A fluorescence-based helicase assay: application to the screening of G-quadruplex ligands

International audienceHelicases, enzymes that unwind DNA or RNA structure , are present in the cell nucleus and in the mito-chondrion. Although the majority of the helicases unwind DNA or RNA duplexes, some of these proteins are known to resolve unusual structures such as G-quadruplexes (G4) in vitro. G4 may form stable barrier to the progression of molecular motors tracking on DNA. Monitoring G4 unwinding by these enzymes may reveal the mechanisms of the enzymes and provides information about the stability of these structures. In the experiments presented herein, we developed a reliable, inexpensive and rapid fluorescence-based technique to monitor the activity of G4 heli-cases in real time in a 96-well plate format. This system was used to screen a series of G4 structures and G4 binders for their effect on the Pif1 enzyme, a 5 to 3 DNA helicase. This simple assay should be adaptable to analysis of other helicases and G4 structures

G-quadruplex denaturation and stabilization : interaction with helicases and screening of G4 ligands

Les quadruplexes de guanines (G4) sont des structures polymorphiques adoptées in vitro par les séquences d’ADN et d’ARN riches en guanines. L’utilisation d’anticorps et de ligands spécifiques des structures G4 a permis leur détection au niveau cellulaire. Des études computationnelles ont prédit des séquences possédant une signature G4 au niveau de régions génomiques capitales comme les télomères ou les promoteurs de certains oncogènes. De plus, de nombreuses protéines impliquées dans des processus cellulaires comme la réplication, la transcription ou encore la réparation, peuvent interagir directement avec des G4, en facilitant leur formation ou au contraire leur dénaturation. C’est notamment le cas d’hélicases impliquées dans des pathologies humaines, comme BLM, WRN, FANC J ou PIF1. Ce sont des enzymes capables de dénaturer des G4 et dont l'inactivation induit une instabilité génomique, en particulier au niveau de régions susceptibles de former un G4. Dans ce travail, nous présentons la mise au point d’un test de criblage à moyen débit pour le suivi des interactions G4/hélicases en temps réel. Ce test nous a permis de définir les conditions favorisant ou inhibant l’interaction d’une hélicase vis-à-vis de son substrat G4. Nous avons démontré que ces conditions pouvaient différer d’une hélicase à une autre, notamment les conditions salines optimales nécessaires aux activités hélicases de ScPif1 et de RHAU. Nous avons également prouvé, à travers ce test, que l’utilisation de ligands capables de stabiliser les G4 n’induisait pas forcément d’inhibition de l’activité hélicase de ScPif1. Enfin, nous avons également pu définir la directionnalité de la protéine RPA, ce qui fait de notre test une technique prometteuse pour la caractérisation de nouvelles protéines pouvant dérouler des structures G4.G-quadruplexes are highly polymorphic non-canonical nucleic acid structures adopted by both DNA and RNA guanine-rich sequences in vitro. They have been detected at the cellular level using structure specific antibodies and small molecule ligands. Computational studies demonstrated that G4-prone sequences are located in key genomic regions such as telomeres and oncogene promoters. Numerous studies showed that G4 sequences can interact with proteins involved in cellular processes, including replication, transcription or reparation. Those interactions include binding, G4 folding promotion or in contrary unwinding. Indeed, WRN, BLM, FANC J or Pif1 are helicases associated with human-diseases. They can unwind G4 forming sequences; mutation of these helicases lead to genomic instability of G4-prone motifs when mutated. Here, we present a medium-throughput technique to monitor G4-helicase interactions in real time. We were able to determine both favourable and deleterious conditions for G4 unwinding by a given helicase. We show that these conditions differ from one helicase to another as exemplified with the optimal salt conditions required for both ScPif1 and RHAU activities. We also reveal that the G4 ligands that stabilize G4 structures do not necessarily induce an inhibition of their unwinding by ScPif1 helicase. Finally, we also prove that our assay is adapted to clear up RPA directionality, making it an attractive technique to screen for new proteins able to unwind G4 structures

Stabilisation physico-chimique des sols gonflants (sable de dune + sel)

Cette étude a été réalisée sur deux sols expansifs provenant de deux sites différents du sud de l’Algérie, où des dégâts ont été enregistrés (endommagement des structures, fissuration et destruction partielle des chaussées). Dans la première étape, le gonflement des sols est estimé par des méthodes indirectes basées sur les caractéristiques géotechniques. Par la suite, des mesures directes des paramètres de gonflement (taux et pression de gonflement) ont été effectués. La seconde étape de cette investigation porte sur l’effet de l’ajout du sable dunaire sur le potentiel de gonflement. L’ajout de sable a conduit à une diminution des limites de consistance et à une réduction importante des paramètres de gonflement. Enfin, une technique de stabilisation combinée, par l’association de sel plus sable, a été réalisée. Les résultats sont très encourageants et montrent que pour certaines combinaisons, le taux de réduction du potentiel de gonflement est très important (environ 95 %)

Hydraulic properties of dune sand–bentonite mixtures of insulation barriers for hazardous waste facilities

This paper presents a study on the valorization of local materials such as desert dune sand obtained from Laghouat region in the South Algeria and mine bentonite intended for the realization of liner base layers in the conception of insulation barriers for hazardous waste facilities. In practice, an economical mixture satisfying the hydraulic requirements is generally concerned. First, in order to get an adequate dune sand–bentonite mixture compacted to the optimum Proctor condition, an investigation on saturated hydraulic behavior is carried out in this study for different mixtures. Using oedometer test (indirect measurement), the adequate mixture of 85% dune sand and 15% bentonite satisfies the conditions of saturated hydraulic conductivity (k  3 MPa). This technique is conducted based on the exploitation of the water retention curve in order to establish the relationships between hydraulic conductivity, degree of saturation, and suction. It shows that the hydraulic conductivity increases with the degree of saturation and decreases with the suction. However, the hydraulic conductivity has a constant value for suctions larger than 20 MPa. The selected dune sand–bentonite mixture satisfies the regulation requirements and hence constitutes a good local and economical material for the conception of barrier base liners

Effect of bentonite on the saturated hydraulic conductivity of landfill liners based on dune sand

Sand-bentonite (SB) mixtures have been used successfully for construction of hydraulic barriers when clayey soils are not available. Compacted layers of SB mixtures have been proposed and used in a variety of geotechnical structures as engineered barriers for the enhancement of impervious landfill liners. In the practice we will try to get an economical mixture that satisfies the hydraulic and mechanical requirements. The effects of the bentonite additions are reflected in lower water permeability, and acceptable shear strength. In order to get an adequate dune sand bentonite mixtures, an investigation relative to the hydraulic and mechanical behavior is carried out in this study for different mixtures. According to the results obtained, the adequate percentage of bentonite should be between 12% and 15 %, which result in a hydraulic conductivity less than 10E–6 cm/s, and good shear strength

Experimental investigation on effects of calcined bentonite on fresh, strength and durability properties of sustainable self-compacting concrete

With the current focus on sustainable development in the civil engineering field, it is necessary to develop construction and building materials with reasonable costs and low environmental impacts in order to reduce CO2 emissions during the production of concrete, and from the cement industry as a whole. This research studies the effect of using calcined bentonite (CB) as partial replacement of Ordinary Portland Cement (OPC) on the sustainability of self-compacting concrete (SCC). The cement in SCC mixes has been replaced with two different types of CB at 0, 5, 10, 15, 20, 25 and 30% by weight. Slump flow, V-funnel flow time, L-box test and sieve stability tests are performed to evaluate the fresh properties of SCC mixtures. Various tests are used to assess the performance of SCC mixtures in hardened states, such as compressive strength, porosity accessible to water, chloride-ions penetration and gas permeability. The results showed that the use of CB in SCC mixes reduced the fresh properties of SCC and the slump flows, flow times, and segregation tests are good enough for SCC production. At a hardened state, SCC with 10–15% of CB had a higher compressive strength up to 90 days, as well as improved porosity, chloride-ions penetration and gas permeability properties. These results indicate that a CB solution will reduce CO2 emissions and make durable and eco-friendly SCC at a low cost

Potential pozzolanicity of Algerian calcined bentonite used as cement replacement: optimisation of calcination temperature and effect on strength of self-compacting mortars

The effect of using calcined bentonite (CB) as a partial replacement for Ordinary Portland cement (OPC) in self-compacting mortar (SCM) is investigated. The pozzolanicity of this calcined clay is evaluated using the strength activity index and TG/ATD analysis. The cement in SCM has been replaced with CB at 0, 5, 10, 15, 20, 25 and 30% by mass of cement. The effect of CB on fresh SCM properties is examined using mini-slump flow and V-funnel flow time. The compressive strength is determined at the age of 3, 7 and 28days, and the ultrasonic pulse velocity (UPV), hardened density and water absorption are determined at the age of 28days. The behaviour of SCM exposed to high temperature is also studied. The results indicate that CB significantly decreased the flowability of SCM, but these results are good enough for SCM and SCC production. Incorporating 10 and 15% of CB improves the compressive strength and UPV. Water absorption tends to increase slightly with an increase in CB content and there is a decrease in density as the amount of CB increases. SCM containing CB is stronger when exposed to high temperature than those exposed to normal temperature (23C)

5′ to 3′ Unfolding Directionality of DNA Secondary Structures by Replication Protein A

International audienceThe replication protein A (RPA) is a single-stranded DNA-binding protein that plays an essential role in DNA metabolism. RPA is able to unfold G-quadruplex (G4) structures formed by telomeric DNA sequences, a function important for telomere maintenance. To elucidate the mechanism through which RPA unfolds telomeric G4s, we studied its interaction with oligonucleotides that adopt a G4 structure extended with a single-stranded tail on either side of the G4. Binding and unfolding was characterized using several biochemical and biophysical approaches and in the presence of specific G4 ligands, such as telomestatin and 360A. Our data show that RPA can bind on each side of the G4 but it unwinds the G4 only from 5′ toward 3′. We explain the 5′ to 3′ unfolding directionality in terms of the 5′ to 3′ oriented laying out of hRPA subunits along single-stranded DNA. Furthermore, we demonstrate by kinetics experiments that RPA proceeds with the same directionality for duplex unfolding