The signature motif of the Saccharomyces cerevisiae Pif1 DNA helicase is essential in vivo for mitochondrial and nuclear functions and in vitro for ATPase activity

Pif1 family DNA helicases are conserved from bacteria to humans and have critical and diverse functions in vivo that promote genome integrity. Pif1 family helicases share a 23 amino acid region, called the Pif1 signature motif (SM) that is unique to this family. To determine the importance of the SM, we did mutational and functional analysis of the SM from the Saccharomyces cerevisiae Pif1 (ScPif1). The mutations deleted portions of the SM, made one or multiple single amino acid changes in the SM, replaced the SM with its counterpart from a bacterial Pif1 family helicase and substituted an α-helical domain from another helicase for the part of the SM that forms an α helix. Mutants were tested for maintenance of mitochondrial DNA, inhibition of telomerase at telomeres and double strand breaks, and promotion of Okazaki fragment maturation. Although certain single amino acid changes in the SM can be tolerated, the presence and sequence of the ScPif1 SM were essential for all tested in vivo functions. Consistent with the in vivo analyses, in vitro studies showed that the presence and sequence of the ScPif1 SM were critical for ATPase activity but not substrate binding

Method for size optimisation of large wind\u2013hydrogen systems with high penetration on power grids

Wind power generation is growing rapidly in many locations around the world. Power systems are able to absorb large amounts of wind capacity, but operational problems arise when the wind power penetration becomes high. Such factors as voltage dips, frequency variations, low power system stability, low reactive power and power flow imbalances reduce the economic value and represent a barrier to the unlimited development of wind energy. Hydrogen production from wind power that is not matched with hourly electricity demand appears to be an attractive storage option capable of providing a balancing service to the electricity generators and suppliers for mitigation of the negative impacts due to the random nature of wind. Because of its multi-functionality, hydrogen can be used directly as a fuel, mixed with methane, or transmitted through pipelines to the users. The aim of this paper is to produce useful suggestions for the planning, development and sizing of wind\u2013hydrogen systems by taking into account the local and regional resources, demands, constraints and opportunities. This study considers both the economic and technological variables and describes an optimisation method (OM) for analysing power systems in which part of the electricity generated by a grid-connected wind plant is used to produce hydrogen by electrolysis. An example application of this OM has been developed for a specific geographical area located in central Sicily. Our results identify the potential and the limitations connected to cases that use excess wind power to produce hydrogen for civil applications

Procedimiento conciliatorio en Colombia

La conciliación es uno de los mecanismos alternativos de solución de conflictos más importantes y desarrollados en Colombia. Pese a que las normas legales que rigen la materia son las mismas, en la práctica parece que los conciliadores y centros de conciliación aplican el procedimiento de manera diferente. El presente texto tiene como objetivo poner a disposición de las personas interesadas en la conciliación una descripción de las etapas que integran el procedimiento conciliatorio. El análisis jurídico del procedimiento empieza con los requisitos de la solicitud de conciliación y termina con el seguimiento que se debe hacer al resultado del servicio ofrecido. Para el desarrollo de la presente obra, se integra la legislación, la jurisprudencia y los conceptos de línea institucional del Ministerio del Interior y de Justicia con ejemplos sencillos que permiten un mejor entendimiento de los conceptos que se quieren dar a conocer

Rrm3 and Pif1 division of labor during replication through leading and lagging strand G-quadruplex

Members of the conserved Pif1 family of 5\u27-3\u27 DNA helicases can unwind G4s and mitigate their negative impact on genome stability. In Saccharomyces cerevisiae, two Pif1 family members, Pif1 and Rrm3, contribute to the suppression of genomic instability at diverse regions including telomeres, centromeres and tRNA genes. While Pif1 can resolve lagging strand G4s in vivo, little is known regarding Rrm3 function at G4s and its cooperation with Pif1 for G4 replication. Here, we monitored replication through G4 sequences in real time to show that Rrm3 is essential for efficient replisome progression through G4s located on the leading strand template, but not on the lagging strand. We found that Rrm3 importance for replication through G4s is dependent on its catalytic activity and its N-terminal unstructured region. Overall, we show that Rrm3 and Pif1 exhibit a division of labor that enables robust replication fork progression through leading and lagging strand G4s, respectively

The mitochondrial single-stranded DNA binding protein from S. cerevisiae, Rim1, does not form stable homo-tetramers and binds DNA as a dimer of dimers

Rim1 is the mitochondrial single-stranded DNA binding protein in Saccharomyces cerevisiae and functions to coordinate replication and maintenance of mtDNA. Rim1 can form homo-tetramers in solution and this species has been assumed to be solely responsible for ssDNA binding. We solved structures of tetrameric Rim1 in two crystals forms which differ in the relative orientation of the dimers within the tetramer. In testing whether the different arrangement of the dimers was due to formation of unstable tetramers, we discovered that while Rim1 forms tetramers at high protein concentration, it dissociates into a smaller oligomeric species at low protein concentrations. A single point mutation at the dimer–dimer interface generates stable dimers and provides support for a dimer–tetramer oligomerization model. The presence of Rim1 dimers in solution becomes evident in DNA binding studies using short ssDNA substrates. However, binding of the first Rim1 dimer is followed by binding of a second dimer, whose affinity depends on the length of the ssDNA. We propose a model where binding of DNA to a dimer of Rim1 induces tetramerization, modulated by the ability of the second dimer to interact with ssDNA

Pif1 removes a Rap1-dependent barrier to the strand displacement activity of DNA polymerase δ

Using an in vitro reconstituted system in this work we provide direct evidence that the yeast repressor/activator protein 1 (Rap1), tightly bound to its consensus site, forms a strong non-polar barrier for the strand displacement activity of DNA polymerase δ. We propose that relief of inhibition may be mediated by the activity of an accessory helicase. To this end, we show that Pif1, a 5′–3′ helicase, not only stimulates the strand displacement activity of Pol δ but it also allows efficient replication through the block, by removing bound Rap1 in front of the polymerase. This stimulatory activity of Pif1 is not limited to the displacement of a single Rap1 molecule; Pif1 also allows Pol δ to carry out DNA synthesis across an array of bound Rap1 molecules that mimics a telomeric DNA-protein assembly. This activity of Pif1 represents a novel function of this helicase during DNA replication

Second harmonic generation response by gold nanoparticles at the polarized water/2-octanone interface: from dispersed to aggregated particles

Gold nanoparticles with a diameter of approximately 20 nm have been observed at the polarized water/2-octanone interface by the nonlinear optical technique of second harmonic generation. Electric field induced adsorption of the gold particles at this liquid/liquid interface is clearly observed and confirms that these are negatively charged. The process is quasi-reversible at high potential sweep rates, but aggregation at the interface is observed at slower sweep rates through the loss of the nonlinear optical signal. The time evolution of the second harmonic signal is also reported during potential step experiments. After a rapid increase due to adsorption, a continuous decrease in the nonlinear optical signal intensity is observed due to aggregation of the particles into large islands at the interface. Diffusion of these large islands at the interface was observed for a longer timescale through large signal fluctuations