Biology subjects

Croquette, V.

Publications and source records attributed to Croquette, V..

7 recordsLinked to original sources

bioRxiv2022

Displacement and dissociation of oligonucleotidesduring DNA hairpin closure under strain

The simple 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 approaches exist that address 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 where the oligonucleotide is displaced by the refolding of a dsDNA fork. In this second situation, the fork is expelling the oligonucleotide thus reducing significantly its residence time. To account for our data in these two situations, we have constructed a mathematical model, based on the known nearest neighbor dinucleotide free energies, and provided a good estimate of the residence times of different oligonucleotides (DNA, RNA, LNA) with various length 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 a number of biological and bioengineering contexts.

biophysics↗

bioRxiv2022

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 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 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.

biophysics↗

bioRxiv2021

Nucleosome positioning on large tandem DNA repeats of the '601' sequence engineered in Saccharomyces cerevisiae

The so-called 601 DNA sequence is often used to constrain the position of nucleosomes on a DNA molecule in vitro. Although the ability of the 147 base pair sequence to precisely position a nucleosome in vitro is well documented, in vivo application of this property has been explored only in a few studies and yielded contradictory conclusions. Our goal in the present study was to test the ability of the 601 sequence to dictate nucleosome positioning in Saccharomyces cerevisiae in the context of a long tandem repeat array inserted in a yeast chromosome. We engineered such arrays with three different repeat size, namely 167, 197 and 237 base pairs. Although our arrays are able to position nucleosomes in vitro as expected, analysis of nucleosome occupancy on these arrays in vivo revealed that nucleosomes are not preferentially positioned as expected on the 601-core sequence along the repeats and that the measured nucleosome repeat length does not correspond to the one expected by design. Altogether our results demonstrate that the rules defining nucleosome positions on this DNA sequence in vitro are not valid in vivo, at least in this chromosomal context, questioning the relevance of using the 601 sequence in vivo to achieve precise nucleosome positioning on designer synthetic DNA sequences.

synthetic biology↗

bioRxiv2021

Folding and Persistence Time of Intramolecular G-Quadruplexes Transiently Embedded in a DNA duplex

G-quadruplex (G4) DNA structures have emerged as important regulatory elements during DNA replication, transcription or repair. 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 pairing between the two complementary strands. Since the energy of hybridization of Watson-Crick structures dominates the energy of G4 folding, this competition should play a critical role on the persistence of G4 in vivo. To address this issue, we designed a single molecule assay allowing measuring G4 folding and persistence while the structure is periodically challenged by the complementary strand. We quantified both the folding rate and the persistence time of biologically relevant G4 structures and showed that the dynamics of G4 formation depends strongly on the genomic location. G4 are found much more stable in promoter regions and replication origins than in telomeric regions. In addition, we characterized how G4 dynamics was affected by G4 ligands and showed that both folding rate and persistence increased. Our assay opens new perspectives for the measurement of G4 dynamics, which is critical to understand their role in genetic regulation.

biophysics↗

bioRxiv2020

Fluorescence-free quantification of protein/nucleic-acid binding through single-molecule kinetic locking

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 new 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 proteins 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 E. coli RecQ helicase with its DNA substrate.

biophysics↗

bioRxiv2020

Detecting genetic variation and base modifications together in the same single molecules of DNA and RNA at base pair resolution using a magnetic tweezer platform.

Accurate decoding of nucleic acid variation is important to understand the complexity and regulation of genome function. Here we introduce a single-molecule platform based on magnetic tweezer (MT) technology that can identify and map the positions of sequence variation and multiple base modifications together in the same single molecules of DNA or RNA at single base resolution. Using synthetic templates, we demonstrate that our method can distinguish the most common epigenetic marks on DNA and RNA with high sensitivity, specificity and precision. We also developed a highly specific CRISPR-Cas enrichment strategy to target genomic regions in native DNA without amplification. We then used this method to enrich native DNA from E. coli and characterized the differential levels of adenine and cytosine base modifications together in molecules of up to 5 kb in length. Finally, we enriched the 5 UTR of FMR1 from cells derived from a Fragile X carrier and precisely measured the repeat expansion length and methylation status of each molecule. These results demonstrate that our platform can detect a variety of genetic, epigenetic and base modification changes concomitantly within the same single molecules.

genomics↗

bioRxiv2017

Asymmetric adhesion of rod-shaped bacteria controls microcolony morphogenesis

Bacterial biofilms are spatially structured communities, within which bacteria can differentiate depending on environmental conditions. During biofilm formation, bacteria attach to a surface and use cell-cell contacts to convey the signals required for the coordination of biofilm morphogenesis. How bacteria can maintain both substrate adhesions and cell-cell contacts during the expansion of a microcolony is still a critical yet poorly understood phenomenon. Here, we describe the development of time-resolved methods to measure substrate adhesion at the single cell level during the formation of E. coli and P. aeruginosa microcolonies. We show that bacterial adhesion is asymmetrically distributed along the cell body. Higher adhesion forces at old poles put the daughter cells under tension and force them to slide along each other. These rearrangements increase cell-cell contacts and the circularity of the colony. We propose a mechanical model based on the microscopic details of adhesive links, which recapitulates microcolony morphogenesis and quantitatively predicts bacterial adhesion from simple time lapse movies. These results explain how the distribution of adhesion forces at the subcellular level directs the shape of bacterial colonies, which ultimately dictates the circulation of secreted signals.

biophysics↗