Unraveling the Complexities of DNA-Dependent Protein Kinase Autophosphorylation

DNA-dependent protein kinase (DNA-PK) orchestrates DNA repair by regulating access to breaks through autophosphorylations within two clusters of sites (ABCDE and PQR). Blocking ABCDE phosphorylation (by alanine mutation) imparts a dominant negative effect, rendering cells hypersensitive to agents that cause DNA double-strand breaks. Here, a mutational approach is used to address the mechanistic basis of this dominant negative effect. Blocking ABCDE phosphorylation hypersensitizes cells to most types of DNA damage (base damage, cross-links, breaks, and damage induced by replication stress), suggesting that DNA-PK binds DNA ends that result from many DNA lesions and that blocking ABCDE phosphorylation sequesters these DNA ends from other repair pathways. This dominant negative effect requires DNA-PK's catalytic activity, as well as phosphorylation of multiple (non-ABCDE) DNA-PK catalytic subunit (DNA-PKcs) sites. PSIPRED analysis indicates that the ABCDE sites are located in the only contiguous extended region of this huge protein that is predicted to be disordered, suggesting a regulatory role(s) and perhaps explaining the large impact ABCDE phosphorylation has on the enzyme's function. Moreover, additional sites in this disordered region contribute to the ABCDE cluster. These data, coupled with recent structural data, suggest a model whereby early phosphorylations promote initiation of nonhomologous end joining (NHEJ), whereas ABCDE phosphorylations, potentially located in a “hinge” region between the two domains, lead to regulated conformational changes that initially promote NHEJ and eventually disengage NHEJ

Pso2 (SNM1) is a DNA structure-specific endonuclease

Many types of DNA structures are generated in response to DNA damage, repair and recombination that require processing via specialized nucleases. DNA hairpins represent one such class of structures formed during V(D)J recombination, palindrome extrusion, DNA transposition and some types of double-strand breaks. Here we present biochemical and genetic evidence to suggest that Pso2 is a robust DNA hairpin opening nuclease in budding yeast. Pso2 (SNM1A in mammals) belongs to a small group of proteins thought to function predominantly during interstrand crosslink (ICL) repair. In this study, we characterized the nuclease activity of Pso2 toward a variety of DNA substrates. Unexpectedly, Pso2 was found to be an efficient, structure-specific DNA hairpin opening endonuclease. This activity was further shown to be required in vivo for repair of chromosomal breaks harboring closed hairpin ends. These findings provide the first evidence that Pso2 may function outside ICL repair and open the possibility that Pso2 may function at least in part during ICL repair by processing DNA intermediates including DNA hairpins or hairpin-like structures

Investigación de mercados I

La investigación de mercados es el proceso mediante el cual las empresas buscan hacer una recolección de datos de manera sistemática para poder tomar mejores decisiones, pero su verdadero valor reside en la manera en que se usa todos los datos obtenidos para poder lograr un mejor conocimiento del consumidor. Se trata, en definitiva, de una potente herramienta, que debe permitir a la empresa obtener la información necesaria para establecer las diferentes políticas, objetivos, planes y estrategias más adecuadas a sus intereses. La investigación de mercados resulta benéfica en diversas situaciones; sin embargo, la decisión de realizarla no es automática. Esta decisión debe sustentarse en consideraciones como la comparación entre costos y beneficios, los recursos disponibles para realizar la investigación y para llevar a la práctica sus hallazgos, así como la actitud de la administración hacia su realización. La investigación de mercados debería realizarse cuando el valor de la información que genera supera los costos de llevarla a cabo. En general, cuanto más importante sea la decisión que enfrenta la administración y mayor sea la incertidumbre o el riesgo que ésta implica, mayor será el valor de la información obtenida. Se dispone de procedimientos formales para cuantificar tanto el valor esperado como los costos de un proceso de investigación de mercados. El investigador debe considerar todos esos factores al decidir si va a realizar experimentos de laboratorio o de campo. Los experimentos de campo son menos comunes que los experimentos de laboratorio en la investigación de mercados, aunque ambos desempeñan funciones complementarias

Structural analysis of the carboxy terminal PH domain of pleckstrin bound to D-myo-inositol 1,2,3,5,6-pentakisphosphate

<p>Abstract</p> <p>Background</p> <p>Pleckstrin homology (PH) domains are one of the most prevalent domains in the human proteome and represent the major phosphoinositide-binding module. These domains are often found in signaling proteins and function predominately by targeting their host proteins to the cell membrane. Inositol phosphates, which are structurally similar to phosphoinositides, are not only known to play a role as signaling molecules but are also capable of being bound by PH domains.</p> <p>Results</p> <p>In the work presented here it is shown that the addition of commercial <it>myo</it>-inositol hexakisphosphate (IP₆) inhibited the binding of the carboxy terminal PH domain of pleckstrin (C-PH) to phosphatidylinositol 3,4-bisphosphate with an IC₅₀of 7.5 μM. In an attempt to characterize this binding structurally, C-PH was crystallized in the presence of IP₆and the structure was determined to 1.35 Å. Examination of the resulting electron density unexpectedly revealed the bound ligand to be D-<it>myo</it>-inositol 1,2,3,5,6-pentakisphosphate.</p> <p>Conclusion</p> <p>The discovery of D-<it>myo</it>-inositol 1,2,3,5,6-pentakisphosphate in the crystal structure suggests that the inhibitory effects observed in the binding studies may be due to this ligand rather than IP₆. Analysis of the protein-ligand interaction demonstrated that this <it>myo</it>-inositol pentakisphosphate isomer interacts specifically with protein residues known to be involved in phosphoinositide binding. In addition to this, a structural alignment of other PH domains bound to inositol phosphates containing either four or five phosphate groups revealed that the majority of phosphate groups occupy conserved locations in the binding pockets of PH domains. These findings, taken together with other recently reported studies suggest that <it>myo-</it>inositol pentakisphosphates could act to regulate PH domain-phosphoinositide interactions by directly competing for binding, thus playing an important role as signaling molecules.</p

Inositol pentakisphosphate isomers bind PH domains with varying specificity and inhibit phosphoinositide interactions

<p>Abstract</p> <p>Background</p> <p>PH domains represent one of the most common domains in the human proteome. These domains are recognized as important mediators of protein-phosphoinositide and protein-protein interactions. Phosphoinositides are lipid components of the membrane that function as signaling molecules by targeting proteins to their sites of action. Phosphoinositide based signaling pathways govern a diverse range of important cellular processes including membrane remodeling, differentiation, proliferation and survival. <it>Myo-</it>Inositol phosphates are soluble signaling molecules that are structurally similar to the head groups of phosphoinositides. These molecules have been proposed to function, at least in part, by regulating PH domain-phosphoinositide interactions. Given the structural similarity of inositol phosphates we were interested in examining the specificity of PH domains towards the family of <it>myo-</it>inositol pentakisphosphate isomers.</p> <p>Results</p> <p>In work reported here we demonstrate that the C-terminal PH domain of pleckstrin possesses the specificity required to discriminate between different <it>myo-</it>inositol pentakisphosphate isomers. The structural basis for this specificity was determined using high-resolution crystal structures. Moreover, we show that while the PH domain of Grp1 does not possess this high degree of specificity, the PH domain of protein kinase B does.</p> <p>Conclusions</p> <p>These results demonstrate that some PH domains possess enough specificity to discriminate between myo-inositol pentakisphosphate isomers allowing for these molecules to differentially regulate interactions with phosphoinositides. Furthermore, this work contributes to the growing body of evidence supporting <it>myo</it>-inositol phosphates as regulators of important PH domain-phosphoinositide interactions. Finally, in addition to expanding our knowledge of cellular signaling, these results provide a basis for developing tools to probe biological pathways.</p

Estéticas de lo cotidiano: aislamiento social en la pintura argentina contemporánea

La presente investigación realiza un análisis iconológico sobre la influencia ejercida por el contexto histórico y social de la reciente pandemia de COVID-19 en las temáticas de un grupo de pinturas creadas en la Ciudad Autónoma de Buenos Aires (CABA) y La Plata entre el año 2020 y 2021. Además, el estudio de las temáticas, así como de las formas de producción y circulación de estas obras, nos entrega las cualidades que nos permiten situarlas dentro del arte contemporáneo. En estas imágenes podemos observar la importancia de captar la naturaleza del tiempo y la esfera relacional de la vida como puntos de partida para la producción plástica.Facultad de Arte

Structural and phylogenetic analysis of a conserved actinobacteria-specific protein (ASP1; SCO1997) from Streptomyces coelicolor

<p>Abstract</p> <p>Background</p> <p>The Actinobacteria phylum represents one of the largest and most diverse groups of bacteria, encompassing many important and well-characterized organisms including <it>Streptomyces, Bifidobacterium, Corynebacterium </it>and <it>Mycobacterium</it>. Members of this phylum are remarkably diverse in terms of life cycle, morphology, physiology and ecology. Recent comparative genomic analysis of 19 actinobacterial species determined that only 5 genes of unknown function uniquely define this large phylum <abbrgrp><abbr bid="B1">1</abbr></abbrgrp>. The cellular functions of these actinobacteria-specific proteins (ASP) are not known.</p> <p>Results</p> <p>Here we report the first characterization of one of the 5 actinobacteria-specific proteins, ASP1 (Gene ID: SCO1997) from <it>Streptomyces coelicolor</it>. The X-ray crystal structure of ASP1 was determined at 2.2 Ǻ. The overall structure of ASP1 retains a similar fold to the large NP-1 family of nucleoside phosphorylase enzymes; however, the function is not related. Further comparative analysis revealed two regions expected to be important for protein function: a central, divalent metal ion binding pore, and a highly conserved elbow shaped helical region at the C-terminus. Sequence analyses revealed that ASP1 is paralogous to another actinobacteria-specific protein ASP2 (SCO1662 from <it>S. coelicolor</it>) and that both proteins likely carry out similar function.</p> <p>Conclusion</p> <p>Our structural data in combination with sequence analysis supports the idea that two of the 5 actinobacteria-specific proteins, ASP1 and ASP2, mediate similar function. This function is predicted to be novel since the structures of these proteins do not match any known protein with or without known function. Our results suggest that this function could involve divalent metal ion binding/transport.</p

Investigación de mercados I

La investigación de mercados es el proceso mediante el cual las empresas buscan hacer una recolección de datos de manera sistemática para poder tomar mejores decisiones, pero su verdadero valor reside en la manera en que se usa todos los datos obtenidos para poder lograr un mejor conocimiento del consumidor. Se trata, en definitiva, de una potente herramienta, que debe permitir a la empresa obtener la información necesaria para establecer las diferentes políticas, objetivos, planes y estrategias más adecuadas a sus intereses. La investigación de mercados resulta benéfica en diversas situaciones; sin embargo, la decisión de realizarla no es automática. Esta decisión debe sustentarse en consideraciones como la comparación entre costos y beneficios, los recursos disponibles para realizar la investigación y para llevar a la práctica sus hallazgos, así como la actitud de la administración hacia su realización. La investigación de mercados debería realizarse cuando el valor de la información que genera supera los costos de llevarla a cabo. En general, cuanto más importante sea la decisión que enfrenta la administración y mayor sea la incertidumbre o el riesgo que ésta implica, mayor será el valor de la información obtenida. Se dispone de procedimientos formales para cuantificar tanto el valor esperado como los costos de un proceso de investigación de mercados. El investigador debe considerar todos esos factores al decidir si va a realizar experimentos de laboratorio o de campo. Los experimentos de campo son menos comunes que los experimentos de laboratorio en la investigación de mercados, aunque ambos desempeñan funciones complementarias