passive character of helicases. past years, several studies have used theoretical framework proposed by Betterton and Julicher [Betterton, M.D. Julicher, F. (2005) Opening nucleic-acid double strands helicases: versus opening. Phys. Rev. E, 71, 11904-11911.] to analyse unwinding data assess mechanism helicase under study (active passive). However, procedure has given rise apparently contradictory interpretations: helicases exhibiting similar behaviour...

The restart of a stalled replication fork is major challenge for DNA replication. Depending on the nature damage, different repair processes might be triggered; one template switching, which bypass leading-strand lesion via regression. Using magnetic tweezers to study T4 bacteriophage enzymes, we have reproduced in vitro complete process switching. We show that UvsW helicase cooperation with holoenzyme can overcome damage by pseudostochastic process, periodically forming and migrating...

Abstract Helicases that both unwind and rewind DNA have central roles in repair genetic recombination. In contrast to unwinding, rewinding by helicases has proved difficult characterize biochemically because of its thermodynamically downhill nature. Here we use single-molecule assays mechanically destabilize a molecule follow, real time, unwinding two helicases, bacteriophage T4 UvsW Escherichia coli RecG. We find enzymes are robust enzymes, which can work against opposing forces as large 35...

Replicative holoenzymes exhibit rapid and processive primer extension DNA synthesis, but inefficient strand displacement synthesis. We investigated the bacteriophage T4 T7 activity on a hairpin substrate manipulated by magnetic trap. Holoenzyme is moderately hindered applied force. In contrast, strongly stimulated force; polymerization favoured at high force, while exonuclease triggered low propose that fork upstream of holoenzyme generates regression pressure which inhibits...

Rapid and processive leading-strand DNA synthesis in the bacteriophage T4 system requires functional coupling between helicase holoenzyme, consisting of polymerase trimeric clamp loaded by loader. We investigated mechanism this on a hairpin substrate manipulated magnetic trap. In stark contrast to isolated enzymes, coupled synthesized at maximum rate without exhibiting fork regression or pauses. unwinding activities were low forces, but became uncoupled displaying separate high forces dNTP...

We present a method for determining the free energy of coexisting states from irreversible work measurements. Our approach is based on fluctuation relation that valid dissipative transformations in partially equilibrated systems. To illustrate validity and usefulness approach, we use optical tweezers to determine branches native unfolded two-state molecule as function pulling control parameter. determine, within $0.6{k}_{B}T$ accuracy, transition point where energies are equal.

Most DNA processes are governed by molecular interactions that take place in a sequence-specific manner. Determining the sequence selectivity of ligands is still challenge, particularly for small drugs where labeling or sequencing methods do not perform well. Here, we present fast and accurate method based on parallelized single molecule magnetic tweezers to detect characterize thermodynamics kinetics binding assay. Mechanical manipulation hairpins with an engineered used ligand as blocking...

We apply Kramers theory to investigate the dissociation of multiple bonds under mechanical force and interpret experimental results for unfolding refolding distributions an RNA hairpin pulled at different loading rates using laser tweezers. identify two kinetic regimes depending on range forces explored during process. The present approach extends validity two-states approximation by providing a theoretical framework reconstruct free-energy landscapes force-induced structural changes in...

We investigate the thermodynamics and kinetics of DNA hairpins that fold/unfold under action applied mechanical force. introduce concept molecular free energy landscape derive simplified expressions for force dependent Kramers-Bell rates. To test theory we have designed a specific hairpin sequence shows two-state cooperative folding tension carried out pulling experiments using optical tweezers. show how can determine parameters characterize such from rupture kinetic studies. Finally combine...

DNA bis-intercalators are widely used in molecular biology with applications ranging from imaging to anticancer pharmacology. Two fundamental aspects of these ligands the lifetime bis-intercalated complexes and their sequence selectivity. Here, we perform single-molecule optical tweezers experiments peptide Thiocoraline showing, for first time, that bis-intercalation is driven by a very slow off-rate steeply decreases applied force. This feature reveals existence long-lived (minutes)...

The Escherichia coli single stranded DNA binding protein (SSB) is crucial for replication, recombination and repair. Within each process, it has two seemingly disparate roles: stabilizes single-stranded (ssDNA) intermediates generated during processing and, forms complexes with a group of proteins known as the SSB-interactome. Key to both roles C-terminal, one-third protein, in particular intrinsically disordered linker (IDL). Previously, they have shown using series deletion mutants that...

Most RecQ DNA helicases share a conserved domain arrangement that mediates their activities in genomic stability. This comprises helicase motor domain, C-terminal (RecQ-C) region including winged-helix (WH) and 'Helicase RNase D C-terminal' (HRDC) domain. Single-molecule real-time translocation unwinding by full-length Escherichia coli variants lacking either the HRDC or both WH domains was analyzed. operated under two interconvertible kinetic modes, 'slow' 'normal', as it unwound duplex...

We investigate irreversibility and dissipation in single molecules that cooperatively fold/unfold a two-state manner under the action of mechanical force. apply path thermodynamics to derive analytical expressions for average dissipated work hopping number systems. It is shown how these quantities only depend on two parameters characterize folding/unfolding kinetics molecule: fragility coexistence rate. The latter has be rescaled take into account appropriate experimental set-up. Finally we...

Abstract 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, origami, etc. Although single descriptions exist for special cases this problem, there are no simple general prediction schemes. In work, we have measured experimentally, with fluorescent labelling, the displacement from substrate two situations: one corresponding binding/unbinding on ssDNA and which...

The folding of biological macromolecules is a fundamental process which we lack full comprehension. Mostly studied in proteins and RNA, single-stranded DNA (ssDNA) also folds, at physiological salt conditions, by forming nonspecific secondary structures that are difficult to characterize with biophysical techniques. Here, present helix-coil model for secondary-structure formation, where ssDNA bases organized two different types domains (compact free). contains parameters: the energy gain per...

Stochastic resonance (SR) is a well-known phenomenon in dynamical systems. It consists of the amplification and optimization response system assisted by stochastic (random or probabilistic) noise. Here we carry out first experimental study SR single DNA hairpins which exhibit cooperatively transitions from folded to unfolded configurations under action an oscillating mechanical force applied with optical tweezers. By varying frequency oscillation, investigate folding unfolding kinetics...