Different Quaternary Structures of Human RECQ1 Are Associated with Its Dual Enzymatic Activity

RecQ helicases are essential for the maintenance of chromosome stability. In addition to DNA unwinding, some RecQ enzymes have an intrinsic DNA strand annealing activity. The function of this dual enzymatic activity and the mechanism that regulates it is, however, unknown. Here, we describe two quaternary forms of the human RECQ1 helicase, higher-order oligomers consistent with pentamers or hexamers, and smaller oligomers consistent with monomers or dimers. Size exclusion chromatography and transmission electron microscopy show that the equilibrium between the two assembly states is affected by single-stranded DNA (ssDNA) and ATP binding, where ATP or ATPγS favors the smaller oligomeric form. Our three-dimensional electron microscopy reconstructions of human RECQ1 reveal a complex cage-like structure of approximately 120 Å × 130 Å with a central pore. This oligomeric structure is stabilized under conditions in which RECQ1 is proficient in strand annealing. In contrast, competition experiments with the ATPase-deficient K119R and E220Q mutants indicate that RECQ1 monomers, or tight binding dimers, are required for DNA unwinding. Collectively, our findings suggest that higher-order oligomers are associated with DNA strand annealing, and lower-order oligomers with DNA unwinding

Utilizzo della logica fuzzy per la valutazione del rischio di un processo produttivo aziendale

Il seguente lavoro di tesi è stato realizzato a seguito di un stage avvenuto presso la funzione “Qualificazione e Risk Assessment Processi Produttivi” di AleniaAermacchi, azienda leader nella progettazione, produzione e supporto di aerei per l'addestramento di piloti militari, allo scopo di applicare la logica fuzzy alla valutazione del rischio di un processo produttivo aziendale. In particolare, il processo produttivo aziendale analizzato è una parte del processo di lavorazione di parti in composito di AleniaAermacchi, dalla fase di consegna del materiale preimpregnato al reparto, fino ad arrivare alla fase di preparazione del sacco a vuoto finale necessario per la successiva polimerizzazione della parte in autoclave. Oltre alla logica fuzzy gli strumenti utilizzati per realizzare questo lavoro di tesi sono essenzialmente due: Business Process Modeling Notation (BPMN) e mappe cognitive fuzzy (FCM). Il BPMN (Business Process Modelling Notation) è una notazione grafica standard che consente di modellare i processi di business attraverso una tecnica di flowcharting. Una mappa cognitiva fuzzy, invece, è un grafo orientato i cui nodi rappresentano concetti e gli archi rappresentano le relazioni causali tra concetti, il cui scopo principale è di fornire una rappresentazione grafica di quali concetti influenzano altri concetti

Opportunities Offered by Graphene Nanoparticles for MicroRNAs Delivery for Amyotrophic Lateral Sclerosis Treatment

Amyotrophic lateral sclerosis (ALS) is characterized by the degeneration and death of motor neurons. This neurodegenerative disease leads to muscle atrophy, paralysis, and death due to respiratory failure. MicroRNAs (miRNAs) are small non-coding ribonucleic acids (RNAs) with a length of 19 to 25 nucleotides, participating in the regulation of gene expression. Different studies have demonstrated that miRNAs deregulation is critical for the onset of a considerable number of neurodegenerative diseases, including ALS. Some studies have underlined how miRNAs are deregulated in ALS patients and for this reason, design therapies are used to correct the aberrant expression of miRNAs. With this rationale, delivery systems can be designed to target specific miRNAs. Specifically, these systems can be derived from viral vectors (viral systems) or synthetic or natural materials, including exosomes, lipids, and polymers. Between many materials used for non-viral vectors production, the two-dimensional graphene and its derivatives represent a good alternative for efficiently delivering nucleic acids. The large surface-to-volume ratio and ability to penetrate cell membranes are among the advantages of graphene. This review focuses on the specific pathogenesis of miRNAs in ALS and on graphene delivery systems designed for gene delivery to create a primer for future studies in the field

Surface Rendering of 3D EM Reconstructions of RECQ1 Oligomers

<div><p>Untagged wild-type RECQ1 + ssDNA (red); wild-type RECQ1+ssDNA (pink); K119R mutant RECQ1 + ATPγS (gold); and K119R mutant RECQ1 + ssDNA (white). All maps are shown using an arbitrary density threshold level of 3σ. The general shape of RECQ1 in the four functional states shows an overall similarity and consists of two ring-like densities “A” and “B,” interconnected by a larger ring-like structure “C.”</p> <p>(A) Side-on views showing the basic three-ring structure with the middle ring “C” connecting the two outer rings with diagonally spanning densities.</p> <p>(B) Head-on views of ring “A,” which has an outer diameter of 8 nm.</p> <p>(C) Side-on views rotated by 90° about the <i>y</i>-axis relative to (B).</p> <p>(D) Head-on views of ring “B,” which has approximately the same outer diameter as ring “A.”</p> <p>The resolutions of the maps range from 22 Å for the tagged RECQ1 with ssDNA reconstruction to approximately 26 Å for the untagged RECQ1 with ssDNA reconstruction. Scale bar represents 10 nm [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0050020#pbio-0050020-b056" target="_blank">56</a>].</p></div

Analysis of the Unwinding and Strand Annealing Activities of RECQ1

<div><p>(A) Kinetics of unwinding performed using 5 nM RECQ1.</p> <p>(B) RECQ1 unwinding at varying enzyme concentrations (15-min reaction).</p> <p>(C) Kinetics of strand annealing in the presence of 20 nM RECQ1 and in the absence of enzyme (open diamond [◊]).</p> <p>(D) Analysis of RECQ1 strand annealing activity as a function of protein concentration (15-min reaction). Data points were the mean of three independent experiments with the standard deviation indicated by error bars. The concentration of ATP in all the experiments varied from 0 to 5 mM (filled circle [•] = 0 mM, open inverted triangle [▿] = 0.5 mM, filled triangle [▴] = 1 mM, open circle [○] = 2 mM, and filled square [▪] = 5 mM).</p></div

Image Processing and 3D Reconstructions of RECQ1 Oligomers

<div><p>Representative images from the various stages of image-processing procedures using negatively stained untagged RECQ1 + ssDNA sample as an example.</p> <p>(A) Examples of individual particle images after band-pass filtering and alignment. These images are members of the corresponding class averages shown in the row below (protein is white).</p> <p>(B) Class averages obtained after classification.</p> <p>(C) Re-projections of the 3D density map in orientations corresponding to the Euler angles assigned to the class averages in the row above.</p> <p>(D) Surface renderings of the density map at a threshold level of σ = 3 in orientations corresponding to the Euler angles assigned to the class averages in (B). The left- and right-most images correspond to approximately diametrically opposite views of the molecule, whereas the images in-between correspond to views that are rotated by approximately 90° with respect to these, and also with respect to each other. Scale bar represents 10 nm.</p></div

Characterization of the Enzymatic Activity of the K119R and E220Q Mutants

<div><p>(A) ATPase assays for wild-type (wt) RECQ1 and its mutants. Thin-layer chromatography assays were performed using M13 ssDNA (32 μM) and 20 nM RECQ1 in buffer A as previously described [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0050020#pbio-0050020-b030" target="_blank">30</a>]. The plots are the average of three independent experiments. The insert shows the ATP binding assays for wild-type, K119R, and E220Q proteins performed by nitrocellulose filter binding at three different ATP concentrations.</p> <p>(B) DNA binding assay at increasing protein concentrations using the forked duplex probe. EMSA experiments were performed in buffer 20 mM Tris-HCl (pH 7.5), 50 mM NaCl, 2 mM MgCl₂, 1 mM DTT, and 0.1 mg/ml of BSA as previously described [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0050020#pbio-0050020-b030" target="_blank">30</a>]. The plots are the average of three independent experiments.</p> <p>(C) Unwinding assay at varying enzyme concentrations. The fractions of protein-dependent unwound product formation for the wild-type RECQ1 and its mutants are plotted.</p> <p>(D) Strand annealing assays with two fully complementary oligonucleotides that form a blunt-ended duplex of 42 bp upon annealing. The reaction was carried out for 15 min at 37 °C using 20 nM wild-type, K119R, or E220Q plus or minus the indicated concentrations of ATP or ATPγS. The plots are the average of three independent experiments with the standard deviation indicated by error bars.</p></div

Competition Experiments with the K119R and E220Q Mutants

<div><p>(A) Time courses of the unwinding reactions with 5 nM RECQ1 containing 0–100 nM K119R. The fraction of ssDNA released as a product of the unwinding reaction is plotted versus time.</p> <p>(B) Plot of the unwinding rate versus the concentration of K119R. The solid line represents the fit of the experimental data to <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0050020#pbio-0050020-e001" target="_blank">Equation 1</a> with <i>n</i> = 0.6 ± 0.1. The dashed-dotted, dashed, and dotted lines simulate the expected variation of unwinding rate for <i>n</i> = 1, 2, or 6.</p> <p>(C) Rates of unwinding by wild-type RECQ1 in the presence of increasing concentrations of K119R (filled circle [•]) and E220Q (open circle [○]) under single-turnover conditions. A total of 50 nM oligo(dT)₄₀ is utilized to trap the helicase that dissociates from the substrate during the reaction.</p></div

Size Exclusion Chromatography Experiments

<p>Chromatographic profiles of the untagged RECQ1 (solid lane) and His₆-RECQ1 (dashed line) eluting from the Superdex200 HR 10/30 gel filtration column. The protein species were detected by protein fluorescence (λ_excitation = 290 nm and λ_emission = 340 nm). Approximately 40 μg of recombinant RECQ1 were loaded at a final concentration of 1 μM. The protein eluted in two main peaks. The first peak corresponds to a calculated molecular mass of approximately 400 kDa, whereas the second corresponds to a calculated molecular weight of approximately 155 kDa. The absence of protein eluting with the void volume highlights the lack of nonspecific aggregates. (A) RECQ1 alone. (B) RECQ1 + ssDNA (oligo(dT)₄₀). (C) RECQ1 + ATPγS. (D) RECQ1 + ssDNA (oligo(dT)₄₀) + ATPγS. Analogous results were obtained using oligo(dT)₃₀ (unpublished data).</p