Shared Active Site Architecture between the Large Subunit of Eukaryotic Primase and DNA Photolyase

DNA synthesis during replication relies on RNA primers synthesised by the primase, a specialised DNA-dependent RNA polymerase that can initiate nucleic acid synthesis de novo. In archaeal and eukaryotic organisms, the primase is a heterodimeric enzyme resulting from the constitutive association of a small (PriS) and large (PriL) subunit. The ability of the primase to initiate synthesis of an RNA primer depends on a conserved Fe-S domain at the C-terminus of PriL (PriL-CTD). However, the critical role of the PriL-CTD in the catalytic mechanism of initiation is not understood.Here we report the crystal structure of the yeast PriL-CTD at 1.55 A resolution. The structure reveals that the PriL-CTD folds in two largely independent alpha-helical domains joined at their interface by a [4Fe-4S] cluster. The larger N-terminal domain represents the most conserved portion of the PriL-CTD, whereas the smaller C-terminal domain is largely absent in archaeal PriL. Unexpectedly, the N-terminal domain reveals a striking structural similarity with the active site region of the DNA photolyase/cryptochrome family of flavoproteins. The region of similarity includes PriL-CTD residues that are known to be essential for initiation of RNA primer synthesis by the primase.Our study reports the first crystallographic model of the conserved Fe-S domain of the archaeal/eukaryotic primase. The structural comparison with a cryptochrome protein bound to flavin adenine dinucleotide and single-stranded DNA provides important insight into the mechanism of RNA primer synthesis by the primase

Flexible tethering of primase and DNA Pol α in the eukaryotic primosome

The Pol α/primase complex or primosome is the primase/polymerase complex that initiates nucleic acid synthesis during eukaryotic replication. Within the primosome, the primase synthesizes short RNA primers that undergo limited extension by Pol α. The resulting RNA-DNA primers are utilized by Pol δ and Pol ε for processive elongation on the lagging and leading strands, respectively. Despite its importance, the mechanism of RNA-DNA primer synthesis remains poorly understood. Here, we describe a structural model of the yeast primosome based on electron microscopy and functional studies. The 3D architecture of the primosome reveals an asymmetric, dumbbell-shaped particle. The catalytic centers of primase and Pol α reside in separate lobes of high relative mobility. The flexible tethering of the primosome lobes increases the efficiency of primer transfer between primase and Pol α. The physical organization of the primosome suggests that a concerted mechanism of primer hand-off between primase and Pol α would involve coordinated movements of the primosome lobes. The first three-dimensional map of the eukaryotic primosome at 25 Å resolution provides an essential structural template for understanding initiation of eukaryotic replication.Spanish Ministry of Science and Innovation (SAF2008-00451 to O.L.); the ‘Red Temática de Investigación Cooperativa en Cáncer (RTICC)’ from the ‘Instituto de Salud Carlos III’ (RD06/0020/1001 to O.L.); the Human Frontiers Science Program (RGP39/2008 to O.L. and E.N.); a Wellcome Trust Senior Fellowship award in Basic Biomedical Sciences (to L.P.); a FPI fellowship from the Spanish Ministry of Science and Innovation to MAR-C; a JAE-DOC contract of the ‘Consejo Superior de Investigaciones Científicas (CSIC)’ and a ‘Juan de la Cierva’ contract from the Spanish Ministry of Science to BGA; EN is a Howard Hughes Medical Institute Investigator. Funding for open access charge: Spanish Ministry of Science and Innovation (SAF2008-00451 to O.L.)

The Emergence of Emotions

Emotion is conscious experience. It is the affective aspect of consciousness. Emotion arises from sensory stimulation and is typically accompanied by physiological and behavioral changes in the body. Hence an emotion is a complex reaction pattern consisting of three components: a physiological component, a behavioral component, and an experiential (conscious) component. The reactions making up an emotion determine what the emotion will be recognized as. Three processes are involved in generating an emotion: (1) identification of the emotional significance of a sensory stimulus, (2) production of an affective state (emotion), and (3) regulation of the affective state. Two opposing systems in the brain (the reward and punishment systems) establish an affective value or valence (stimulus-reinforcement association) for sensory stimulation. This is process (1), the first step in the generation of an emotion. Development of stimulus-reinforcement associations (affective valence) serves as the basis for emotion expression (process 2), conditioned emotion learning acquisition and expression, memory consolidation, reinforcement-expectations, decision-making, coping responses, and social behavior. The amygdala is critical for the representation of stimulus-reinforcement associations (both reward and punishment-based) for these functions. Three distinct and separate architectural and functional areas of the prefrontal cortex (dorsolateral prefrontal cortex, orbitofrontal cortex, anterior cingulate cortex) are involved in the regulation of emotion (process 3). The regulation of emotion by the prefrontal cortex consists of a positive feedback interaction between the prefrontal cortex and the inferior parietal cortex resulting in the nonlinear emergence of emotion. This positive feedback and nonlinear emergence represents a type of working memory (focal attention) by which perception is reorganized and rerepresented, becoming explicit, functional, and conscious. The explicit emotion states arising may be involved in the production of voluntary new or novel intentional (adaptive) behavior, especially social behavior

Copolymères fluorés à base de fluorure de vinylidène porteur de groupements acide sulfonique ou acide phosphoniques pour membranes de piles à combustible

The goal of this PhD concerns the synthesis and the electro-chemical characterization of a new generation of membranes obtained from perfluorinated polymers incorporating vinylidene fluoride (VDF) and functionnalized by sulfonic acids termonomers, as electrolyte for Proton Exchange Membranes for Fuel Cells (PEMFC). Two processes were investigated to obtain original macromolecular architecture. The first way concerns the preparation of membranes from ionomers obtained by direct radical copolymerization of perfluoro(4-methyl-3,6-dioxaoct-7-ene) sulfonyl fluoride (PFSVE) with VDF, hexafluoropropene (HFP), chlorotrifluoroethylene (CTFE) or 8-bromo-1H,1H,2H-perfluorooct-1-ène (BDFO). The Ionic Exchange Capacity (IEC) and the proton conductivity () were assessed. The second way of PEMFC synthesis deals with the chemical grafting of styrene by atom transfer polymerization (ATRP) starting from poly(VDF-co-BDFO) copolymersL'objectif de cette étude consiste à synthétiser et à évaluer les performances électrochimiques d'une nouvelle génération d'électrolyte pour piles à combustible à membrane échangeuse de protons (PEMFC), à partir de copolymères fluorés incorporant du fluorure de vinylidène (VDF) et des comonomères fonctionnalisés par des acides sulfoniques. Dans cette optique, deux stratégies de synthèse pour l'obtention d'architectures macromoléculaires originales ont été réalisées. La première, s'appuie sur la co- et terpolymérisation radicalaire directe d'un monomère fluoré aliphatique fonctionnalisé fluorure de sulfonyle avec des oléfines fluorées (VDF, hexafluoropropène (HFP), chlorotrifluoroethylène (CTFE), bromotrifluoroethylène (BrTFE) et 8-bromo-1H,1H,2H-perfluorooct-1-ène (BDFO)) conduisant à des copolymères statistiques. La seconde est basée sur la modification chimique de copolymères à base de VDF et BDFO conduisant à l'obtention de copolymères fluorés greffés PVDF-g-PS et/ou réticulés

Fluorinated, Crosslinkable Terpolymers Based on Vinylidene Fluoride and Bearing Sulfonic Acid Side Groups for Fuel-Cell Membranes

International audienceThe radical terpolymerization of 8-bromo-1H,1H,2H-perfluorooct-1-ene with vinylidene fluoride (VDF) and perfluoro(4-methyl-3,6-dioxaoct-7-ene) sulfonyl fluoride is presented. Changing the feed compositions of these three fluorinated comonomers enabled us to obtain different random-type poly[vinylidene fluoride-ter-perfluoro(4- methyl-3,6-dioxaoct-7-ene) sulfonyl fluoride-ter-8-bromo-1H,1H,2H-perfluorooct-1-ene] terpolymers containing various sulfonyl fluoride and brominated side groups. Yields higher than 70% were reached in all cases. The hydrolysis of the sulfonyl fluoride group into the SO3Li function in the presence of lithium carbonate was quantitatively achieved without the content of VDF being affected, and so dehydrofluorination of the VDF base unit was avoided. These original terpolymers were then crosslinked via dangling bromine atoms in the presence of a peroxide/triallyl isocyanurate system, which produced films insoluble in organic solvents such as acetone and dimethylformamide (which totally dissolved uncured terpolymers). The acidification of SO3Li into the SO3H function enabled protonic membranes to be obtained. The thermal stabilities of the crosslinked materials were higher than those of the uncured terpolymers, and their electrochemical performances were investigated. According to the contents of the sulfonic acid side functions, the ion-exchange capacities ranged from 0.6 to 1.5 mequiv of Hþ/g, whereas the water uptake and conductivities ranged from 5–26% (611%) and from 0.5 to 6.0 mS/cm, respectively

Fluorinated Copolymers and Terpolymers Based on Vinylidene Fluoride and Bearing Sulfonic Acid Side-Group

International audienceThe radical co- and terpolymerization of perfluoro(4-methyl-3,6-dioxaoct- 7-ene) sulfonyl fluoride (PFSVE) with 1,1-difluoroethylene (or vinylidene fluoride, VDF or VF2), hexafluoropropene (HFP), chlorotrifluoroethylene (CTFE), and bromotrifluoroethylene (BrTFE) is presented. Although PFSVE could not homopolymerize under radical initiation, it could be copolymerized in solution under a radical initiator with VDF, while its copolymerizations with HFP or CTFE led to oligomers in low yields. The terpolymerizations of PFSVE with VDF and HFP, with VDF and CTFE, or with VDF and BrTFE also led to original fluorinated terpolymers bearing sulfonyl fluoride side-groups. The conditions of co- and terpolymerization were optimized in terms of the nature and the amount of the radical initiators, of the nature of solvents (fluorinated or nonhalogenated), and of the initial amounts of fluorinated comonomers. The different mol % contents of comonomers in the co- and terpolymers were assessed by 19F NMR spectroscopy. A wide range of co- and terpolymers containing mol % of PFSVE functional monomer ranging from 10 to 70% was produced. The kinetics of copolymerization of VDF with PFSVE enabled to assess the reactivity ratios of both comonomers: rVDF ¼ 0.57 6 0.15 and rPFSVE ¼ 0.07 6 0.04 at 120 8C. The thermal and physicochemical properties were also studied. Moreover, the glass transition temperatures (Tgs) of poly(VDF-co-PFSVE) copolymers containing different amounts of VDF and PFSVE were determined and the theoretical Tg of poly(PFSVE) homopolymer was deduced. Then, the hydrolysis of the SO2F into SO3H function was investigated and enabled the synthesis of fluorinated copolymers bearing sulfonic acid functions

Synthesis and Characterization of Poly(vinylidene fluoride)-g-poly(styrene) Graft Polymers Obtained by Atom Transfer Radical Polymerization of Styrene

International audiencePoly(vinylidene fluoride)-g-poly(styrene) graft copolymers (PVDF-g-PS) were synthesized by the “grafting from” method from a PVDF macroinitiator bearing bromine side groups. This fluorinated macroinitiator was obtained from the radical copolymerization of VDF with 8-bromo-1H,1H,2H-perfluorooct-1-ene (BDFO), and then it was used in the atom transfer radical polymerization (ATRP) of styrene initiated by CuIBr/1,1,4,7,- 10,10-hexamethyltriethylenetetramine (HMTETA) catalyst. First, the synthesis of a model poly(styrene) was investigated starting from 1-bromoperfluorooctane (C8F17Br) as the initiator to check the reactivity of -CF2-Br in ATRP process. Successful ATRP of styrene in the presence of 1-bromoperfluorooctane was observed from a kinetic study and NMR spectroscopy, and the activation rate constant of this initiator (kact ) 35 10-3 M-1 s-1 at 35 °C in acetonitrile) was assessed for the first time. In a second part, ATRP of styrene was also studied from poly(VDF-co-BDFO) copolymers as the macroinitiators, taking into account: (i) the effect of the polymerization temperature, (ii) the ligand concentration in the ATRP catalyst, and (iii) the amount of solvent vs the conversion of poly(styrene). The formation of graft copolymers was confirmed by size exclusion chromatography and by 1H and 19F NMR spectroscopies. Interestingly, the linear dependences of both the evolutions of ln([M]0/[M]) vs time and of the molecular weights of the resulting graft copolymers vs the styrene conversions, and the decrease of their dispersity indexes vs the styrene conversions evidenced the controlled behavior of that graft polymerization