RESUMEN
SignificanceBiomolecular condensates are intracellular organelles that are not bounded by membranes and often show liquid-like, dynamic material properties. They typically contain various types of proteins and nucleic acids. How the interaction of proteins and nucleic acids finally results in dynamic condensates is not fully understood. Here we use optical tweezers and fluorescence microscopy to study how the prototypical prion-like protein Fused-in-Sarcoma (FUS) condenses with individual molecules of single- and double-stranded DNA. We find that FUS adsorbs on DNA in a monolayer and hence generates an effectively sticky FUS-DNA polymer that collapses and finally forms a dynamic, reversible FUS-DNA co-condensate. We speculate that protein monolayer-based protein-nucleic acid co-condensation is a general mechanism for forming intracellular membraneless organelles.
Asunto(s)
ADN de Cadena Simple/química , ADN/química , Proteína FUS de Unión a ARN/química , Humanos , Microscopía FluorescenteRESUMEN
Optical tweezers enable the isolation and mechanical manipulation of individual nucleoprotein complexes. Here, we describe how to use this technique to interrogate the mechanical properties of individual protein-DNA complexes and extract information about their overall structural organization.
Asunto(s)
ADN de Cadena Simple/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas Mitocondriales/metabolismo , Nucleoproteínas/química , Replicación del ADN , ADN de Cadena Simple/química , Proteínas de Unión al ADN/química , Humanos , Proteínas Mitocondriales/química , Conformación Molecular , Pinzas ÓpticasRESUMEN
Single-stranded DNA-binding proteins (SSBs) play a key role in genome maintenance, binding and organizing single-stranded DNA (ssDNA) intermediates. Multimeric SSBs, such as the human mitochondrial SSB (HmtSSB), present multiple sites to interact with ssDNA, which has been shown in vitro to enable them to bind a variable number of single-stranded nucleotides depending on the salt and protein concentration. It has long been suggested that different binding modes might be used selectively for different functions. To study this possibility, we used optical tweezers to determine and compare the structure and energetics of long, individual HmtSSB-DNA complexes assembled on preformed ssDNA and on ssDNA generated gradually during 'in situ' DNA synthesis. We show that HmtSSB binds to preformed ssDNA in two major modes, depending on salt and protein concentration. However, when protein binding was coupled to strand-displacement DNA synthesis, only one of the two binding modes was observed under all experimental conditions. Our results reveal a key role for the gradual generation of ssDNA in modulating the binding mode of a multimeric SSB protein and consequently, in generating the appropriate nucleoprotein structure for DNA synthetic reactions required for genome maintenance.
Asunto(s)
ADN Mitocondrial/genética , ADN de Cadena Simple/genética , Proteínas de Unión al ADN/genética , Mitocondrias/genética , Proteínas Mitocondriales/genética , Sitios de Unión , ADN Mitocondrial/biosíntesis , ADN de Cadena Simple/biosíntesis , Proteínas de Unión al ADN/metabolismo , Genoma Mitocondrial , Humanos , Cinética , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Pinzas Ópticas , Unión Proteica , Cloruro de Sodio/farmacología , TermodinámicaRESUMEN
Ligands binding to polymers regulate polymer functions by changing their physical and chemical properties. This ligand regulation plays a key role in many biological processes. We propose here a model to explain the mechanical, thermodynamic, and kinetic properties of the process of binding of small ligands to long biopolymers. These properties can now be measured at the single molecule level using force spectroscopy techniques. Our model performs an effective decomposition of the ligand-polymer system on its covered and uncovered regions, showing that the elastic properties of the ligand-polymer depend explicitly on the ligand coverage of the polymer (i.e., the fraction of the polymer covered by the ligand). The equilibrium coverage that minimizes the free energy of the ligand-polymer system is computed as a function of the applied force. We show how ligands tune the mechanical properties of a polymer, in particular its length and stiffness, in a force dependent manner. In addition, it is shown how ligand binding can be regulated applying mechanical tension on the polymer. Moreover, the binding kinetics study shows that, in the case where the ligand binds and organizes the polymer in different modes, the binding process can present transient shortening or lengthening of the polymer, caused by changes in the relative coverage by the different ligand modes. Our model will be useful to understand ligand-binding regulation of biological processes, such as the metabolism of nucleic acid. In particular, this model allows estimating the coverage fraction and the ligand mode characteristics from the force extension curves of a ligand-polymer system.
Asunto(s)
Biopolímeros/química , Biopolímeros/metabolismo , Cinética , Ligandos , Fenómenos Mecánicos , Modelos Químicos , TermodinámicaRESUMEN
During DNA replication replicative polymerases move in discrete mechanical steps along the DNA template. To address how the chemical cycle is coupled to mechanical motion of the enzyme, here we use optical tweezers to study the translocation mechanism of individual bacteriophage Phi29 DNA polymerases during processive DNA replication. We determine the main kinetic parameters of the nucleotide incorporation cycle and their dependence on external load and nucleotide (dNTP) concentration. The data is inconsistent with power stroke models for translocation, instead supports a loose-coupling mechanism between chemical catalysis and mechanical translocation during DNA replication. According to this mechanism the DNA polymerase works by alternating between a dNTP/PPi-free state, which diffuses thermally between pre- and post-translocated states, and a dNTP/PPi-bound state where dNTP binding stabilizes the post-translocated state. We show how this thermal ratchet mechanism is used by the polymerase to generate work against large opposing loads (â¼50 pN).
Asunto(s)
Replicación del ADN , ADN Polimerasa Dirigida por ADN/metabolismo , Transporte Biológico , CinéticaAsunto(s)
Fagos de Bacillus/genética , Replicación del ADN , ADN Viral/metabolismo , ADN Polimerasa Dirigida por ADN/metabolismo , Desnaturalización de Ácido Nucleico , Fagos de Bacillus/enzimología , Secuencia de Bases , ADN Helicasas/genética , ADN Helicasas/metabolismo , ADN Viral/genética , ADN Polimerasa Dirigida por ADN/genética , Activación Enzimática , Modelos Genéticos , Origen de Réplica , Proteínas Virales/genética , Proteínas Virales/metabolismoRESUMEN
Duplication of double-stranded DNA (dsDNA) requires a fine-tuned coordination between the DNA replication and unwinding reactions. Using optical tweezers, we probed the coupling dynamics between these two activities when they are simultaneously carried out by individual Phi29 DNA polymerase molecules replicating a dsDNA hairpin. We used the wild-type and an unwinding deficient polymerase variant and found that mechanical tension applied on the DNA and the DNA sequence modulate in different ways the replication, unwinding rates, and pause kinetics of each polymerase. However, incorporation of pause kinetics in a model to quantify the unwinding reaction reveals that both polymerases destabilize the fork with the same active mechanism and offers insights into the topological strategies that could be used by the Phi29 DNA polymerase and other DNA replication systems to couple unwinding and replication reactions.
Asunto(s)
Fagos de Bacillus/genética , ADN Helicasas/genética , Replicación del ADN/genética , ADN Viral/genética , ADN Polimerasa Dirigida por ADN/genética , ADN Helicasas/metabolismo , ADN Viral/química , ADN Polimerasa Dirigida por ADN/metabolismo , Regulación Viral de la Expresión Génica/fisiología , Proteínas Motoras Moleculares/fisiología , Conformación de Ácido Nucleico , Estrés Mecánico , Proteínas Virales/genética , Proteínas Virales/metabolismoRESUMEN
La Revista Médica Electrónica, de Matanzas, dando cumplimiento a su objetivo estratégico de ampliar las posibilidades de publicación a los profesionales y técnicos de la salud, deja inaugurada, con este nuevo número, la sección de Artículos de Opinión. El artículo de opinión ofrece la oportunidad para que los autores expresen y argumenten sus consideraciones acerca de algún problema, hecho o circunstancia de interés científico en el ámbito de las Ciencias Médicas y de la Salud Pública. Puede abarcar aspectos de los servicios asistenciales, educacionales y de la investigación, que se extiendan a temas de promoción, prevención, diagnóstico, terapéutica, pronóstico y otros de interés clínico-epidemiológico y administrativo. También en temas de pedagogía y didáctica en el campo de la educación de las ciencias médicas, así como en aspectos del desarrollo de investigaciones en sus diferentes enfoques ontológicos, epistemológicos y metodológicos, y en temas del desarrollo e introducción de tecnologías y productos
Electronic Medical Journal of Matanzas, in compliance with its strategic goal of expanding the possibilities of publishing professionals and health technicians, left opened, with this new number, the Opinion section. The review article provides an opportunity for authors to express and argue their considerations about an issue, event or circumstance of scientific interest in the field of Medical Sciences and Public Health. May include elements of health care services, education and research, which extend to issues of promotion, prevention, diagnosis, treatment, prognosis and other clinical and epidemiological interest and administrative staff. Also on matters of pedagogy and didactics in the field of medical science education and development aspects of research in their different approaches ontological, epistemological and methodological issues in the development and introduction of technologies and products
Asunto(s)
Promoción de la Salud/métodos , Opinión Pública , Artículo Introductorio de RevistaRESUMEN
Se realiza una descripción de los antecedentes de las unidades hospitalarias y el cambio de su función de acuerdo a las transformaciones demográficas, epidemiológicas, políticas, económicas y sociales. Se analiza los retos que enfrentan los sistemas de salud en el nuevo siglo y la necesidad de la reorientación de las instituciones hospitalarias dentro de la red de servicios de salud para lograr una mayor eficiencia y accesibilidad de la atención médica. Por último se analiza como está conformada la red de servicios de salud en nuestro país...(AU)
Asunto(s)
Hospitales/historia , Hospitales/tendencias , Sistemas de Salud , Servicios Centralizados de Hospital/organización & administraciónRESUMEN
Se realiza una descripción de los antecedentes de las unidades hospitalarias y el cambio de su función de acuerdo a las transformaciones demográficas, epidemiológicas, políticas, económicas y sociales. Se analiza los retos que enfrentan los sistemas de salud en el nuevo siglo y la necesidad de la reorientación de las instituciones hospitalarias dentro de la red de servicios de salud para lograr una mayor eficiencia y accesibilidad de la atención médica. Por último se analiza como está conformada la red de servicios de salud en nuestro país...