Differential Interaction Kinetics of a Bipolar Structure-Specific Endonuclease with DNA Flaps Revealed by Single-Molecule Imaging

As DNA repair enzymes are essential for preserving genome integrity, understanding their substrate interaction dynamics and the regulation of their catalytic mechanisms is crucial. Using single-molecule imaging, we investigated the association and dissociation kinetics of the bipolar endonuclease NucS from Pyrococcus abyssi (Pab) on 5′ and 3′-flap structures under various experimental conditions. We show that association of the PabNucS with ssDNA flaps is largely controlled by diffusion in the NucS-DNA energy landscape and does not require a free 5′ or 3′ extremity. On the other hand, NucS dissociation is independent of the flap length and thus independent of sliding on the single-stranded portion of the flapped DNA substrates. Our kinetic measurements have revealed previously unnoticed asymmetry in dissociation kinetics from these substrates that is markedly modulated by the replication clamp PCNA. We propose that the replication clamp PCNA enhances the cleavage specificity of NucS proteins by accelerating NucS loading at the ssDNA/dsDNA junctions and by minimizing the nuclease interaction time with its DNA substrate. Our data are also consistent with marked reorganization of ssDNA and nuclease domains occurring during NucS catalysis, and indicate that NucS binds its substrate directly at the ssDNA-dsDNA junction and then threads the ssDNA extremity into the catalytic site. The powerful techniques used here for probing the dynamics of DNA-enzyme binding at the single-molecule have provided new insight regarding substrate specificity of NucS nucleases.     

The invasive brown seaweed Sargassum muticum as new resource for alginate in Morocco: Spectroscopic and rheological characterization

The Japanese brown seaweed Sargassum muticum, recently invaded several shorelines worldwide including the Atlantic coast of Morocco with large well‐established populations. Within the framework of a sustainable strategy to control this invasive seaweed, we report on extraction yield, spectroscopic characterization and rheological properties of alginate, a commercially valuable colloid, from harvested biomass of S. muticum. Extraction yield was about 25.6% on dry weight basis. Infrared spectroscopy analysis shows that the obtained Fourier transform infrared spectra of the extracted biopolymer exhibit strong similarities with that of the commercial alginate. Furthermore, Proton nuclear magnetic resonance spectroscopy revealed that S. muticum alginate has almost equal amounts of β‐D‐mannuronic acid (M; 49%) and α‐L‐guluronic acid (G; 51%) with an M/G ratio of 1.04 and a high content of heteropolymeric MG GM diads suggesting a sequence distribution of an alternated polymer type. Rheological measurements were performed at different sodium alginate concentrations, temperatures and shear rates. The hydrocolloid exhibited pseudoplastic behavior and showed shear thinning, particularly at high solution concentration and low temperature which is consistent with the rheological behavior reported for commercial alginates. Considering the abundance of S. muticum in the Northwestern Atlantic coast of Morocco and the quality of the extracted hydrogel, this invasive species could be considered as a potential source of alginates.     

Conserved Amphipathic Helices Mediate Lipid Droplet Targeting of Perilipins 1–3

Perilipins (PLINs) play a key role in energy storage by orchestrating the activity of lipases on the surface of lipid droplets. Failure of this activity results in severe metabolic disease in humans. Unlike all other lipid droplet-associated proteins, PLINs localize almost exclusively to the phospholipid monolayer surrounding the droplet. To understand how they sense and associate with the unique topology of the droplet surface, we studied the localization of human PLINs in Saccharomyces cerevisiae, demonstrating that the targeting mechanism is highly conserved and that 11-mer repeat regions are sufficient for droplet targeting. Mutations designed to disrupt folding of this region into amphipathic helices (AHs) significantly decreased lipid droplet targeting in vivo and in vitro. Finally, we demonstrated a substantial increase in the helicity of this region in the presence of detergent micelles, which was prevented by an AH-disrupting missense mutation. We conclude that highly conserved 11-mer repeat regions of PLINs target lipid droplets by folding into AHs on the droplet surface, thus enabling PLINs to regulate the interface between the hydrophobic lipid core and its surrounding hydrophilic environment.     

Electrostatic interactions in ionic homopolypeptides in solutions of moderate ionic strength

At sufficiently high ionic strength, long-range electrostatic interactions in a polyelectrolyte such as poly(L -glutamic acid) might be adequately approximated in matrix calculations by use of statistical weights representing second-order interactions. The validity of this assumption has been investigated making use of experimental observations (CD spectra and titration curves) for poly(L -glutamic acid) as a function of temperature in 0.1–0.5M sodium chloride. Theoretical analysis, using a statistical weight matrix proposed by Warashina and Ikegami, is based on the Zimm-Rice theory. Implementation differs from that of Warashina and Ikegami in one respect. Refinement of the initial estimates is achieved using a form of the configuration partition function which does not assume diagonalization of the statistical weight matrix. This difference is of no consequence for the values of σ and s, but it does produce somewhat different values for the statistical weights used to represent the electrostatic interactions. The method used to treat electrostatic interactions in poly(L -glutamic acid) in 0.1M sodium chloride can be viewed as successful in that it properly reproduces the helix–coil transition and titration curves in this solvent and the molecular-weight dependence of the titration curves yields values for s in harmony with those obtained using a treatment which is independent of model, and gives a reasonable ionic-strength dependence for the electrostatic parameters. Furthermore, the model can account for measured helix–coil transitions and titration curves in homopolypeptides in which the side chain is —(CH₂)_xNHCO(CH₂)_yCOOH. The model, however, is not exact. It does not properly account for the molecular-weight dependence of the helical content for polymers of low degree of polymerization.     

Fat inclusions strongly alter membrane mechanics

Neutral lipids (NLs) are apolar oil molecules synthesized in the endoplasmic reticulum bilayer upon diverse biological stimuli. NLs synthesized are released in the hydrophobic core of the bilayer. At a critical concentration, NLs condense by phase separation and nucleate a lipid droplet (LD). After an LD forms, a fraction of NLs can be present in the bilayer but at a concentration below that of the nucleation. Here, we study whether and how the accumulation of NLs alters a lipid bilayer’s mechanical properties. In synthetic systems, we found that NLs proffer unusual bilayer stretching capacities, especially in the presence of negatively curved phospholipids. This impact becomes spectacular when an LD is contiguous with the bilayer and supplies it with NLs. The tested NLs markedly decrease the bilayer area expansion modulus and significantly increase lysis tension but had opposite effects on membrane bending rigidity. Our data unveil how NL molecules modify overall membrane mechanics, the alteration of which may be linked to pathologies or anticancer treatments targeting NLs.     

Environmental Control on Fish and Macrocrustacean Spring Community-Structure,on an Intertidal Sandy Beach

The inter-annual variability of the fish and macrocrustacean spring community on an intertidal sandy beach near the Canche estuary (North of France) was studied from 2000 to 2013 based on weekly spring sampling over an 11-year period. Twenty-eight species representing 21 families were collected during the course of the study. The community was dominated by a few abundant species accounting for > 99% of the total species densities. Most individuals caught were young-of-the-year indicating the importance of this ecosystem for juvenile fishes and macrocrustaceans. Although standard qualitative community ecology metrics (species composition, richness, diversity, evenness and similarity) indicated notable stability over the study period, community structure showed a clear change since 2009. Densities of P. platessa, P. microps and A. tobianus decreased significantly since 2009, whereas over the period 2010-2013, the contribution of S. sprattus to total species density increased 4-fold. Co-inertia and generalised linear model analyses identified winter NAO index, water temperature, salinity and suspended particular matter as the major environmental factors explaining these changes. Although the recurrent and dense spring blooms of the Prymnesiophyte Phaeocystis globosa is one of the main potential threats in shallow waters of the eastern English Channel, no negative impact of its temporal change was detected on the fish and macrocrustacean spring community structure.     

Chelation-controlled regioselective alkylation of pyrimidine 2′-deoxynucleosides

Protection-deprotection steps, which are usually needed for regioselective alkylation of pyrimidine deoxynucleosides, can be avoided by choosing the appropriate solvent. The combined effects of low dielectric constant and possible sodium chelation by pyrimidine nucleosides may account for the unexpected regioselectivity observed in THF.     

Characterization of a human coagulation factor Xa-binding site on <Emphasis Type="Italic">Viperidae</Emphasis> snake venom phospholipases A<Subscript>2</Subscript> by affinity binding studies and molecular bioinformatics

Background  

The snake venom group IIA secreted phospholipases A₂ (SVPLA₂), present in the Viperidae snake family exhibit a wide range of toxic and pharmacological effects. They exert their different functions by catalyzing the hydrolysis of phospholipids (PL) at the membrane/water interface and by highly specific direct binding to: (i) presynaptic membrane-bound or intracellular receptors; (ii) natural PLA₂-inhibitors from snake serum; and (iii) coagulation factors present in human blood.