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In 2002 the United States of America started the process to implement a Free Trade Agreement with Central America and Dominican Republic (CAFTA). From the beginning CAFTA met the resistance of the different expressions of the social movement in Costa Rica, since it steered away from the historical conquests that conformed the socially conscious state. Given the heavy polarization of opinions around the agreement a national referendum was called upon. This article focuses on the way the women's movement strategically used digital technologies during the struggle and the role these technologies enabled women to have within the social movement as a whole. ; En el 2002 Estados Unidos comenzó el proceso para implementar un Tratado de Libre Comercio con América Central y República Dominicana (TLC). Desde el principio, el TLC se topó con la resistencia de las diferentes expresiones del movimiento social en Costa Rica, dado que se alejaba de las conquistas históricas del Estado social de Derecho. Dada la gran polarización de opiniones en torno al tratado, se convocó a un referendo nacional. Este artículo se enfoca en la forma en que el movimiento de mujeres utilizó estratégicamente las tecnologías digitales durante la lucha y el papel que estas tecnologías permitieron tener a las mujeres en el movimiento social amplio.

The bacteriophage φ29 replication protein p1 self-interacts in vitro, generating highly ordered structures. Specifically, the 53-amino acid protein p1ΔN33, which retains the sequence of p1 spanning amino acids Met34 to Lys85, assembles into two-dimensional protofilament sheets. The region of protein p1 located between residues Glu38 and Asn65 presumably forms an α-helical coiled-coil structure. Here we have examined the role of this coiled-coil sequence in the formation of protofilament sheets. Using sedimentation assays and negative-stain electron microscopy analysis, we demonstrate that residues Leu46, Met53, and Leu60, but not Leu39, are essential for p1ΔN33 assembly into sheets. Remarkably, replacement of Leu46 by Val shifts the pathway of molecular assembly, leading to the formation of filamentous polymers ∼10 nm in diameter. These results show, for the first time, that a short coiled-coil motif can mediate protein assembly into protofilament sheet structures. ; This work was supported by National Institutes of Health Grant 2R01 GM27242-21, Dirección General de Investigación Cientı́fica y Técnica Grant PB98-0645, and by the European Union Grants BIO4-CT98-0250 and ERBFMX-CT97-0125. The institutional help of Fundación Ramón Areces is acknowledged.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ; Peer reviewed

DNA replication of ϕ29 and related phages takes place via a strand displacement mechanism, a process that generates large amounts of single-stranded DNA (ssDNA). Consequently, phage-encoded ssDNA-binding proteins (SSBs) are essential proteins during phage ϕ29-like DNA replication. In the present work we analyze the helix-destabilizing activity of the SSBs of ϕ29 and the related phages Nf and GA-1, their ability to eliminate non-productive binding of ϕ29 DNA polymerase to ssDNA and their stimulatory effect on replication by ϕ29 DNA polymerase in primed M13 ssDNA replication, a situation that resembles type II replicative intermediates that occur during ϕ29-like DNA replication. Significant differences have been appreciated in the functional behavior of the three SSBs. First, the GA-1 SSB is able to display helix-destabilizing activity and to stimulate dNTP incorporation by ϕ29 DNA polymerase in the M13 DNA replication assay, even at SSB concentrations at which the ϕ29 and Nf SSBs do not show any effect. On the other hand, the ϕ29 SSB is the only one of the three SSBs able to increase the replication rate of ϕ29 DNA polymerase in primed M13 ssDNA replication. From the fact that the ϕ29 SSB, but not the Nf SSB, stimulates the replication rate of Nf DNA polymerase we conclude that the different behaviors of the SSBs on stimulation of the replication rate of ϕ29 and Nf DNA polymerases is most likely due to formation of different nucleoprotein complexes of the SSBs with the ssDNA rather than to a specific interaction between the SSB and the corresponding DNA polymerase. A model that correlates the thermodynamic parameters that define SSB–ssDNA nucleoprotein complex formation with the functional stimulatory effect of the SSB on ϕ29-like DNA replication has been proposed. ; This investigation was aided by research grant 2RO1 GM27242-20 from the National Institutes of Health, by grant PB93-0173 from the Dirección General de Investigación Científica y Técnica, by grant ERBFMRX CT970115 from the European Union and by an institutional grant from the Fundación Ramón Areces. I.G. was holder of a pre-doctoral fellowship from the Fundación Ramón Areces. ; Peer reviewed

Phage phi 29 encodes a DNA-dependent DNA polymerase belonging to the eukaryotic-type (family B) subgroup of DNA polymerases that use a protein as the primer for initiation of DNA synthesis. In one of the most important motifs present in the 3'5' exonucleolytic domain of proofreading DNA polymerases, the ExoII motif, phi 29 DNA polymerase contains three amino acid residues, Y59, H61 and F69, which are highly conserved among most proofreading DNA polymerases. These residues have recently been shown to be involved in proper stabilization of the primer terminus at the 3'5' exonuclease active site. Here we investigate by means of site-directed mutagenesis the role of these three residues in reactions that are specific for DNA polymerases utilizing a protein-primed DNA replication mechanism. Mutations introduced at residues Y59, H61 and F69 severely affected the protein-primed replication capacity of phi 29 DNA polymerase. For four of the mutants, namely Y59L, H61L, H61R and F69S, interaction with the terminal protein was affected, leading to few initiation and transition products. These findings, together with the specific conservation of Y59, H61 and F69 among DNA polymerases belonging to the protein-primed subgroup, strongly suggest a functional role of these amino acid residues in the DNA polymerase–terminal protein interaction ; This investigation was aided by research grant 5R01 GM27242-22 from the National Institutes of Health, by grant PB98-0645 from the Dirección General de Investigacíon Científica y Técnica, by grant ERBFMX CT97 0125 from the European Union and by an institutional grant from Fundación Ramón Areces. R.E. was a post-doctoral fellow of the European Union ; Peer reviewed

Alignment of the protein sequence of DNA-dependent DNA polymerases has allowed the definition of a new motif, lying adjacent to motif B in the direction of the N-terminus and therefore named pre-motif B. Both motifs are located in the fingers subdomain, shown to rotate towards the active site to form a dNTP-binding pocket in several DNA polymerases in which a closed ternary complex pol:DNA:dNTP has been solved. The functional significance of pre-motif B has been studied by site-directed mutagenesis of phi 29 DNA polymerase. The affinity for nucleotides of phi 29 DNA polymerase mutant residues Ile364 and Lys371 was strongly affected in DNA- and terminal protein-primed reactions. Additionally, mutations in Ile364 affected the DNA-binding capacity of phi 29 DNA polymerase. The results suggest that Lys371 of phi 29 DNA polymerase, highly conserved among families A and B, interacts with the phosphate groups of the incoming nucleotide. On the other hand, the role of residue Ile364 seems to be structural, being important for both DNA and dNTP binding. Pre-motif B must therefore play an important role in binding the incoming nucleotide. Interestingly, the roles of Lys371 and Ile364 were also shown to be important in reactions without template, suggesting that phi 29 DNA polymerase can achieve the closed conformation in the absence of a DNA template ; This investigation has been aided by research grant 2R01 GM27242-22 from the National Institutes of Health, by grant PB98-0645 from the Dirección General de Investigación Científica y Técnica, by grant ERBFMRX CT97 0125 from the European Union and by an institutional grant from Fundación Ramón Areces. V.T. was a post-doctoral fellow of the Comunidad Autónoma de Madrid. ; Peer reviewed

8 pags, 7 figs, 2 tab ; The single-stranded DNA-binding protein (SSB) of Bacillus subtilis phage φ29 is absolutely required for viral DNA replication in vivo. About ~95% of the intrinsic tyrosine fluorescence of φ29 SSB is quenched upon binding to ssDNA, making tyrosine residues strong candidates to be directly involved in complex formation with ssDNA. Thus, we have studied the spectroscopic properties of the φ29 SSB tyrosines (Tyr-50, Tyr-57, and Tyr-76) using steady-state and time-resolved fluorescence measurements. φ29 SSB tyrosines do not seem to be highly restricted by strong interactions with neighbor residues, as suggested by (i) the high value of the average quantum yield of the φ29 SSB fluorescence emission (φ(F) = 0.067 ± 0.010), (ii) the fast motions of the tyrosine side chains (φ(short) = 0.14 ± 0.06 ns), and (iii) the lack of tyrosinate emission at neutral pH. Stern-Volmer analysis of the quenching by acrylamide and I- indicates that φ29 SSB tyrosines are surrounded by a negatively charged environment and located in a relatively exposed protein domain, accessible to the solvent and, likely, to ssDNA. Changes in the intrinsic fluorescence upon ssDNA binding allowed us to determine that temperature has an opposite effect on the thermodynamic parameters K (intrinsic binding constant) and ω (cooperativity) defining φ29 SSB-poly(dT) interaction, the effective DNA binding constant, K(eff) = Kω, being largely independent of temperature. Altogether, the fluorescent properties of φ29 SSB tyrosines are consistent with a direct participation in complex formation with ssDNA. ; This work was supported in part by Grant 5RO1 GM 27242-16 from the National Institutes of Health, CHRX-CT93-0248 from the European Union, and PB93-0176 from DGICYT (to M. S.), and PB93-126 from DIGYCT and Acción Integrada Hispano-Francesa HF 94-183 (to A. U. A.). An institutional grant from Fundación Ramón Areces to Centro de Biología Molecular "Severo Ochoa" is also acknowledged. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Family B DNA polymerases (PolBs) play a central role during replication of viral and cellular chromosomes. Here, we report the discovery of a third major group of PolBs, which we denote primer-independent PolB (piPolB), that might be a link between the previously known protein-primed and RNA/DNA-primed PolBs. PiPolBs are encoded by highly diverse mobile genetic elements, pipolins, integrated in the genomes of diverse bacteria and also present as circular plasmids in mitochondria. Biochemical characterization showed that piPolB displays efficient DNA polymerization activity that can use undamaged and damaged templates and is endowed with proofreading and strand displacement capacities. Remarkably, the protein is also capable of template-dependent de novo DNA synthesis, i.e., DNApriming activity, thereby breaking the long-standing dogma that replicative DNA polymerases require a pre-existing primer for DNA synthesis. We suggest that piPolBs are involved in self-replication of pipolins and may also contribute to bacterial DNA damage tolerance ; Spanish Ministry of Economy and Competitiveness (BFU2014-52656P). M.R.-R. was supported by a ComFuturo Grant (NewPols4Biotech) from Fundacio´ n General CSIC. C.D.O. was holder of a ''Plan de Empleo Juvenil'' contract from Madrid Regional Government (funded by YEI program from European Social Fund, EC). An institutional grant from Fundación Ramón Areces to the Centro de Biología Molecular Severo Ochoa ; Peer Reviewed

Family B DNA polymerases (PolBs) play a central role during replication of viral and cellular chromosomes. Here, we report the discovery of a third major group of PolBs, which we denote primer-independent PolB (piPolB), that might be a link between the previously known protein-primed and RNA/DNA-primed PolBs. PiPolBs are encoded by highly diverse mobile genetic elements, pipolins, integrated in the genomes of diverse bacteria and also present as circular plasmids in mitochondria. Biochemical characterization showed that piPolB displays efficient DNA polymerization activity that can use undamaged and damaged templates and is endowed with proofreading and strand displacement capacities. Remarkably, the protein is also capable of template-dependent de novo DNA synthesis, i.e., DNA-priming activity, thereby breaking the long-standing dogma that replicative DNA polymerases require a pre-existing primer for DNA synthesis. We suggest that piPolBs are involved in self-replication of pipolins and may also contribute to bacterial DNA damage tolerance. Redrejo-Rodríguez et al. report and characterize a DNA polymerase group (piPolB) from the B family that can perform primer-independent DNA replication. PiPolBs are encoded by Pipolins, diverse self-replicating genetic elements that are widespread among bacterial phyla and in mitochondria. ; M.S.'s lab is funded by the Spanish Ministry of Economy and Competitiveness (BFU2014-52656P). M.R.-R. was supported by a ComFuturo Grant (NewPols4Biotech) from Fundación General CSIC. C.D.O. was holder of a ''Plan de Empleo Juvenil'' contract from Madrid Regional Government (funded by YEI program from European Social Fund, EC). An institutional grant from Fundación Ramón Areces to the Centro de Biología Molecular Severo Ochoa is also acknowledged. P.F. was funded by the European Research Council under the European Union's Seventh Framework Program (FP/2007-2013)/ Project EVOMOBIL - ERC Grant Agreement 340440.