Functional characterization of highly processive protein-primed DNA polymerases from phages Nf and GA-1, endowed with a potent strand displacement capacity

This paper shows that the protein-primed DNA polymerases encoded by bacteriophages Nf and GA-1, unlike other DNA polymerases, do not require unwinding or processivity factors for efficient synthesis of full-length terminal protein (TP)-DNA. Analysis of their polymerization activity shows that both DNA polymerases base their replication efficiency on a high processivity and on the capacity to couple polymerization to strand displacement. Both enzymes are endowed with a proofreading activity that acts coordinately with the polymerization one to edit polymerization errors. Additionally, Nf double-stranded DNA binding protein (DBP) greatly stimulated the in vitro formation of the TP-dAMP initiation complex by decreasing the K(m) value for dATP of the Nf DNA polymerase by >20-fold. Whereas Nf DNA polymerase, as the φ29 enzyme, is able to use its homologous TP as well as DNA as primer, GA-1 DNA polymerase appears to have evolved to use its corresponding TP as the only primer of DNA synthesis. Such exceptional behaviour is discussed in the light of the recently solved structure of the DNA polymerase/TP complex of the related bacteriophage φ29

DNA-Binding Proteins Essential for Protein-Primed Bacteriophage Φ29 DNA Replication

Bacillus subtilis phage Φ29 has a linear, double-stranded DNA 19 kb long with an inverted terminal repeat of 6 nucleotides and a protein covalently linked to the 5′ ends of the DNA. This protein, called terminal protein (TP), is the primer for the initiation of replication, a reaction catalyzed by the viral DNA polymerase at the two DNA ends. The DNA polymerase further elongates the nascent DNA chain in a processive manner, coupling strand displacement with elongation. The viral protein p5 is a single-stranded DNA binding protein (SSB) that binds to the single strands generated by strand displacement during the elongation process. Viral protein p6 is a double-stranded DNA binding protein (DBP) that preferentially binds to the origins of replication at the Φ29 DNA ends and is required for the initiation of replication. Both SSB and DBP are essential for Φ29 DNA amplification. This review focuses on the role of these phage DNA-binding proteins in Φ29 DNA replication both in vitro and in vivo, as well as on the implication of several B. subtilis DNA-binding proteins in different processes of the viral cycle. We will revise the enzymatic activities of the Φ29 DNA polymerase: TP-deoxynucleotidylation, processive DNA polymerization coupled to strand displacement, 3′–5′ exonucleolysis and pyrophosphorolysis. The resolution of the Φ29 DNA polymerase structure has shed light on the translocation mechanism and the determinants responsible for processivity and strand displacement. These two properties have made Φ29 DNA polymerase one of the main enzymes used in the current DNA amplification technologies. The determination of the structure of Φ29 TP revealed the existence of three domains: the priming domain, where the primer residue Ser232, as well as Phe230, involved in the determination of the initiating nucleotide, are located, the intermediate domain, involved in DNA polymerase binding, and the N-terminal domain, responsible for DNA binding and localization of the TP at the bacterial nucleoid, where viral DNA replication takes place. The biochemical properties of the Φ29 DBP and SSB and their function in the initiation and elongation of Φ29 DNA replication, respectively, will be described.This work has been supported by grants from the Spanish Ministry of Economy and Competitiveness (BFU2014-52656-P to MS) and (BFU2014-53791-P to MV), ComFuturo Grant from Fundación General CSIC (to MR) and by an Institutional grant from Fundación Ramón Areces to the Centro de Biología Molecular “Severo Ochoa.”Peer reviewedPeer Reviewe

Efecto de la aplicación de un protocolo de intervención sobre las variables fuerza, resistencia y funcionalidad en la extremidad superior no dominante asociado a inmovilización contralateral: un estudio de caso

Tesis (Kinesiólogo)Antecedentes: Existen distintos tipos de lesiones en el sistema musculoesquelético (SME). En el caso de las fracturas, y dependiendo del tipo, se establece los tiempos de consolidación con un mínimo de 21 días de inmovilización. En caso de ser en el hemicuerpo dominante, la pérdida funcional es aún mayor. Esta funcionalidad, bajo un enfoque de la Clasificación Internacional del funcionamiento, de la Discapacidad y la Salud debiera ser mantenida durante todo el período, incluso en fase aguda, por ende y con el fin de conocer los efectos de la aplicación de un protocolo de intervención sobre las variables fuerza, resistencia y funcionalidad de la extremidad superior no dominante, simulando una inmovilización contralateral se ha creado un diseño de estudio que permite conocer la aplicación de la técnica en sujetos con lesión en SME Objetivo: Determinar los cambios sobre las variables fuerza, resistencia y funcionalidad mediante la aplicación de un protocolo de intervención en la extremidad superior no dominante en paralelo a la simulación de una lesión mediante una inmovilización y restricción del movimiento del miembro superior dominante por un periodo de 21 días. Metodología: Se evaluó un sujeto sexo femenino, 22 años, sano. Se aplicó un protocolo de evaluación de fuerza y resistencia a través de un dinamómetro prensil y la valoración de funcionalidad mediante el test de 400 puntos. Se realizó una intervención durante 21 días que incluía una férula y cabestrillo para la inmovilización simulada de su extremidad superior dominante y un entrenamiento diario de las distintas variables en su extremidad superior no dominante. Resultados: Se generó un aumento de la fuerza prensil, con un porcentaje de cambio de 8,33% respecto a la etapa previo vs durante y un 16,67% en la etapa previo vs posterior. Se produjo un aumento de la resistencia en contracción isométrica con un porcentaje de cambio de 88,67% en la etapa previo vs durante y 138,67% en la etapa previo vs posterior. Se evidenció un aumento en la funcionalidad con un porcentaje de cambio de 11,32% en la etapa previo vs durante, un 13,01% en la etapa previo vs posterior y un cambio de 1,51% en la etapa durante vs posterior. Conclusión: La aplicación de un protocolo de entrenamiento en extremidad superior no dominante, genera cambios favorables en las variables fuerza, resistencia y funcionalidad del sujeto en este estudio

Involvement of φ29 DNA polymerase thumb subdomain in the proper coordination of synthesis and degradation during DNA replication

φ29 DNA polymerase achieves a functional coupling between its 3′–5′ exonuclease and polymerization activities by means of important contacts with the DNA at both active sites. The placement and orientation of residues Lys538, Lys555, Lys557, Gln560, Thr571, Thr573 and Lys575 in a modelled φ29 DNA polymerase–DNA complex suggest a DNA-binding role. In addition, crystal structure of φ29 DNA polymerase–oligo (dT)(5) complex showed Leu567, placed at the tip of the thumb subdomain, lying between the two 3′-terminal bases at the exonuclease site. Single replacement of these φ29 DNA polymerase residues by alanine was made, and mutant derivatives were overproduced and purified to homogeneity. The results obtained in the assay of their synthetic and degradative activities, as well as their coordination, allow us to propose: (1) a primer-terminus stabilization role at the polymerase active site for residues Lys538, Thr573 and Lys575, (2) a primer-terminus stabilization role at the exonuclease active site for residues Leu567 and Lys555 and (3) a primer-terminus binding role in both editing and polymerization modes for residue Gln560. The results presented here lead us to propose φ29 DNA polymerase thumb as the main subdomain responsible for the coordination of polymerization and exonuclease activities

Incidencia del cálculo de reservas en la viabilidad de explotación de la concesión San Juan, empresa Calinor S.A.C, Cajamarca, 2018

RESUMEN Esta tesis tiene como objetivo general determinar el nivel de incidencia del cálculo de reservas en la viabilidad de explotación de la concesión San Juan, Empresa Calinor S.A.C, Cajamarca, 2018. Para lo cual se debe determinar la influencia de la clasificación de la calidad de la roca de la concesión, definir el efecto de cubicar las reservas de la concesión y finalmente especificar el grado de incidencia del análisis de la viabilidad técnica - económica (flujo de caja) en la explotación de la concesión. El cálculo de reservas además permite hacer un planeamiento en un futuro próximo, dependiendo los intereses. Para ello se deben tener muchos aspectos en consideración como son los estratos de la minería, resoluciones dictaminadas por organismos competentes como el Ministerio de Energía y Minas o Ministerio de la Industria. El tipo de investigación es descriptiva ya que reúne un conjunto de procesos y procedimientos lógicos y prácticos que permiten identificar, el diseño de investigación es descriptivo aplicativo, ya que se van a describir las condiciones del yacimiento de roca caliza a explotar, de la cual se van a calcular reservas. Se concluyó que el método de los perfiles para el cálculo de las reservas, aunque es un método un poco tradicional, tiene buena precisión si es que se toma las distancias adecuadas, tomando en cuenta la variación de la topografía. Se logró determinar que las reservas totales de toda el área es 5 523 753.348 tn. La producción mensual en promedio es de 45,000 TM. El método de explotación en el área se estableció el banqueo por derribo, los cuales tiene como elementos constituyentes a un ángulo de talud final igual a 21°, ángulo de banco igual a 60° y un ancho de banco establecido en 7.5m. Las normas de gestión de seguridad, salud ocupacional y medio ambiente se rigen a las normas y a la ley general de minería según el decreto supremo 024 EM. PALABRAS CLAVE: Cálculo de reservas, viabilidad de explotación, cantera, caliza, banqueo.ABSTRACT The general objective of this thesis is to determine the level of impact of the calculation of reserves on the operating viability of the San Juan concession, Calinor SAC Company, Cajamarca, 2018. For this purpose, the influence of the classification of the quality of the the concession rock, define the effect of cubing the reserves of the concession and finally specify the degree of impact of the analysis of the technical - economic feasibility (cash flow) in the exploitation of the concession. The calculation of reserves also allows planning in the near future, depending on the interests. For this, many aspects must be taken into consideration such as the mining strata, resolutions dictated by competent bodies such as the Ministry of Energy and Mines or the Ministry of Industry. The type of research is descriptive since it gathers a set of logical and practical processes and procedures that allow to identify, the research design is descriptive and applicative, since the conditions of the limestone deposit to be exploited will be described, of which they will calculate reserves. It was concluded that the method of the profiles for the calculation of the reserves, although it is a somewhat traditional method, has good precision if the appropriate distances are taken, taking into account the variation of the topography. It was determined that the total reserves of the entire area are 5 523 753.348 tn. The monthly production on average is 45,000 MT. The method of exploitation in the area was established bank by demolition, which has as constituent elements to a final slope angle equal to 21 °, bank angle equal to 60 ° and a bank width established in 7.5m. The safety, occupational health and environmental management standards are governed by the norms and the general mining law according to supreme decree 024 EM. KEYWORDS: calculation of reserves, exploitation feasibility, quarry, limestone, banking

Involvement of residues of the ϕ29 terminal protein intermediate and priming domains in the formation of a stable and functional heterodimer with the replicative DNA polymerase

Bacteriophage ϕ29 genome consists of a linear double-stranded DNA with a terminal protein (TP) covalently linked to each 5′ end (TP-DNA) that together with a specific sequence constitutes the replication origins. To initiate replication, the DNA polymerase forms a heterodimer with a free TP that recognizes the origins and initiates replication using as primer the hydroxyl group of TP residue Ser232. The 3D structure of the DNA polymerase/TP heterodimer allowed the identification of TP residues that could be responsible for interaction with the DNA polymerase. Here, we examined the role of TP residues Arg158, Arg169, Glu191, Asp198, Tyr250, Glu252, Gln253 and Arg256 by in vitro analyses of mutant derivatives. The results showed that substitution of these residues had an effect on either the stability of the TP/DNA polymerase complex (R158A) or in the functional interaction of the TP at the polymerization active site (R169A, E191A, Y250A, E252A, Q253A and R256A), affecting the first steps of ϕ29 TP-DNA replication. These results allow us to propose a role for these residues in the maintenance of the equilibrium between TP-priming domain stabilization and its gradual exit from the polymerization active site of the DNA polymerase as new DNA is being synthesized

Role of the LEXE motif of protein-primed DNA polymerases in the interaction with the incoming nucleotide

The LEXE motif, conserved in eukaryotic type DNA polymerases, is placed close to the polymerization active site. Previous studies suggested that the second Glu was involved in binding a third noncatalytic ion in bacteriophage RB69 DNA polymerase. In the protein-primed DNA polymerase subgroup, the LEXE motif lacks the first Glu in most cases, but it has a conserved Phe/Trp and a Gly preceding that position. To ascertain the role of those residues, we have analyzed the behavior of mutants at the corresponding φ29 DNA polymerase residues Gly-481, Trp-483, Ala-484, and Glu-486. We show that mutations at Gly-481 and Trp-483 hamper insertion of the incoming dNTP in the presence of Mg2+ ions, a reaction highly improved when Mn2+ was used as metal activator. These results, together with previous crystallographic resolution of φ29 DNA polymerase ternary complex, allow us to infer that Gly-481 and Trp-483 could form a pocket that orients Val-250 to interact with the dNTP. Mutants at Glu-486 are also defective in polymerization and, as mutants at Gly-481 and Trp-483, in the pyrophosphorolytic activity with Mg2+. Recovery of both reactions with Mn2+ supports a role for Glu-486 in the interaction with the pyrophosphate moiety of the dNT

Heraldic seizure

AbstractBackground: The term heraldic seizures indicates epileptic seizures caused by cerebrovascular disease, believed to be triggered by silent ischemia and occurring before a stroke. This fact widens the spectrum of possible interrelations between epilepsy and cerebrovascular disease outside the well known context of post-stroke epilepsy. Methods: This is a case report of a healthy 67-year-old male who had a new onset epileptic seizure prior to a lobar intracerebral hemorrhage (ICH). This man began to suffer myoclonic jerks in his left arm which progressed to a generalized tonic–clonic seizure. At the emergency area the physical and neurological examination were unremarkable and a CT scan was normal. The next day the patient developed left hemiparesis, hemianopsia and confusion and a new CT scan showed right parietal–occipital ICH. Conclusions: This case report exemplifies the concept of heraldic seizures, showing a patient who had a focal seizure preceding an intracerebral hemorrhage. Our etiologic diagnostic work led us to a diagnosis of probable amyloid angiopathy. We suggest that cerebral amyloid angiopathy (CAA) may be the underlying cause, since it may be the origin of both the late event (ICH) and the heralding seizures, resulting from concurrent ischemia