SAMHD1 is a single-stranded nucleic acid binding protein with no active site-associated nuclease activity.

The HIV-1 restriction factor SAMHD1 is a tetrameric enzyme activated by guanine nucleotides with dNTP triphosphate hydrolase activity (dNTPase). In addition to this established activity, there have been a series of conflicting reports as to whether the enzyme also possesses single-stranded DNA and/or RNA 3\u27-5\u27 exonuclease activity. SAMHD1 was purified using three chromatography steps, over which the DNase activity was largely separated from the dNTPase activity, but the RNase activity persisted. Surprisingly, we found that catalytic and nucleotide activator site mutants of SAMHD1 with no dNTPase activity retained the exonuclease activities. Thus, the exonuclease activity cannot be associated with any known dNTP binding site. Monomeric SAMHD1 was found to bind preferentially to single-stranded RNA, while the tetrameric form required for dNTPase action bound weakly. ssRNA binding, but not ssDNA, induces higher-order oligomeric states that are distinct from the tetrameric form that binds dNTPs. We conclude that the trace exonuclease activities detected in SAMHD1 preparations arise from persistent contaminants that co-purify with SAMHD1 and not from the HD active site. An in vivo model is suggested where SAMHD1 alternates between the mutually exclusive functions of ssRNA binding and dNTP hydrolysis depending on dNTP pool levels and the presence of viral ssRNA

Bubbles, clusters and denaturation in genomic DNA: modeling, parametrization, efficient computation

The paper uses mesoscopic, non-linear lattice dynamics based (Peyrard-Bishop-Dauxois, PBD) modeling to describe thermal properties of DNA below and near the denaturation temperature. Computationally efficient notation is introduced for the relevant statistical mechanics. Computed melting profiles of long and short heterogeneous sequences are presented, using a recently introduced reparametrization of the PBD model, and critically discussed. The statistics of extended open bubbles and bound clusters is formulated and results are presented for selected examples.Comment: to appear in a special issue of the Journal of Nonlinear Mathematical Physics (ed. G. Gaeta

Total linear polarization in the OH maser W75N: VLBA polarization structure

W75N is a star-forming region containing various ultracompact HII regions and OH, water, and methanol maser emission. Our VLBA map shows that the OH masers are located in a thin disk rotating around an O-star which is the exciting star of the ultracompact HII region VLA1. A separate set of maser spots is connected with the ultracompact HII region VLA2. The radial velocity of OH maser spots varies across the disk from 3.7 km/s to 10.9 km/s. The diameter of the disk is 4000 A.U. All maser spots are strongly polarized. This are the first OH masers showing nearly 100 per cent linear polarization in several spots. Two maser spots seem to be Zeeman pairs corresponding to a magnetic field of 5.2 mgauss and 7.7 mgauss, and in one case we tentatively found a Zeeman pair consisting of two linearly polarized components. The linearly polarized maser spots are shown to be sigma-components which is the case of the magnetic field being perpendicular to the line of sight. The direction of the magnetic field as determined from linearly polarized spots is perpendicular to the plane of the disk, although the galactic Faraday rotation may significantly affect this conclusion.Comment: 14 figures, 1 table, 27 pages. accepted for publication in ApJ, scheduled for v.564, N1, 200

Tuning the translational freedom of DNA for high speed AFM

Direct observation is arguably the preferred way to investigate the interactions between two molecular complexes. With the development of high speed atomic force microscopy it is becoming possible to observe directly DNA protein interactions with relevant spatial and temporal resolutions. These interactions are of central importance to biology, bio-nanotechnology but also functional biologically inspired materials. Critically, sample preparation plays a central role in all microscopy studies and minimal perturbation of the sample is desired. Here, we demonstrate the ability to tune the interactions of DNA molecules with the surface such that an association strong enough to enable high resolution AFM imaging while providing sufficient translational freedom to allow the relevant protein DNA interactions to take place, can be maintained. Furthermore, we describe a quantitative method for measuring the DNA mobility, which also allows the dissection of the different contributions to the overall movement of the DNA molecules. We find that for weak surface association, a significant contribution to the movement arises from the interaction of the AFM tip with the DNA. In combination, these methods enable the tuning of the surface translational freedom of DNA molecules to allow the direct study of a wide range of nucleo-protein interactions by high speed atomic force microscopy

The Role of Histone H4 Biotinylation in the Structure of Nucleosomes

Background: Post-translational modifications of histones play important roles in regulating nucleosome structure and gene transcription. It has been shown that biotinylation of histone H4 at lysine-12 in histone H4 (K12Bio-H4) is associated with repression of a number of genes. We hypothesized that biotinylation modifies the physical structure of nucleosomes, and that biotin-induced conformational changes contribute to gene silencing associated with histone biotinylation. Methodology/Principal Findings: To test this hypothesis we used atomic force microscopy to directly analyze structures of nucleosomes formed with biotin-modified and non-modified H4. The analysis of the AFM images revealed a 13% increase in the length of DNA wrapped around the histone core in nucleosomes with biotinylated H4. This statistically significant (p,0.001) difference between native and biotinylated nucleosomes corresponds to adding approximately 20 bp to the classical 147 bp length of nucleosomal DNA. Conclusions/Significance: The increase in nucleosomal DNA length is predicted to stabilize the association of DNA with histones and therefore to prevent nucleosomes from unwrapping. This provides a mechanistic explanation for the gene silencing associated with K12Bio-H4. The proposed single-molecule AFM approach will be instrumental for studying the effects of various epigenetic modifications of nucleosomes, in addition to biotinylation

Осложнение силикотуберкулеза амилоидозом внутренних органов

.

Значение видеоторакоскопии в диагностике сложных случаев профессиональных болезней легких

.

Enzymatic Digestion of Single DNA Molecules Anchored on Nanogold-Modified Surfaces

To study enzyme–DNA interactions at single molecular level, both the attachment points and the immediate surroundings of surfaces must be carefully considered such that they do not compromise the structural information and biological properties of the sample under investigation. The present work demonstrates the feasibility of enzymatic digestion of single DNA molecules attached to nanoparticle-modified surfaces. With Nanogold linking DNA to the mica surface by electrostatic interactions, advantageous conditions with fewer effects on the length and topography of DNA are obtained, and an appropriate environment for the activities of DNA is created. We demonstrate that by using Dip-Pen Nanolithography, individual DNA molecules attached to modified mica surfaces can be efficiently digested by DNase I

Effect of Spermidine on Misfolding and Interactions of Alpha-Synuclein

Alpha-synuclein (α-Syn) is a 140 aa presynaptic protein which belongs to a group of natively unfolded proteins that are unstructured in aqueous solutions. The aggregation rate of α-Syn is accelerated in the presence of physiological levels of cellular polyamines. Here we applied single molecule AFM force spectroscopy to characterize the effect of spermidine on the very first stages of α-Syn aggregation – misfolding and assembly into dimers. Two α-Syn variants, the wild-type (WT) protein and A30P, were studied. The two protein molecules were covalently immobilized at the C-terminus, one at the AFM tip and the other on the substrate, and intermolecular interactions between the two molecules were measured by multiple approach-retraction cycles. At conditions close to physiological ones at which α-Syn misfolding is a rare event, the addition of spermidine leads to a dramatic increase in the propensity of the WT and mutant proteins to misfold. Importantly, misfolding is characterized by a set of conformations, and A30P changes the misfolding pattern as well as the strength of the intermolecular interactions. Together with the fact that spermidine facilitates late stages of α-Syn aggregation, our data demonstrate that spermidine promotes the very early stages of protein aggregation including α-Syn misfolding and dimerization. This finding suggests that increased levels of spermidine and potentially other polyamines can initiate the disease-related process of α-Syn

The replication of plastid minicircles involves rolling circle intermediates

Plastid genomes of peridinin-containing dinoflagellates are unique in that its genes are found on multiple circular DNA molecules known as ‘minicircles’ of ∼2–3 kb in size, carrying from one to three genes. The non-coding regions (NCRs) of these minicircles share a conserved core region (250–500 bp) that are AT-rich and have several inverted or direct repeats. Southern blot analysis using an NCR probe, after resolving a dinoflagellate whole DNA extract in pulsed-field gel electrophoresis (PFGE), revealed additional positive bands (APBs) of 6–8 kb in size. APBs preferentially diminished from cells treated with the DNA-replication inhibitor aphidicolin, when compared with 2–3 kb minicircles, implicating they are not large minicircles. The APBs are also exonuclease III-sensitive, implicating the presence of linear DNA. These properties and the migration pattern of the APBs in a 2D-gel electrophoresis were in agreement with a rolling circle type of replication, rather than the bubble-forming type. Atomic force microscopy of 6–8 kb DNA separated by PFGE revealed DNA intermediates with rolling circle shapes. Accumulating data thus supports the involvement of rolling circle intermediates in the replication of the minicircles