Computational insight into the broad substrate specificity of enzymes that process nucleic acids

Many enzymes that bind DNA and RNA possess broad substrate specificity and play diverse roles in biology. Three classes of enzymes with broad substrate specificity are nucleoside hydrolases that salvage nucleic acid building blocks, and alkyladenine DNA glycosylases (AAG) and AlkB enzymes, which repair alkylated and/or deaminated DNA damage. This thesis uses advanced computational techniques to examine how enzymes process structurally diverse substrates. Specifically, structural and energetic information is provided by molecular dynamics (MD) simulations, quantum mechanics (QM) and hybrid quantum mechanics/molecular mechanics (QM/MM) calculations, which provide insight into how each enzyme active site changes to accommodate unique substrates and quantify the impact that these changes have on catalyzed reactions. From these results, atomistic explanations for the activity of these enzymes is obtained, which can be used to develop new treatments for diseases. The computational approach presented can be applied to other enzymes that exhibit broad substrate specificity

Effect of Naturally Occurring DNA Modifications on DNA Structure and Packaging

In eukaryotes, the genomic double-stranded DNA (dsDNA) coils around histones to form nucleosomes. Arrays of these nucleosomes bundle together to generate chromatin. Most DNA-related processes require interactions between chromatin-protected DNA and cellular machinery. Access of cell machinery to genomic DNA is partially regulated by the position and stability of nucleosomes, which may be influenced by changes in nucleosomal DNA. DNA is composed of adenine (A), guanine (G), cytosine (C), thymine (T) nucleotides and their derivatives. It has been shown that some C derivatives participate in directing multiple biological processes, and aberrant modification patterns are often linked to diseases. It has been proposed that T derivatives exhibit similar effects. This thesis focuses on elucidating the effect of naturally occurring DNA modifications on the properties of dsDNA and nucleosomes. dsDNA sequences systematically modified with various T derivatives were characterized using classical biophysical techniques to assess the effect of these DNA modifications. The results indicate that in the sequence context studied, 5-hydroxymethyluracil modifications destabilize dsDNA, while dense symmetrical 5-formyluracil (fU) modifications alter the dsDNA structure. These effects may provide clues to the differential protein recruitment observed in previous research. In vitro studies on nucleosome occupancy and stability revealed that 5-formylcytosine (fC) modifications have positive effects on nucleosome formation and stability compared to the unmodified counterpart by influencing the intrinsic biochemical and biophysical properties of the nucleosomes. These results provide casual links for the observation in vivo between fC and the increased nucleosome occupancy and positioning. In order to further understand the positional effect of fC on the nucleosomes, a method was developed for quick and reliable incorporation of C derivatives into dsDNA at desired positions. The positive effect of fC modifications on nucleosome occupancy and stability observed here has necessitated further studies to gain deeper insights into the biological functions of fC in the nucleosome context. Cryo-EM can be used to elucidate the structural foundation for the changes fC posts to nucleosome, and protein interacting assays will identify the cellular machineries specifically recruited/repulsed by fC-modified nucleosomes. The effect of DNA modifications elucidated by the above studies advances our understanding on the role that DNA modifications play in regulating cellular processes.Chemicals were covered by Wellcome Trust (grant no. 099232/z/12/z) and the core funding from Cancer Research UK (C14303/A17197). Z.Li was also supported by a studentship from A*STAR (Singapore)

Caracterización molecular y funcional del gen AtMBD4L, codificante de una nueva DNA glicosilasa de Arabidopsis Thaliana

Tesis (Doctora en Ciencias Químicas) - - Universidad Nacional de Córdoba. Facultad de Ciencias Químicas, 2014La DNA glicosilasa METHYL BINDING DOMAIN 4 (MBD4) al igual que el gen codificante para la misma están muy bien caracterizados en animales. Sin embargo, al presente no existen estudios sobre el gen que codifica para esta enzima en plantas, donde se desconoce su patrón de expresión, participación en procesos biológicos y de desarrollo y sus efectos sobre respuestas al estrés. En bacterias y animales MBD4 forma parte de uno de los sistemas de reparación del DNA donde actúa como DNA glicosilasa monofuncional. Estructuralmente MBD4 pertenece a la superfamilia HhH-GPD y actúa sobre diversos sustratos, constituyendo una enzima multifacética implicada en diversos procesos celulares. En este trabajo de Tesis doctoral se seleccionó y caracterizó un homólogo de MBD4 de humanos en Arabidopsis thaliana (Arabidopsis), llamada METHYL BINDING DOMAIN 4- LIKE (MBD4L). Los resultados contenidos en este trabajo indican que AtMBD4L presenta dos transcriptos alternativos, uno de ellos no descripto anteriormente. Ambos transcriptos codifican para dos proteínas nucleares que conservan intacto el dominio DNA glicosilasa y difieren en la región N-terminal. Se observó, además que el dominio de MBD4L presenta una estructura similar a la descripta para la superfamilia HhH-GPD DNA glicosilasa, a la cual también pertenece MBD4 de animales y está muy conservado desde bacterias hasta humanos, aunque no presenta proteínas homólogas en Arabidopsis. Para contribuir a la caracterización funcional del gen se estudió la participación de AtMBD4L en procesos de desarrollo y en respuestas al estrés biótico y abiótico. Se determinó que MBD4L participa en la decondensación de heterocromatina centromérica que sufre el genoma de Arabidopsis en infecciones con Pseudomonas syringaae pv. tomato DC3000 (P. syringae pv. tomato) y favorece el crecimiento del patógeno en la planta. Además, el análisis de fenotipos en plantas mutantes y silenciadas para AtMBD4L indicó que dicho gen participa en procesos del desarrollo vegetativo y reproductivo de Arabidopsis. Por otra parte, para abordar la participación de MBD4L en respuestas al estrés abiótico se utilizaron plantas mutantes y líneas transgénias sobre-expresantes, las cuales demostraron que MBD4L estimula la tolerancia al estrés oxidativo y salino en plantas adultas, pero no durante la germinación. Finalmente, durante este trabajo de Tesis doctoral se propusieron diferentes blancos para MBD4L, aunque por el momento se desconoce le mecanismo de acción. En condiciones de infección con el patógeno bacteriano, MBD4L podría actuar sobre las repeticiones y transposones centroméricos, permitiendo la activación de reguladores negativos de la defensa. Durante el desarrollo, MBD4L podría regular la expresión de FLC, determinando el tiempo de floración, posiblemente mediante mecanismos epigenéticos. En condiciones de estrés oxidativo y salino en plantas adultas, MBD4L podría participar en la reparación del genoma de Arabidopsis, removiendo mutaciones tales como U:G, T:G y 5-hmU.Fil: Nota, María Florencia. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas; Argentina.Fil: Alvarez, María Elena. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Química Biológica; Argentina.Fil: Alvarez, María Elena. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro de Investigaciones en Química Biológica de Córdoba; Argentina.Fil: Paraje, María Gabriela. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas, Físicas y Naturales. Escuela de Biología; Argentina.Fil: Paraje, María Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto Multidisciplinario de Biología Vegetal; Argentina.Fil: Genti de Raimondi, Susana Del Valle. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Bioquímica Clínica; Argentina.Fil: Genti de Raimondi, Susana Del Valle. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina.Fil: Argaraña, Carlos Enrique. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Química Biológica; Argentina.Fil: Argaraña, Carlos Enrique. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro de Investigaciones en Química Biológica de Córdoba; Argentina.Fil: Casati, Paula. Universidad Nacional de Rosario; Argentina.Fil: Casati, Paula. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro de Estudios Fotosintéticos y Bioquímicos; Argentina

Molecular simulations of conformational transitions in biomolecules using a novel computational tool

The function of biological macromolecules is inherently linked to their complex conformational behaviour. As a consequence, the corresponding potential energy landscape encompasses multiple minima. Some of the intermediate structures between the initial and final states can be characterized by experimental techniques. Computer simulations can explore the dynamics of individual states and bring these together to rationalize the overall process. A novel method based on atomistic structure-based potentials in combination with the empirical valence bond theory (EVB-SBP) has been developed and implemented in the Amber package. The method has been successfully applied to explore various biological processes. The first application of the EVB-SBP approach involves the study of base flipping in B-DNA. The use of simple structurebased potentials are shown to reproduce structural ensembles of stable states obtained by using more accurate force field simulations. Umbrella sampling in conjunction with the energy gap reaction coordinate enables the study of alternative molecular pathways efficiently. The main application of the method is the study of the switching mechanism in a short bistable RNA. Molecular pathways, which connect the two stable states, have been elucidated, with particular interest to the characterisation of the transition state ensemble. In addition, NMR experiments have been performed to support the theoretical findings. Finally, a recent study of large-scale conformational transitions in protein kinases shows the general applicability of the method to different biomolecules

Concepts, perspectives and implications of a hybrid system made of nucleic acids biopolymers and hydroxyapatite mineral

The origin of building blocks of life and how life thrived on Earth remains a topic of high interest for researchers of the Origin of Life. In this thesis, we deal with concepts, perspectives and implications of the system termed hydroxyolite, a combination of outstanding biopolymers (nucleic acids such as DNA and RNA) and an exceptional mineral (hydroxyapatite). First we study, based on Revilla et al. (2013) and Bertran et al. (2014), how hydroxyapatite forms crystals able to encapsulate DNA or RNA when nucleic acids are used as a nucleating template. Later, in Bertran et al., (2016), we reported the mechanism of how the encapsulated nucleic acid is released to the surroundings when environmental conditions change, for instance becoming more acidic. As a consequence, we postulated that DNA existing in cells can be encapsulated and protected by hydroxyapatite against environmental attacks (i.e. poisonous gases, gamma radiation or enzymatic degradation) until they change, making feasible the reintroduction of nucleic acids in the mainstream of life. We hypothesized about the implications of such a system in the early history of life when mass extinction events occurred on Earth (Turon et al., 2015). Moreover, we extended the hydroxyolite concept, borrowed from the materials chemistry, to other disciplines such as paleontology, biology, biotechnology and medicine by considering hydroxyolites as equivalents to non-viral vectors that can introduce and release DNA into a cell (transfection). Such nucleic acid triggers the expression of foreign proteins if released in the cytosol or might be recombined with cell genome when DNA is released in the target cell nucleus. In the second part of the thesis, we studied the hydroxyolite system from a complementary perspective. We speculate about the consequences of being hydroxyapatite the first actor and not the nucleic acid. We propose that hydroxyapatite might act as an inorganic mold if considered as a catalytic substrate that facilitates the synthesis of simple organic molecules as the building blocks of life. Thus, we identified a prebiotic scenario, a volcanic eruption under lightning, where a phenomenon known as dirty storm usually occurs under certain conditions. Hydroxyapatite is known in nature to be part of igneous rocks and volcanic ash in small but significant concentrations. We replicated in the laboratory such extreme conditions by developing a thermally and electrically stimulated polarization. A process performed at 1000 ºC and under a difference of potential of 300 kV·m-1, to obtain permanently polarized hydroxyapatite (Turon et al., 2016; PCT/EP2017/069437) that turned out to be an enhanced catalyst compared to hydroxyapatite able to fix nitrogen and carbon from a gas mixture of N2, CO2 and CH4 (Rivas et al., 2018). The catalyst, under UV light, converts them into amino acids (Glycine and D/L-Alanine) and small organic molecules by means of a new inorganic photosynthetic process. In this work, we develop an integrative prebiotic model that describes how simple molecules might be synthesized from mildly reducing atmospheres by combining previous models such as volcanos as giant reactors, minerals as catalysts and photochemical reactions in the atmosphere under prebiotic sun light. All of them under the framework of a prebiotic inorganic photosynthesis, a process that might be considered the corner stone of the rise of the building blocks of life.L’origen de les molècules que van donar lloc a la vida i com la vida va prosperar a la Terra segueix essent un tema del màxim interès pels investigadors de l’origen de la vida. En aquesta tesi, discutim conceptes, perspectives de futur i implicacions del sistema que hem anomenat hidroxiolita (hydroxyolite), una combinació de biopolímers amb característiques molt especials (àcids nucleics com l’ADN i l’ARN) i un mineral excepcional (hidroxiapatita). En primer lloc, en els treballs Revilla et al., (2013) i Bertran et al., (2014) estudiem com els cristalls d’hidroxiapatita tenen la capacitat d’encapsular ADN o ARN quan l’àcid nucleic es comporta com agent nucleant. Reportem com l’àcid nucleic prèviament encapsulat pot ser alliberat si les condicions ambientals canvien, per exemple tornant-se lleugerament més àcides (Bertran et al., 2016). Com a conseqüència, postulem que l’ADN existent a les cèl·lules pot ser encapsulat per la hidroxiapatita protegint-lo contra atacs de l’entorn (per exemple, la influència de gasos tòxics, la radiació gamma o la degradació enzimàtica) fins que les condicions externes canvien i els àcids nucleics poder ser reintroduïts en el torrent principal de la vida. Discutim les implicacions d’aquest sistema híbrid a la història primitiva de la vida a la Terra, quan van ocórrer les grans catàstrofes que van donar lloc a extincions massives d’éssers vius. Tanmateix, estenem el concepte d’hidroxiolita a altres disciplines com la paleontologia, la biologia cel·lular, la biotecnologia i la medicina, considerant les hidroxiolites com a vectors no virals que poden introduir i alliberar ADN dins una cèl·lula (transfecció). Aquest àcid nucleic, si s’allibera en el citosol pot desencadenar l’expressió de proteïnes codificades en l’ADN introduït, o si s’allibera en el nucli podria recombinar-se amb el propi ADN de la cèl·lula diana de manera transitòria o permanent. A la segona part de la tesi, estudiem el sistema hidroxiolita des d’una perspectiva complementària. Especulem sobre les conseqüències de ser la hidroxiapatita l’actor principal del sistema i no l’àcid nucleic. Proposem que la hidroxiapatita pot actuar com un motlle inorgànic si es comporta com a substrat catalític que facilita la síntesi de molècules orgàniques, com les molècules que van donar lloc a la vida. A partir d’aquest concepte hem identificat un escenari prebiòtic, una erupció volcànica acompanyada de descàrregues elèctriques, fenomen que succeeix amb certa freqüència en funció de les característiques de l’erupció. La hidroxiapatita a la natura és coneguda per formar part de la composició de roques ígnies i de la cendra volcànica en petites però significatives quantitats. Al laboratori hem replicat aquestes condicions extremes i hem desenvolupat un procés de polarització mitjançant estimulació elèctrica i tèrmica, aplicant 1000 ºC i una diferència de potencial de 300 kV·m-1, que dóna com a resultat hidroxiapatita polaritzada permanentment que converteix el mineral en un catalitzador extraordinari comparat amb la hidroxiapatita i que té la capacitat de fixar nitrogen i carboni a partir d’una mescla de gasos composada per N2, CO2 i CH4 en presència d’aigua. El catalitzador, sota il·luminació de llum UV facilita la conversió d’aquests gasos en aminoàcids (Glicina i D/L-Alanina) i en molècules orgàniques simples a través d’un procés fotosintètic inorgànic. En aquest treball, desenvolupem un model prebiòtic que descriu com molècules senzilles van poder ser sintetitzades a partir d’atmosferes suaument reductores combinant models prebiòtics previs (volcans que es comporten com grans reactors, reaccions fotoquímiques que succeeixen a l’atmosfera sota el sol prebiòtic i minerals que actuen com a catalitzadors) sota el marc de la fotosíntesi inorgànica prebiòtica, un procés que podria ser considerat la pedra angular de l’aparició de les molècules que van donar lloc a la vida.El origen de las moléculas que dieron lugar a la vida y como la vida prosperó en la Tierra sigue siendo un tema del máximo interés para los investigadores del Origen de la Vida. En esta tesis discutimos conceptos, perspectivas de futuro e implicaciones del sistema que hemos denominado hidroxiolita (hydroxyolite), una combinación de biopolímeros con características muy especiales (ácidos nucleicos tales como el ADN y el ARN) y un mineral excepcional (hidroxiapatita). En primer lugar, en nuestros trabajos Revilla et al. (2013) y Bertrán et al. (2014) estudiamos como los cristales de hidroxiapatita tienen la capacidad de encapsular ADN o ARN cuando el ácido nucleico se comporta como un agente nucleante. A continuación, reportamos como el ácido nucleico previamente encapsulado puede ser liberado cuando las condiciones ambientales cambian, por ejemplo, cuando se vuelven ligeramente más ácidas (Bertrán et al., 2016). A consecuencia, postulamos que el ADN existente en las células puede ser encapsulado por la hidroxiapatita protegiéndolo contra ataques del entorno (por ejemplo, la influencia de gases tóxicos, la radiación gamma o la degradación enzimática) hasta que cambian las condiciones externas y los ácidos nucleicos pueden ser reintroducidos de nuevo en el torrente principal de la vida. A continuación, discutimos las implicaciones de este sistema híbrido en la historia primitiva de la vida en la Tierra, cuando ocurrieron las grandes catástrofes que dieron lugar a extinciones masivas de seres vivos (Turon et al., 2015). Asimismo, extendemos el concepto hidroxiolita, acuñado en la ciencia de materiales, a otras disciplinas como la paleontología, la biología celular, la biotecnología y la medicina, considerando las hidroxiolitas como vectores no virales que pueden introducir y liberar ADN dentro de una célula (transfección). Este ácido nucleico, si es liberado en el citosol puede desencadenar la expresión de proteínas codificadas en el ADN introducido, o si se libera en el núcleo podría recombinarse con el propio ADN de la célula diana de forma transitoria o permanente. En la segunda parte de la tesis, estudiamos el sistema hidroxiolita desde una perspectiva complementaria. Especulamos sobre las consecuencias de ser la hidroxiolita el actor principal y no el ácido nucleico. Proponemos que la hidroxiolita puede actuar como un molde inorgánico si se comporta como un sustrato catalítico que facilita la síntesis de moléculas orgánicas, como las moléculas que dieron lugar a la vida. Hemos identificado un escenario prebiótico basado en una erupción volcánica con descargas eléctricas, fenómeno que ocurre con cierta frecuencia en función de las características de la erupción. La hidroxiapatita es conocida en la naturaleza por formar parte de la composición de rocas ígneas y ceniza volcánica en bajas pero significativas concentraciones. Hemos replicado en el laboratorio estas condiciones extremas y hemos desarrollado un proceso de polarización mediante estimulación térmica y eléctrica, aplicando 1000ºC y una diferencia de potencial de 300 kV·m-1, que da como resultado hidroxiapatita permanentemente polarizada (Turón et al, 2016; PCT/EP2017/069437). Este proceso convierte el mineral en un catalizador extraordinario comparado con la hidroxiapatita y tiene la capacidad de fijar nitrógeno y carbono a partir de una mezcla de gases compuesta por N2, CO2 y CH4 (Rivas et al., 2018) en presencia de agua. El catalizador, bajo iluminación de luz UV, facilita la conversión de estos gases en aminoácidos (Glicina y D/L-Alanina) y en ácidos orgánicos simples a través de un proceso de fotosíntesis inorgánica. En este trabajo desarrollamos un modelo prebiótico que describe como moléculas sencillas pudieron ser sintetizadas a partir de atmósferas suavemente reductores combinando modelos prebióticos ya existentes (volcanes que se comportan grandes reactores, reacciones fotoquímicas que ocurren en la atmosfera bajo el sol prebiótico y minerales que actúan como catalizadores) bajo el marco de una fotosíntesis prebiótica inorgánica, un proceso que podría ser considerado la piedra angular en la que se basó la aparición de las moléculas que dieron lugar a la vida.Postprint (published version

Détection d'ADN par spectroscopie SERRS et interactions entre nucléotides et surfaces des minéraux phyllosilicatés ferromagnésiens dans le contexte de l'origine de la Vie

Cette thèse a premièrement permis le développement d une méthode de détection non-enzymatique de l ADN. Les techniques enzymatiques couramment utilisées, comme la Polymerase Chain Reaction (PCR), échouent souvent dans l analyse d échantillons anciens ou transformés. L ADN subit en effet de nombreuses dégradations post mortem, parmi lesquelles certaines bloquent les enzymes ADN-polymérases. Notre méthode combine hybridation et détection par SERRS (Surface Enhanced Resonant Raman Scattering), permettant la détection et la quantification de séquences d ADN dégradées réfractaires à l analyse par PCR. De nouvelles perspectives s ouvrent donc en paléogénétique. Cette thèse aborde également le rôle des surfaces minérales dans l origine des acides nucléiques. Les surfaces minérales pourraient avoir piégé et concentré les briques élémentaires de ces biopolymères, contribuant ainsi à leur construction. Les travaux existants se sont concentrés sur des minéraux comme la montmorillonite, qui n était cependant pas abondante à l Hadéen/Archéeen. La minéralogie de la Terre primitive aurait plutôt été dominée par les phyllosilicates ferromagnésiens. Nous avons étudié l adsorption de nucléotides sur des minéraux plus cohérents avec le contexte géologique, en variant les paramètres environnementaux. Ce travail permet de préciser le mécanisme d adsorption des nucléotides sur les surfaces minérales, ainsi que les conditions de l origine du matériel génétique.The first goal of this thesis was the development of a non-enzymatic DNA detection method. Current enzymatic techniques such as Polymerase Chain Reaction (PCR) often fail in analyzing ancient or processed samples. Indeed DNA undergoes numerous post-mortem degradations, among which some are known to block the bypass of DNA-polymerases. Our method combines hybridization and SERRS (Surface Enhanced Resonant Raman Scattering) spectroscopy, and allows the detection and quantification of degraded DNA sequences that are refractory to PCR analysis. This novel detection method therefore opens new perspectives, especially in paleogenetics. This thesis also aims at studying the role of mineral surfaces in the origin of nucleic acids. Mineral surfaces might have trapped and concentrated the elementary bricks of those biopolymers, thus contributing in their formation. Previous work has focused on minerals such as montmorillonite, although it might not have been abundant during the Hadean/Archean. The primitive Earth s mineralogy would have been preferentially dominated by Fe-Mg rich phyllosilicates. We have therefore studied the adsorption of nucleotides on minerals we think are relevant to the geological context, and have varied the environmental conditions. This work allows characterizing the adsorption mechanism of nucleotides on mineral surfaces, as well as environmental conditions of the origin of genetic material.LYON-ENS Sciences (693872304) / SudocSudocFranceF

Détection d'ADN par spectroscopie SERRS et interactions entre nucléotides et surfaces des minéraux phyllosilicatés ferromagnésiens dans le contexte de l'origine de la Vie

The first goal of this thesis was the development of a non-enzymatic DNA detection method. Current enzymatic techniques such as Polymerase Chain Reaction (PCR) often fail in analyzing ancient or processed samples. Indeed DNA undergoes numerous post-mortem degradations, among which some are known to block the bypass of DNA-polymerases. Our method combines hybridization and SERRS (Surface Enhanced Resonant Raman Scattering) spectroscopy, and allows the detection and quantification of degraded DNA sequences that are refractory to PCR analysis. This novel detection method therefore opens new perspectives, especially in paleogenetics. This thesis also aims at studying the role of mineral surfaces in the origin of nucleic acids. Mineral surfaces might have trapped and concentrated the elementary bricks of those biopolymers, thus contributing in their formation. Previous work has focused on minerals such as montmorillonite, although it might not have been abundant during the Hadean/Archean. The primitive Earth’s mineralogy would have been preferentially dominated by Fe-Mg rich phyllosilicates. We have therefore studied the adsorption of nucleotides on minerals we think are relevant to the geological context, and have varied the environmental conditions. This work allows characterizing the adsorption mechanism of nucleotides on mineral surfaces, as well as environmental conditions of the origin of genetic material.Cette thèse a premièrement permis le développement d’une méthode de détection non-enzymatique de l’ADN. Les techniques enzymatiques couramment utilisées, comme la Polymerase Chain Reaction (PCR), échouent souvent dans l’analyse d’échantillons anciens ou transformés. L’ADN subit en effet de nombreuses dégradations post mortem, parmi lesquelles certaines bloquent les enzymes ADN-polymérases. Notre méthode combine hybridation et détection par SERRS (Surface Enhanced Resonant Raman Scattering), permettant la détection et la quantification de séquences d’ADN dégradées réfractaires à l’analyse par PCR. De nouvelles perspectives s’ouvrent donc en paléogénétique. Cette thèse aborde également le rôle des surfaces minérales dans l’origine des acides nucléiques. Les surfaces minérales pourraient avoir piégé et concentré les briques élémentaires de ces biopolymères, contribuant ainsi à leur construction. Les travaux existants se sont concentrés sur des minéraux comme la montmorillonite, qui n’était cependant pas abondante à l’Hadéen/Archéeen. La minéralogie de la Terre primitive aurait plutôt été dominée par les phyllosilicates ferromagnésiens. Nous avons étudié l’adsorption de nucléotides sur des minéraux plus cohérents avec le contexte géologique, en variant les paramètres environnementaux. Ce travail permet de préciser le mécanisme d’adsorption des nucléotides sur les surfaces minérales, ainsi que les conditions de l’origine du matériel génétique

Structural Role of Uracil DNA Glycosylase for the Recognition of Uracil in DNA Duplexes. Clues from Atomistic Simulations

In the \ufb01rst stage of the base excision repair pathway the enzyme uracil DNA glycosylase (UNG) recognizes and excises uracil (U) from DNA \ufb01laments. U repair is believed to occur via a multistep base-\ufb02ipping process, through which the damaged U base is initially detected and then engulfed into the enzyme active site, where it is cleaved. The subtle recognition mechanism by which UNG discriminates between U and the other similar pyrimidine nucleobases is still a matter of active debate. Detailed structural information on the di\ufb00erent steps of the base-\ufb02ipping pathway may provide insights on it. However, to date only two intermediates have been trapped crystallographically thanks to chemical modi\ufb01cations of the target and/or of its complementary base. Here, we performed force-\ufb01eld based molecular dynamics (MD) simulations to explore the structural and dynamical properties of distinct UNG/dsDNA adducts, containing A:U, A:T, G:U, or G:C base pairs, at di\ufb00erent stages of the base-\ufb02ipping pathway. Our simulations reveal that if U is present in the DNA sequence a shortlived extra-helical (EH) intermediate exists. This is stabilized by a water-mediated H-bond network, which connects U with His148, a residue pointed out by mutational studies to play a key role for U recognition and catalysis. Moreover, in this EH intermediate, UNG induces a remarkable overall axis bend to DNA. We believe this aspect may facilitate the \ufb02ipping of U, with respect to other similar nucleobases, in the latter part of the base-extrusion process. In fact, a large DNA bend has been demonstrated to be associated with a lowering of the free energy barrier for base-\ufb02ipping. A detailed comparison of our results with partially \ufb02ipped intermediates identi\ufb01ed crystallographically or computationally for other base-\ufb02ipping enzymes allows us to validate our results and to formulate hypothesis on the recognition mechanism of UNG. Our study provides a \ufb01rst ground for a detailed understanding of the UNG repair pathway, which is necessary to devise new pharmaceutical strategies for targeting DNA-related pathologies

Structural Role of Uracil DNA Glycosylase for the Recognition of Uracil in DNA Duplexes. Clues from Atomistic Simulations

In the first stage of the base excision repair pathway the enzyme uracil DNA glycosylase (UNG) recognizes and excises uracil (U) from DNA filaments. U repair is believed to occur via a multistep base-flipping process, through which the damaged U base is initially detected and then engulfed into the enzyme active site, where it is cleaved. The subtle recognition mechanism by which UNG discriminates between U and the other similar pyrimidine nucleobases is still a matter of active debate. Detailed structural information on the different steps of the base-flipping pathway may provide insights on it. However, to date only two intermediates have been trapped crystallographically thanks to chemical modifications of the target and/or of its complementary base. Here, we performed force-field based molecular dynamics (MD) simulations to explore the structural and dynamical properties of distinct UNG/dsDNA adducts, containing A:U, A:T, G:U, or G:C base pairs, at different stages of the base-flipping pathway. Our simulations reveal that if U is present in the DNA sequence a short-lived extra-helical (EH) intermediate exists. This is stabilized by a water-mediated H-bond network, which connects U with His148, a residue pointed out by mutational studies to play a key role for U recognition and catalysis. Moreover, in this EH intermediate, UNG induces a remarkable overall axis bend to DNA. We believe this aspect may facilitate the flipping of U, with respect to other similar nucleobases, in the latter part of the base-extrusion process. In fact, a large DNA bend has been demonstrated to be associated with a lowering of the free energy barrier for base-flipping. A detailed comparison of our results with partially flipped intermediates identified crystallographically or computationally for other base-flipping enzymes allows us to validate our results and to formulate hypothesis on the recognition mechanism of UNG. Our study provides a first ground for a detailed understanding of the UNG repair pathway, which is necessary to devise new pharmaceutical strategies for targeting DNA-related pathologies

APS Science 2007.

This report provides research highlights from the Advanced Photon Source (APS). Although these highlights represent less than 10% of the published work from the APS in 2007, they give a flavor of the diversity and impact of user research at the facility. In the strategic planning the aim is to foster the growth of existing user communities and foresee new areas of research. This coming year finds the APS engaged in putting together, along with the users, a blueprint for the next five years, and making the case for a set of prioritized investments in beamlines, the accelerator, and infrastructure, each of which will be transformational in terms of scientific impact. As this is written plans are being formulated for an important user workshop on October 20-21, 2008, to prioritize strategic plans. The fruit from past investments can be seen in this report. Examples include the creation of a dedicated beamline for x-ray photon correlation spectroscopy at Sector 8, the evolution of dedicated high-energy x-ray scattering beamlines at sectors 1 and 11, a dedicated imaging beamline at Sector 32, and new beamlines for inelastic scattering and powder diffraction. A single-pulse facility has been built in collaboration with Sector 14 (BioCARS) and Phil Anfinrud at the National Institutes of Health, which will offer exceptionally high flux for single-pulse diffraction. The nanoprobe at Sector 26, built and operated jointly by the Argonne Center for Nanoscale Materials and the X-ray Operations and Research (XOR) section of the APS X-ray Science Division, has come on line to define the state of the art in nanoscience