Dissertationen zum Thema „DNA hybrides“

Les gènes transcriptionellement actifs peuvent être la source de l'instabilité du génome via de nombreux mécanismes. Ces gènes sont caractérisés par la formation de structures secondaires telles que les hybrides ADN : ARN. Ils se forment lorsque l'ARN sortant l'ARN polymérase II s'hybride au simple brin d'ADN. De nombreuses études ont montrées que l'accumulation de ces hybrides peut mener à la création de dommages à l'ADN. Parmi ces dommages, les Cassures Double Brins (CDB) sont les plus dangereuses pour la cellule puisqu'elles peuvent produire des mutations et des réarrangements chromosomiques. Il existe deux mécanismes de réparation majeurs dans la cellule : la Jonction Non-Homologue des Extrémités (NHEJ) et la Recombinaison Homologue (HR). Mon équipe a récemment montré que les CDB localisées dans les gènes transcrits sont préférentiellement réparés par HR. De plus, de nombreuses études ont montrées une interaction entre transcription et réparation des CDB. Au vue de ces résultats, nous avons donc émis l'hypothèse que les gènes transcriptionellement actifs pourraient être réparés par un mécanisme spécifique nécessitant l'activité de protéines associées à la transcription : "Réparation couplée à la transcription". Durant ma thèse, je me suis intéressée au rôle de deux protéines dans la réparation des régions transcrites en utilisant la lignée cellulaire DIvA (DSB Induction via AsiSI) qui permet l'induction de cassures annotées sur tout le génome. Premièrement, nous avons montré que la réparation des CDB dans des loci transcrits nécessitent une hélicase ADN : ARN connue : sénataxine (SETX). Après induction d'une cassure dans un gène, SETX est recrutée ce qui permet la résolution d'hybride ADN : ARN (cartographié par DRIP-seq). Nous avons aussi montré que SETX permet le recrutement de RAD51 et limite les jonctions illégitimes des CDB et par conséquent promeut la survie des cellules après induction des cassures. Cette étude montre que les CDB dans les loci transcrits requièrent la résolution spécifique des hybrides ADN : ARN par SETX pour permettre une réparation précise et est absolument indispensable pour la survie cellulaire. Deuxièmement, nous avons montré une interaction entre SETX et Bloom (BLM) une G4 DNA hélicase dans la réparation des CDB dans les régions transcrites. Nous avons montré que BLM est aussi recrutée au CDB dans les loci transcrits où elle est nécessaire à la résection et à la fidélité de réparation. De façon importante, nous avons montré que la déplétion de BLM restaure le défaut de survie cellulaire observé dans les cellules déplétées pour SETX après induction des CDB. La déplétion d'autres hélicases G4 (RTEL1, FANCJ) promeut aussi la survie des cellules déplétées pour SETX après dommages. Ces résultats suggèrent une interaction entre les hélicases G4 et la résolution des hybrides ADN : ARN dans la réparation des gènes actifs. En conclusion, ces études permettent une meilleure compréhension de la spécificité de la réparation des régions transcrites du génome, et notamment l'identification de protéines impliquées dans la "Réparation couplée à la Transcription"
Actively transcribed genes can be the source of genome instability through numerous mechanisms. Those genes are characterized by the formation of secondary structures such as RNA-DNA hybrids. They are formed when nascent RNA exiting RNA polymerase II hybridizes single stranded DNA. Numerous studies have shown that RNA-DNA hybrids accumulation can lead to DNA damages. Among those damages, DNA double strand breaks (DSB) are the most deleterious for cells since they can generate mutations and chromosomal rearrangements. Two major repair mechanisms exist in the cell: Non-Homologous End-Joining (NHEJ) and Homologous recombination (HR). My lab showed recently that DSB occurring in transcribed genes are preferentially repaired by HR. Moreover, multiple studies have shown a cross talk between transcription and DSB repair. Those results led us to propose that actively transcribed genes could be repaired by a specific mechanism implicating proteins associated with transcription: "Transcription-coupled DSB repair". During my PhD, using the DIvA (DSB Induction via AsiSI) cell line allowing the induction of annotated DSB through the genome, I worked on 2 projects focusing on DSB repair in transcribed genes. First, we showed that DSB repair in transcribed loci requires a known RNA: DNA helicase: senataxin (SETX). After DSB induction in an active gene, SETX is recruited which allows RNA-DNA hybrid resolution (mapped by DRIP-seq). We also showed that SETX activity allows RAD51 loading and limits DSB illegitimate rejoining and consequently promotes cell survival after DSB induction. This study shows that DSB in transcribed loci require specific RNA-DNA hybrids removal by SETX for accurate repair. Second, we showed an interplay between SETX and Bloom (BLM) a G4 DNA helicase in DSB repair induced in transcribed loci. We showed that BLM is also recruited at DSB in transcribed loci where it promotes resection and repair fidelity. Strikingly, we showed that BLM depletion rescued the survival defects observed in SETX depleted cells following DSB induction. Knock down of other G4-helicases (RTEL1, FANCJ) also promoted cell survival in SETX depleted cells upon damage. Those data suggest an interplay between G4 helicases and RNA: DNA resolution for DSB repair in active genes. Altogether, these studies promote a better understanding of the specificity of DSB repair in transcriptionally active genes, and notably identification of proteins involved in "Transcription-coupled DSB repair"

La compréhension des mécanismes biologiques et l’implication des protéines dans ces mécanismes ont toujours été un enjeu important pour les biologistes. Les zones à risques de l’ADN impliquées dans les cancers, comme les G-quadruplex, les zones riches en Adénine-Thymine et leurs environnements proches sont particulièrement étudiés depuis de nombreuses années. L’essor des techniques d’analyses par fluorescence a permis aux scientifiques de mettre au point des sondes marquant ces domaines avec toujours plus de précision et de sensibilité. Cependant, de nombreuses interrogations existent sur la nature des interactions entre ces zones de l’ADN et les protéines. Afin de répondre à cette problématique, la synthèse d’une sonde pro-fluorescente alliant un ligand de l’ADN (conçu d’après les travaux des équipes de l’Institut Curie, UMR 176) à un piège à protéines (basé sur le squelette de l’epicocconone) a été réalisée et son efficacité biologique a été évaluée. Ces deux parties ont été assemblées en utilisant une réaction de cycloaddition 1,3 dipolaire spontanée entre un azoture et un alcyne contraint (SPAAC). De plus, au cours de ces travaux, une nouvelle bibliothèque de ligands de l’ADN a été synthétisée en utilisant une méthodologie innovante basée sur une réaction de C-H activation « on water »
Understanding biological process and proteins involved in has challenged biologists’ mind for a while. Specific DNA sequences, such as G-quadruplex and Adenine-Thymine rich sequences, have been studied for many years, especially for their involvement in genetic diseases like cancer. Scientists have also been interested in fluorescence monitoring and imaging of these specific sequences for a long time. Indeed, the huge sensitivity of these fluorescent technics and the wide scope of synthetic dyes available allowed several improvements on targeting DNA sequences responsible for genetic disorders. Nonetheless, relation between proteins and these areas remains mostly unknown. In order to answer this question, a pro-fluorescent dye built of two main parts, which are a DNA ligand (designed by Curie Institute teams, UMR 176) and a protein trap (based on epicocconone core). These parts were synthesized, coupled thanks to a Spontaneous Azoture Alkyne Cycloaddition (SPAAC) and the biological properties of the probe were evaluated. Furthermore, new ligands were synthesized using a new and innovating method of “on water” C-H activation reaction

Les machineries de réplication et de transcription peuvent provoquer des conflits entre transcription et réplication (TRC), qui se produisent de manière frontale ou co-directionnelle. La collision frontale est considérée comme étant la plus délétère et peut conduire à de l’instabilité génomique au travers des R-loops qui se composent un hybride ADN-ARN et un brin d'ADN déplacé. En analysant les données multi-omiques, nous avons révélé avec succès que la pause transitoire de la fourche de réplication aux 3' des gènes enrichis en R-loops avec collision frontale affecte la stabilité génomique d'une manière dépendante de Topoisomérase1 (Nat.Communs. 2020) puis j'ai développé le premier outil bio-informatique pour analyser de données de réplication (OKseqHMM, disponible sur GitHub, Liu et al. BioRxiv. 2022). Finalement, il a été montré récemment que dans les cellules cancéreuses du sein, les R-loops colocalisent fortement avec une augmentation des cassures de l'ADN, de manière dépendante de la réplication. Nous visons à étudier le TRC dans des cellules cancéreuses et des échantillons de patients cancéreux pour déterminer comment le stress réplicatif induit de l'instabilité génomique dans le développent de cancer, ce qui pourront contribuer à l’établissement de nouvelles stratégies thérapeutiques contre le cancer
Replication and transcription machinery can cause transcription-replication conflicts (TRCs), which occur either frontally or co-directionally. The head-on collision is considered to be the most deleterious and can lead to genomic instability through R-loops that consist of a DNA-RNA hybrid and a displaced DNA strand. By analyzing multi-omics data, we successfully revealed that transient replication forks pause at the 3' of genes enriched in R-loops with more head-on collisions affects genomic stability in a Topoisomerase1-dependent manner (Nat. Commons . 2020) then I developed the first bioinformatics tool to analyze replication data (OKseqHMM, available on GitHub, Liu et al. BioRxiv. 2022). Finally, it has recently been shown that in breast cancer cells, R-loops strongly colocalize with an increase in DNA breaks, in a replication-dependent manner. We aim to study TRC in cancer cells and samples from cancer patients to determine how replicative stress induces genomic instability in cancer development, which may contribute to the establishment of new therapeutic strategies against cancer

L’approche « bottum-up » via l’auto-assemblage moléculaire est considéré comme une voie prometteuse pour contrôler la fabrication de nouveaux matériaux et leur intégration dans des dispositifs hybrides présentant de propriétés nouvelles. Dans ce travail, nous avons synthétisé plusieurs hybrides à base de molécules organiques (fullerène, porphyrines, phtalocyanine), d’oligonucléotides ou de nanotubes de carbone.Dans un premier temps, nous nous sommes intéressés à la synthèse d’une nouvelle famille de produits constituée d’une unité C60 lié à deux chromophores positionnés face à face et permettant la formation de complexes hôte-invités. Nous avons montré que ces composés s’assemblent pour donner des structures supramoléculaires en solution et sur surface. Les interactions électroniques et la compléxation entre le fullerène et les deux chromophores (porphyrines et phtalocyanines) ont été étudiées par spectroscopie optique et RMN ainsi que par voltammétrie cyclique.Parmi les outils de l’approche « bottom-up », l’ADN a montré son extraordinaire potentiel pour la fabrication d’assemblages bio-dirigés. En effet, la synthèse de matériaux hybrides à base d’ADN permet un contrôle précis (théoriquement à l’échelle d’une base, ~3,4Å) du positionnement des groupements fonctionnels dans un matériau. Dans le but de former des réseaux bi- et tridimensionnels à base d’ADN permettant le positionnement de nano-objets, nous avons synthétisé des hybrides à base d’oligonucléotides et de porphyrines (molécule 2D) ou d’adamantane (molécule 3D). Des édifices supramoléculaires simples ont été réalisés et le travail se poursuit en vue de la réalisation de réseaux fonctionnels.Enfin, dans une dernière partie, nous nous sommes intéressés à la fonctionnalisation des nanotubes de carbone monoparoi (SWNT) avec des chromophores de type porphyrines et phtalocyanines. Alors que les porphyrines présentent une absorption intense presque exclusivement dans le bleu, les phtalocyanines absorbent principalement dans le rouge. Combiner ces deux chromophores à la surface des nanotubes de carbone présente donc un intérêt particulier pour la collecte de lumière car les deux composés absorbent des régions complémentaires du spectre visible. Ce travail ouvre la voie vers l'étude des propriétés optoélectroniques des hybrides à base de nanotubes et en particulier leur utilisation pour la conversion d’énergie lumineuse en énergie électrique (application photovoltaïque)
An Approach "bottum-up" via molecular self-assembly is considered as a promising way to control the manufacture of new materials and their integration into hybrid devices with novel properties. In this work, we have synthesized several hybrids based on organic molecules (fullerene, porphyrin, phthalocyanine), oligonucleotides or carbon nanotubes.At first, we were interested in the synthesis of a new family of products consisting of a unit C60 linked to two chromophores positioned face to face and allowing the formation of host-guest complexes. We have shown that these compounds are combined to give supramolecular structures in solution and on the surface. Electronic interactions and complexation between fullerene and the two chromophores (porphyrins and phthalocyanines) were studied by NMR and optical spectroscopy as well as cyclic voltammetry.Among the tools of the "bottom-up", DNA showed its tremendous potential for the production of bio-directed assembly. Indeed, the synthesis of hybrid materials based DNA allows precise control (theoretically on the scale of a base, ~ 3.4 Å) of the positioning of the functional groups in a material. In order to form networks and bi-dimensional DNA-based for positioning nano-objects, we have synthesized hybrid oligonucleotide-based and porphyrin molecule (2D) or adamantane molecule (3D). Supramolecular structures have been made and this work is ongoing to achieve functional networks.Finally, in a last part, we are interested in the functionalization of single-walled carbon nanotubes (SWNTs) with chromophores like porphyrins and phthalocyanines. While porphyrins exhibit almost exclusively an intense absorption in the blue (around 420-440 nm), phtalocyanines absorb mainly in the red spectral region. Taken together these two chromophores have interesting light harvesting, photophysical and redox properties; the two components will participate independently to increase the overall absorption in the visible range of the solar spectrum. This work opens the route to study the optoelectronic properties of hybrid nanotube and in particular their use for the conversion of light energy into electrical energy (photovoltaic application)

La maintenance de la méthylation ADN sur les sites CG joue un rôle crucial dans le silencing des éléments transposables (TE) et l’expression correcte des gènes. Chez Arabidopsis thaliana, on observe, dans le mutant met1-3 déficient en méthylation CG, une apparition ectopique de méthylation CHG sur de nombreux gènes, ainsi qu’une relocalisation de la diméthylation de la lysine 9 de l’histone H3 (H3K9me2) de l’hétérochromatine vers l’euchromatine. Nous avons démontré que ceci est lié à un défaut de transcription au niveau du grand intron du gène codant la déméthylase H3K9me2 IBM1. Nous avons également constaté que dans les épihybrides F1 issus du croisement de plantes met1-3 avec une plante sauvage la méthylation CHG présente dans l’intron de l’allèle IBM1 hérité du parent mutant était perdue. Afin de définir si la perte de méthylation affecte également d’autres loci génomiques, et plus globalement à l’échelle du génome entier l’impact de la rencontre de deux épigénomes différents, nous avons analysé les profils de méthylation, de siRNAs et de transcription des épihybrides F1. Nos données révèlent que l’union de deux méthylomes distincts au sein d’un même génome provoque une restructuration considérable des profils épigénétiques et transcriptionnels. La méthylation CHG apparaissant sur de nombreux gènes dans met1 tend à persister, créant ainsi de nouveaux épiallèles pouvant être hérités. Du côté des TE, nombre d’entre eux sont déméthylés et réactivés, tandis que d’autres sont immédiatement reméthylés et resilencés. Ainsi, ces résultats contribuent à la compréhension de la stabilité de la méthylation ADN et de son rôle dans le contrôle différentiel des gènes et des TE
Maintenance of DNA methylation at CG sites is crucial for silencing of transposable element (TE) and proper expression of genes. In Arabidopsis thaliana, met1-3 mutant, deficient in CG methylation, shows ectopic appearance of CHG methylation at numerous genes, as well as relocation of H3K9me2, from heterochromatin towards euchromatin. We have shown that this is due to a defect of the transcription of the large intron of the gene encoding the IBM1 H3K9me2 demethylase. We also found that, in the F1 epihybrids from the cross between met1 and WT plants, CHG methylation at the intron of the met1-derived IBM1 allele is lost. In order to define whether the loss of methylation at IBM1 also affects other genomic loci, and the impact of the union of two different epigenomes on methylation and transcription genome-wide, we analyzed the methylation, siRNA and transcription patterns of F1 epihybrids. Our data reveal that the union of two distinct methylomes within the same genome triggers considerable restructuring of epigenetic and transcriptional patterns. CHG methylation appearing in the mutant parent tends to persist in F1, creating new epialleles that can be inherited. On the TE side, lots of them are demethylated and reactivated while others are immediately remethylated and resilenced. Thus, our results provide new insights to the understanding of DNA methylation stability and its role in the differential control of genes and TEs

Le travail de recherche de cette thèse consiste en le développement de nouveaux matériaux hybrides organiques-inorganiques pour des applications en nanotechnologie, nanomédicine et diagnostic. Dans ce contexte, des cristaux poreux de zéolite-L ont été utilisé comme nano-vecteur pour faire de la transfection d’ADN et d’ANP, en combinaison avec le relargage de molécules hôtes placées dans les pores. Des nanoparticules de silice mesoporeuses multifonctionnelles ont été utilisées pour traiter le glioblastome, en combinant la thérapie génique avec l’administration durable d’un principe actif. Des nano-coquilles hybrides biodégradables ont été encore développés pour encapsuler des protéines et les relâcher dans les cellules vivantes. Dans le domaine de la détection d’acides nucléiques, des fibres optiques à cristal photonique, fonctionnalisées avec des sondes d’ANP, ont été exploitées comme plateformes optiques pour faire de la détection ultra-sensible d’oligonucléotides ou d’ADN génomique. Enfin, la squelette de l’ANP a été modifié à créer des sondes fluorescentes pour reconnaître et détecter la présence des séquences cibles spécifiques
The research work presented throughout this thesis focuses on the development of novel organic-inorganichybrid materials for applications in nanotechnology, nanomedicine and diagnostics. In such a context, porous zeolite-L crystals have been used as nanocarriers to deliver either DNA or PNA in live cells, in combination with the release of guest molecules placed into the pores. Multifunctional mesoporous silica nanoparticles have been designed to treat glioblastoma, combining gene therapy with the sustained delivery of a chemotherapy agent. Biodegradable hybrid nano-shells have been furthermore created to encapsulate proteins and release them in living cells upon degradation of the outer structure in reductive environment. In the field of nucleic acid detection, photonic crystal fibers, functionalized with specific PNA probes, have been exploited as optical sensing devices to perform ultra-sensitive detection of DNA oligonucleotides or genomic DNA. Eventually, the PNA backbone has served as scaffold to synthesize fluorescent switching probes able to recognize and to detect the presence of specific target sequences

Le développement de nouvelles molécules capables d’intéragir avec l’ADN, leur mode d’intéraction et leur affinité est un domaine de recherche particulièrement important. Dans ce travail nous avons étudié les interactions avec l’ADN de la molécule Hoechst 33258 connu pour être un ligand du petit sillon ainsi que plusieurs de ces analogues.Dans un premier temps nous avons étudié la stabilité des complexes ADN-Hoechst 33258 en solution avec et sans DMSO comme co-solvant. Deuxièmement, les affinités de dérivés nouvellement conçus et synthétisés du Hoechst 33258 vis-à-vis de l'ADN ont été évaluées. Enfin, nous avons étudiés la capacité d’induction d’excimers en présence d’ADN de différentes molécules pyrénylées. Ces études ont été effectuées par différentes méthodes spectroscopiques, telles que l'absorbance UV-visible, la fluorescence, le dichroïsme circulaire, la spectroscopie de masse ESI et de la modélisation moléculaire
The development of new DNA binders and the evaluation of their affinity toward DNA as well as their mode of binding is an area of research of prime importance. In this thesis we studied the interactions of Hoechst 33258, a well-known groove binder, as well as some of its newly synthesized derivatives with DNA. The stability of DNA-Hoechst 33258 complex in solution with and without DMSO as a co-solvent was evaluated.Secondly, the affinities of newly designed and synthesized derivatives of Hoechst 33258 toward DNA were evaluated. Finally, a set of pyrene derivatives able to induced excimer formation upon binding to DNA were studied. Different spectroscopic methods, such as UV-vis absorbance, fluorescence, circular dichroism, ESI mass spectroscopy and molecular docking were applied for the complete evaluation of the affinity of these ligands toward DNA

Au cours de la phase S, la réplication de l’ADN est initiée au niveau de multiples origines réparties le long du génome. La machinerie de réplication, ou réplisome, peut rencontrer des obstacles ralentissant sa progression, comme des structures secondaires de l’ADN ou des protéines liées à l’ADN telles que les ARN polymérases, générant ainsi ce que l’on appelle un stress réplicatif. Les réplisomes bloqués par un obstacle sont des structures fragiles qui peuvent générer des cassures et conduire à l’instabilité du génome. Lorsque la progression de l’ARN polymérase est ralentie ou arrêtée, le brin d’ARN naissant peut potentiellement s’hybrider avec le brin d’ADN complémentaire en déplaçant le second brin d’ADN, formant ainsi une structure à trois brins appelée R-loop, pouvant entraver la progression du réplisome. La coordination des processus de réplication et de transcription limite les interférences entre la réplication et la transcription. Cependant, cette coordination n’est pas parfaite et même dans des conditions physiologiques, la transcription et l’accumulation de R-loops peuvent conduire à des évènements de recombinaison, notamment au cours de la phase S. Les Ribonucléases H (RNases H) de type 1 et 2 sont des protéines impliquées dans la résolution des R-loops par la dégradation spécifique du brin d’ARN au sein du duplexe ARN:ADN. Les cellules dépourvues de RNases H présentent une accumulation de R-loops et sont extrêmement sensibles à différents agents génotoxiques induisant du stress réplicatif (e.g. MMS : méthanesulfonate de méthyle ou HU : hydroxyurée). Le but de mes travaux de thèse est de déterminer le rôle des RNases H dans la réponse cellulaire au stress réplicatif. Dans deux modèles cellulaires, la levure S. cerevisiae et les cellules humaines, nous avons pu montrer que les cellules déplétées en RNases H présentent des défauts de prise en charge et de redémarrage des fourches de réplication arrêtées en condition de stress réplicatif induit. L'utilisation de mutants de séparation de fonction de RNase H2 suggère que l’élimination défectueuse des hybrides ARN:ADN est responsable de ces défauts. La mesure du taux d’hybrides ARN:ADN au cours du cycle cellulaire montre qu’il augmente en phase S en présence de stress réplicatif exogène dans des cellules sauvages et mutantes pour RNases H. De plus, nos résultats indiquent que l’inhibition de la transcription ou la surexpression de l’hélicase ARN:ADN Sénataxine restaure la prise en charge et le redémarrage des fourches de réplication arrêtées lors d’un stress induit par le MMS et en absence des RNases H. Ainsi, l’ensemble de nos résultats suggère une étroite coopération entre les Ribonucléases H et l’hélicase Sénataxine pour résoudre les interférences entre les ARN polymérases et/ou les hybrides ARN:ADN avec les machineries de réplication
During S phase, DNA replication starts at multiple origins distributed throughout the genome. As the replication machinery (or replisome) progresses throughout the DNA, it often encounters obstacles such as DNA secondary structures or transcription complexes, thereby generating what is called replication stress. Stalled replisomes are fragile structures that can give rise to chromosome breaks and trigger genome instability. When RNA polymerases stall, the nascent RNA can potentially anneal with the template DNA strand, creating a three-strand structure called R-loop. Coordination between replication and transcription in S phase limits the risks of collisions between the replisome and RNA polymerases. Even though, physiological transcription level and R-loops accumulation lead to recombination events in S phase. Type 1 and 2 ribonucleases H (RNase H) are specific proteins involved R-loops’ resolution through the degradation of the RNA strand within the RNA:DNA duplex. In the absence of RNases H, cells accumulate R-loops and are extremely sensitive to different replication stress-inducing genotoxic agents (e.g. MMS: methyl methanesulfonate or HU: hydroxyurea).The goal of my PhD project was to assess the roles of RNases H in the cellular response to replication stress. Using two cellular models, the budding yeast S. cerevisiae and mammalian cells, we demonstrated that RNases H mutations induce HU- and MMS-stalled replication forks processing and restart defects. Analysis of separation-of-function RNase H2 mutants suggests that it is the RNA:DNA hybrids removal activity of RNase H2 that is important for the correct processing of stalled forks experiencing replication stress. Indeed, quantification of RNA:DNA hybrids during the cell cycle reveals a higher level of hybrids in S phase in the presence of exogenous replication stress in both wild-type and RNases H-depleted cells. Moreover, our results demonstrate that the inhibition of transcrip tion or the overexpression of the RNA:DNA helicase Senataxin restore stalled replication fork processing and restart upon MMS treatment when cells lack RNase H2 activities. Altogether, our data indicate that Ribonucleases H1 and 2 and Senataxin helicase cooperate to resolve RNA polymerases and/or RNA:DNA hybrids interferences with replication

Parmi les différents minéraux argileux, la sépiolite, qui est un silicate fibreux naturel, est un potentiel nano-transporteur prometteur pour le transfert non-viral de biomolécules. Il a en effet été montré que la sépiolite interagissait avec des molécules biologiques telles que les lipides, les polysaccharides et les protéines. Dans ce travail, nous démontrons que la sépiolite interagit également efficacement avec différents types de molécules d'ADN (génomique, plasmidique, oligonucléotides simple et double brin), et nous présentons la première étude détaillée sur les mécanismes d'interaction entre la sépiolite et l'ADN, ainsi qu’une caractérisation physico-chimique de bionanocomposites ADN-sepiolite. Une analyse spectroscopique a montré tout d’abord que l’interaction de l'ADN avec la sépiolite était plus forte en présence de polycations, la valence de ces derniers accroissant le rendement d’absorption, et deuxièmement, que l'ADN ainsi adsorbé pouvait être récupéré avec un rendement modulé par la présence d’EDTA, la structure de l'ADN et son activité biologique étant conservées. Par spectroscopie infrarouge à transformée de Fourier (FTIR) nous avons identifié les groupes silanol externes comme les principaux sites d'interaction avec l'ADN. Nous avons ensuite prouvé qu'il est possible d'utiliser la sépiolite pour extraire l'ADN de bactéries, pour la purification de l'ADN et pour la purification de toute contamination bactérienne. En combinant la microscopie à fluorescence, la microscopie électronique à transmission (MET), la vidéo-microscopie et l’analyse par cytométrie en flux (FACS), nous avons montré que la sépiolite peut être spontanément internalisée dans des cellules de mammifère par le biais de deux voies, l’endocytose et la macropinocytose. En tant que preuve de concept, nous montrons que la sépiolite est capable de transférer de manière stable l'ADN de plasmide dans des bactéries et des cellules de mammifères. Il a également été prouvé qu’en incubant des bactéries avec des bionanocomposites ADN-sepiolite, initialement préparés en présence d'une faible concentration en cations divalents et avec de la sépiolite traitée aux ultrasons (sSep), il était possible d'augmenter l'efficacité de la transformation bactérienne 20 à 30 fois par rapport aux méthodes basées sur l'«effet Yoshida». En outre, nous montrons que l'efficacité du transfert de gènes par la sépiolite peut être optimisée : l'utilisation de sSep et l'exposition à la chloroquine augmentent d’un facteur 100 et 2, respectivement, l’efficacité de transfection. Ces résultats ouvrent la voie à l'utilisation de bionanocomposites à base de sépiolite comme de nouveaux potentiels nano-transporteurs hybrides potentiels, à la fois pour la thérapie génique et le développement de nouveaux modèles biologiques en sciences fondamentales et appliquées
Among the various clay minerals, sepiolite, which is a natural fibrous silicate, isa potential promising nanocarrier for the non-viral transfer of bio-molecules. Indeed,sepiolite has been shown to interact with biological molecules such as lipids,polysaccharides and proteins. Here, we show that sepiolite efficiently binds differenttypes of DNA molecules (genomic, plasmid, single strand and double strandoligonucleotides), introducing the first detailed study on the interaction mechanismsbetween sepiolite and DNA, as well as the physicochemical characterization of theresulting DNA-sepiolite bionanocomposites. The interaction mechanisms aresuggested to be electrostatic interactions, van der Waals forces, cation bridges, andhydrogen bonding. Spectroscopy analysis showed that the binding of DNA to sepiolitewas increased by polycations with valence dependent efficiency, and the DNApreviously adsorbed could be recovered with an efficiency that could be modulatedusing a chelating agent (EDTA), preserving the DNA structure and biological activity.Fourier-transform infrared spectroscopy identified the external silanol groups as themain sites of interaction with the DNA. It was proved that it is possible to use sepiolitefor extracting DNA from bacteria, for DNA purification and for purification from bacterialcontamination. By combining fluorescence microscopy, transmission electronmicroscopy (TEM), time-lapse video microscopy and flow cytometry analysis (FACS),we show that sepiolite can be spontaneously internalized into mammalian cells throughboth endocytic and non-endocytic pathways. As a proof of concept, we show thatsepiolite is able to stably transfer plasmid DNA into bacteria and mammalian cells. Itwas also proved that with the incubation of bacteria with the Sep/DNAbionanocomposite initially prepared in the presence of a low concentration of divalentcation, and using sonicated sepiolite (sSep), it is possible to increase the bacterialtransformation efficiency from 20 to 30-fold compared to previously reported methodswhich are based in the “Yoshida effect”. Additionally, we show that the efficiency ofsepiolite-mediated gene transfer can be optimized: the use of sSep and the exposureto the endosome disrupter chloroquine 100-fold and 2-fold stimulated DNA transfectionefficiency, respectively. These results open the way to the use of sepiolite-basedbionanocomposites as a novel class of hybrid nanocarriers for both potential genetherapy and the development of novel biological models of interest for academic andapplied sciences

La transformation naturelle bactérienne permet aux micro-organismes d'échanger des informations génétiques pour promouvoir leurs réponses adaptatives pour faire face aux changements environnementaux. De l'ADN extracellulaire est incorporé et recombiné au génome de l'hôte. Ce processus augmente la plasticité des bactéries. Chez S. pneumoniae, un pathogène majeur chez l'Homme engendrant des infections pouvant être mortelles, la transformation bactérienne accentue la transmission de gènes de résistance aux antibiotiques. Chez les bactéries à Gram positif, l'opéron comF encode l'expression de deux protéines. L'une est démontrée comme étant essentielle à la transformation, est décrite pour être membranaire. La seconde n'a pas été étudiée. Cependant ces protéines n'ont pas été étudiées d'un point de vue structural ou fonctionnel. Des mutagenèse et le double hybride bactérien ont permis de mettre en évidence que ses protéines sont indispensables pour l'expression de la compétence et interagissent avec de nombreuses protéines du transformasome. De plus, l'expression des deux protéines de manière hétérologue prouve qu'elles sont solubles et forment des oligomères. L'analyse structurale de ComFA, atteste de la conformation atypique de cette helicase trimerique et hexamerique. En outre, l'activité ATPasique simple brin DNA-dépendant de cette protéine est démontrée. Finalement un complexe protéique a été révélé entre ComFA et ComFC dont l'étude microscopique à hautes résolutions prouve l'apparition d'un anneau via l'assemblage de deux hexamères. Ces résultats suggèrent que ComFA est le moteur tirant l'ADN dans la cellule. Quant à ComFC, elle semble aider à la stabilisation de ComFA
Bacterial natural transformation allows microorganisms to exchange genetic information to promote their adaptive responses to cope with environmental changes. The extracellular DNA is incorporated and recombined with the genome of the host. This phenomenon increases the plasticity of Gram positive and negative bacteria. S. pneumoniae is a major pathogen for humans, which is causing infections that can be deadly. In this specie, bacterial transformation increases the transmission of antibiotic resistance.In Gram-positive bacteria, comF operon encodes the expression of two proteins. One of them, shown to be essential for natural transformation, is expected to be a membrane protein. The second is not described. However, up to now neither protein has been studied from a structural or functional point of view. Mutagenesis technique and double hybrid bacterial assay allowed to show that both proteins are essential for the expression of the competence and interact with many proteins of the transformasome. In addition, heterologous expresion of both proteins have shown their solubility and the formation of oligomers. Structural analysis of ComFA demonstrates the unique conformation of this hexameric and trimeric helicase. Furthermore, the ATPase single stranded DNA-dependent activity of this protein could be detected. Finally, a protein complex is formed between ComFA and ComF, and high-resolution microscopic study proves the occurrence of a ring via a two-hexamers. These results suggest that ComFA is the engine pulling the DNA in the cell. As for ComFC, this protein seems to help stabilizing of ComFA

La transformation naturelle bactérienne permet aux micro-organismes d'échanger des informations génétiques pour promouvoir leurs réponses adaptatives pour faire face aux changements environnementaux. De l'ADN extracellulaire est incorporé et recombiné au génome de l'hôte. Ce processus augmente la plasticité des bactéries. Chez S. pneumoniae, un pathogène majeur chez l'Homme engendrant des infections pouvant être mortelles, la transformation bactérienne accentue la transmission de gènes de résistance aux antibiotiques. Chez les bactéries à Gram positif, l'opéron comF encode l'expression de deux protéines. L'une est démontrée comme étant essentielle à la transformation, est décrite pour être membranaire. La seconde n'a pas été étudiée. Cependant ces protéines n'ont pas été étudiées d'un point de vue structural ou fonctionnel. Des mutagenèse et le double hybride bactérien ont permis de mettre en évidence que ses protéines sont indispensables pour l'expression de la compétence et interagissent avec de nombreuses protéines du transformasome. De plus, l'expression des deux protéines de manière hétérologue prouve qu'elles sont solubles et forment des oligomères. L'analyse structurale de ComFA, atteste de la conformation atypique de cette helicase trimerique et hexamerique. En outre, l'activité ATPasique simple brin DNA-dépendant de cette protéine est démontrée. Finalement un complexe protéique a été révélé entre ComFA et ComFC dont l'étude microscopique à hautes résolutions prouve l'apparition d'un anneau via l'assemblage de deux hexamères. Ces résultats suggèrent que ComFA est le moteur tirant l'ADN dans la cellule. Quant à ComFC, elle semble aider à la stabilisation de ComFA
Bacterial natural transformation allows microorganisms to exchange genetic information to promote their adaptive responses to cope with environmental changes. The extracellular DNA is incorporated and recombined with the genome of the host. This phenomenon increases the plasticity of Gram positive and negative bacteria. S. pneumoniae is a major pathogen for humans, which is causing infections that can be deadly. In this specie, bacterial transformation increases the transmission of antibiotic resistance.In Gram-positive bacteria, comF operon encodes the expression of two proteins. One of them, shown to be essential for natural transformation, is expected to be a membrane protein. The second is not described. However, up to now neither protein has been studied from a structural or functional point of view. Mutagenesis technique and double hybrid bacterial assay allowed to show that both proteins are essential for the expression of the competence and interact with many proteins of the transformasome. In addition, heterologous expresion of both proteins have shown their solubility and the formation of oligomers. Structural analysis of ComFA demonstrates the unique conformation of this hexameric and trimeric helicase. Furthermore, the ATPase single stranded DNA-dependent activity of this protein could be detected. Finally, a protein complex is formed between ComFA and ComF, and high-resolution microscopic study proves the occurrence of a ring via a two-hexamers. These results suggest that ComFA is the engine pulling the DNA in the cell. As for ComFC, this protein seems to help stabilizing of ComFA

Kinesin is a molecular motor which walks on microtubule tracks in the eukaryotic cytoskeleton. It transports cargo but is also involved in cytoskeletal organisation. This thesis demonstrates fusing kinesin and DNA to construct a molecular transport system using self-organised tracks and to study the mechanics of the minimal motor unit of kinesin. The programmability of DNA allows for the formation of nanostructures with controllable interactions. Kinesin is conjugated to various DNA nanostructures to accomplish different tasks. Instructions encoded into DNA sequences are used to direct the assembly of a polar array of microtubules, to control the loading, active concentration and unloading of cargo on this track network and to trigger the disassembly of the network. Fluorescence microscopy was used to observe these microtubule arrays and the movement of cargo. It was found that the DNA signals used to control the unloading of cargo and the disassembly of the network had to be actively transported, rather than relying on diffusion, for effective delivery of the signal. This work lead to a first author publication, Wollman et al. (2013). DNA was also used to study kinesin by linking defined numbers of minimal functional motor units, single kinesin heads, into teams of 4-12 heads and observing their movement along microtubules via fluorescent labelling. A minimum of 5 heads were required for sustained movement, in agreement with the predictions of Hancock and Howard (1998). The velocity of teams increased with more heads, up to 8, and then a decrease was observed in teams with more heads.

The stability of our genome is constantly challenged by several genotoxic threats. DNA double-strand breaks (DSBs) are the most dangerous DNA lesions that, if not repaired, can lead to cancer initiation and progression and/or ageing. These detrimental consequences can only be avoided if cells promptly recognize the lesions and signal their presence, thus promoting either efficient repair and transient cell cycle arrest or cell death and cellular senescence. This is the role of the DNA damage response (DDR) proteins and the newly identified damage-induced non coding RNAs. We recently discovered that RNA polymerase II is recruited to DSBs and synthetizes damage-induced non-coding RNAs (dilncRNAs). DROSHA- and DICER-mediated processing of dilncRNAs generates small RNA species, named DNA damage response RNA (DDRNAs) (Francia, 2012), that localize to DSBs via pairing with dilncRNAs and promote DDR signaling (Michelini et al., in press). Similar small non-coding RNA species discovered in plants are involved in DNA repair by homologous recombination (HR) (Wei, 2012, Gao, 2014, Wang, 2016). In line with these results, I report that transcriptional inhibition impairs recruitment of the HR proteins BRCA1, BRCA2, and RAD51 to DSBs, while partially promoting DNA end resection. Moreover, I show DNA:RNA hybrids accumulation at DSBs in mammalian cells by both DRIP analyses and imaging techniques. Damage-induced DNA:RNA hybrids form upon the hybridization of RNA species, likely dilncRNAs, to the resected DSBs DNA ends generated during the S/G2 cell cycle phase. I also report that purified recombinant BRCA1 binds DNA:RNA hybrids in vitro; moreover, DNA:RNA hybrids in vivo contribute to BRCA1 recruitment to DSBs. Consistent with the need to tightly regulate DNA:RNA hybrid levels, I demonstrate that RNase H2, the major RNase H activity in mammalian nuclei, is recruited to DSBs through direct interaction with RAD51. In summary, I report for the first time that DNA:RNA hybrids accumulate at DSBs in mammalian cells in a cell-cycle- and DNA end resection-depended way. At DSBs, BRCA1 directly recognizes DNA:RNA hybrids and likely controls their turn-over by mediating the recruitment of RNase H2 via RAD51.

The activation of the innate immune system is the first line of host defence against infection. Nucleic acids can potently stimulate this response and trigger a series of signalling cascades leading to cytokine production and the establishment of an inflammatory state. Mutations in genes encoding nucleases have been identified in patients with autoimmune diseases, including Aicardi-Goutières syndrome (AGS). This rare childhood inflammatory disorder is characterised by the presence of high levels of the antiviral cytokine interferon-α in the cerebrospinal fluid and blood, which is thought to be produced as a consequence of the activation of the innate immunity by unprocessed self-nucleic acids. This thesis therefore aimed to define the role of one of the AGS nucleases, the Ribonuclease H2 (RNase H2) complex, in innate immunity, and to establish if nucleic acid substrates of this enzyme were able to induce type I interferon production in vitro. The AGS nucleases may function as components of the innate immune response to nucleic acids. Consistent with this hypothesis, RNase H2 was constitutively expressed in immune cells, however, its expression was not upregulated in response to type I interferons. RNase H2-deficient cells responded normally to a range of nucleic acid PAMPs, which implied that a role for RNase H2 as a negative regulator of the immune response was unlikely, in contrast to the reported cellular functions of two other AGS proteins, TREX1 and SAMHD1. Therefore, no clear evidence was found for the direct involvement of RNase H2 in the innate immune response to nucleic acids. An alternative model for the pathogenesis of disease hypothesises that decreased RNase H2 activity within the cell results in an accumulation of RNA:DNA hybrids. To investigate the immunostimulatory potential of such substrates, RNA:DNA hybrids with different physiochemical properties were designed and synthesised. Methods to purify the hybrids from other contaminating nucleic acid species were established and their capacity as activators of the innate immune response tested using a range of in vitro cellular systems. A GU-rich 60 bp RNA:DNA hybrid was shown to be an effective activator of a pro-inflammatory cytokine response exclusively in Flt3-L bone marrow cultures. This response was completely dependent on signalling involving MyD88 and/or Trif, however the specific receptor involved remains to be determined. Reduced cellular RNase H2 activity did not affect the ability of Flt3-L cultures to mount a cytokine response against the RNA:DNA hybrid. These in vitro studies suggested that RNA:DNA hybrids may be a novel nucleic acid PAMP. Taken together, the data in this thesis suggest that the cellular function of RNase H2 is in the suppression of substrate formation rather than as a component of the immune response pathways. Future studies to identify endogenous immunostimulatory RNA:DNA hybrids and the signalling pathways activated by them should provide a detailed understanding of the molecular mechanisms involved in the pathogenesis of AGS and related autoimmune diseases.

Pharmacogenomics is the study of how genes affect a individual response to drugs to develop medications tailored to a person’s genetic makeup because the efficacy/safety profile have not be the same way for everyone. Mitochondria are characterized by a unique milieu, with an alkaline and negatively charged interior (pH value 8) due to the proton pumping associated with OXPHOS and a series of specific channels and carrier proteins. As a consequence, mitochondria can easily accumulate lipophilic compounds of cationic character and weak acids in their anionic form, particularly amphiphilic xenobiotics including ethidium bromide, 1-methyl-4 phenylpyridinium (MPP+), paraquat (1,1’-dimethyl-4,4’-bipyridinium dichloride; PQ) and others that can penetrate the inner mitochondrial membrane (IMM) freely since in their undissociated forms. Indeed, it is well understood that many drugs and chemicals can cause mitochondrial dysfunction (mitotoxicity) by interacting with mitochondrial DNA (mtDNA), protein synthesis, respiratory chain, other metabolic processes, channels and transporters Moreover, due to its peculiar uniparental maternal inheritance and high mutation rate, mtDNA presents different clusters of population-specific-polymorphism (SNPs) that characterize different maternal lineages (mitochondrial haplogroups). It has been demonstrated that many non-synonymous SNPs, cause amino acid variations in the mitochondrial-encoded proteins, potentially modifying OXPHOS activity and ROS production. Some of these haplotypes may confer vulnerability to, or protection from, various common diseases. Well-documented examples are the role of mtDNA haplotype in Parkinson’s disease (PD) and Leber’s hereditary optic neuropathy (LHON). It has been proposed that European haplogroups J and K are protective for PD. On the other hand the haplogroups J and T may influence mitochondrial dysfunction, resulting in an increased risk of PD. In addition the 11778/ND4, 14484/ND6 and 3460/ND1 LHON mutations are associated respectively with mitochondrial subhaplogroup J2b, J1c and K, as these mtDNA backgrounds may increase penetrance of LHON mutations. Several reports suggest that environmental factors such as pesticides (e.g. rotenone), herbicides (e.g. paraquat) and MPTP or 1-methyl 4-phenyl 1,2,3,6-tetrahydro-pyridine (contaminant in the illicit synthesis of opiates) increase the risk of PD due to a reduction in ATP synthesis and increase of reactive oxygen species (ROS). Furthermore, tobacco smoking has been proposed as an environmental trigger of visual loss in LHON, due to the presence of substances, contained in the tobacco that can directly act inhibiting CI. Researchers have also associated non synonymous variants in mtDNA with the development of side effects of drugs. Effectively the analysis of mtDNA haplogroup in patients with cancer treated with chemioterapic agent cisplatin (cisPt) revealed an increased incidence of hearing loss in haplogroup J, due to inhibition of mtDNA replication. It has also been shown that patients treated with the antibiotic Linezolid may develop LHON-like optic neuropathy, myelosuppression and lactic acidosis. This is possibly due to the inhibition of mitochondrial protein synthesis, which is modulated by the SNPs at positions 2706 and 3010, in the 16S gene of mtDNA. This sequence region is predicted to be very close to the Peptidyl Transferase Center (PTC) that is the binding site of several antibiotics. To demonstrate that mitochondrial genetic variability may influence individual susceptibility to drugs toxicity (Linezolid and CisPt) or to toxic environmental factors (rotenone, MPP+, paraquat and cigarette smoking) we assessed in vitro cell viability, mitochondrial functions including ATP synthesis, activity of OXPHOS complexes and ROS generation, and biogenesis (mtDNA copy number) in a collection of transmitochondrial cytoplasmic hybrids (cybrids) carrying divergent human mtDNA haplogroups (N1b, H, J, T, U, and K) or LHON mutations, that have been defined by sequencing of D-loop region and then of the entire mtDNA. Cybrids were constructed from fibroblasts obtained, after informed consent, from skin biopsies of unrelated healthy subjects and LHON patients. The results of this study demonstrated that mitochondrial genetic variability may influence individual susceptibility to drugs or environmental factors toxicity, highlighting interesting associations between specific haplogroups, mitochondrial functional alterations, and toxic agent. More in details: 1) haplogroup K1 was found to play a protective role against rotenone toxicity, whereas haplogroup J1 seem to be more susceptible to the action of both rotenone and MPP+; 2) haplogroup T seems to be more susceptible to the action of paraquat; 3) haplogroups H12 and T1 in association with the LHON mutation 3460/ND1, and haplogroups J1c and J2A all increase the susceptibility to mitochondrial damage after smoke exposure. Moreover haplogroup H1, characterized by SNPs 2706A/3010A in 16SrRNA is the most sensitive to Linezolid toxicity and haplogroup J appears to act as risk factor in CisPt toxicity. Even though future studies will be necessary to better understand the mechanism of action of some of these molecules, studying the association between mitochondrial haplogroup and toxicity of drugs and chemicals is extremely useful to prevent toxicity in predisposed subjects. This may avoid the occurrence of adverse reactions leading to the withdrawal of drugs from the market or Black Box warnings by FDA. For these reasons, pharmaceutical companies have introduced early in the drug-development process stringent in vitro studies to evaluate mitochondrial function (respiratory chain, ROS, membrane potential and mtDNA).
La farmacogenomica si occupa di indagare gli effetti di un determinato farmaco o sostanza chimica in base al genotipo dell’individuo con lo scopo di personalizzare le cure e fornire le terapie adeguate. I mitocondri presentano un potenziale di membrana (Δψ) di 180mV, una matrice alcalina (pH 8) con carica negativa e possono accumulare al loro interno sia sostanze cariche positivamente sia acidi deboli in forma anionica, rendendosi bersaglio primario o secondario dell’azione di farmaci e agenti tossici. I mitocondri sono dotati di un proprio corredo genomico (mtDNA) che si caratterizza per un’ereditarietà uniparentale materna un elevato tasso di mutazione e numerose varianti genetiche, distinte in aplogruppi. È noto che variazioni non sinonime nel mtDNA causano alterazioni funzionali di proteine implicate nel processo OXPHOS, tali da supportare studi per comprendere se la variabilità mitocondriale possa svolgere un’azione protettiva o rappresentare un fattore di rischio per l’insorgenza di patologie. In particolare è stato dimostrato che soggetti appartenenti agli aplogruppi J e K e con polimorfismo 10398G nel gene ND3 (CI) si correlano a minore rischio di sviluppare il Morbo di Parkinson (PD). Al contrario la variante 4216C nel gene ND1 (CI), comune al sottogruppo JT, parrebbe correlata a un aumentato rischio di malattia. Inoltre è stato dimostrato un maggiore rischio di Neuropatia Ottica di Leber (LHON) se le mutazioni 11778/ND4 (CI), 14484/ND6 (ND1) e 3460/ND1 (CI) si associano agli aplogruppi J2b, J1c e K rispettivamente. Ulteriormente studi ipotizzano che agenti inquinanti ambientali quali pesticidi (es. rotenone), erbicidi (es. paraquat) e MPTP o 1-metil 4-fenil 1,2,3,6-tetraidro-piridina (composto secondario contaminante nella sintesi illecita di oppiacei) siano causa di un maggiore rischio di PD per un’alterata funzionalità mitocondriale con riduzione della sintesi di ATP e aumento di specie reattive dell’ossigeno (ROS). In altri studi s’ipotizza che il rischio di perdita della vista in carrier di mutazioni LHON, aumenta se il soggetto è fumatore, per azione sul CI della catena respiratoria. Infine ancora dati di letteratura associano numerose variazioni mitocondriali non sinonime allo sviluppo di patologie collaterali a trattamenti farmacologici. E’ stato dimostrato che soggetti appartenenti all’aplogruppo J, presentano un aumento del rischio di sviluppare ototossicità dopo trattamento con l’antitumorale Cisplatino (CisPt) per inibizione della replicazione del mtDNA mentre pazienti con SNPs nella regione 16SrRNA in posizione 2706A e 3010A trattati con l’antibiotico Linezolid, sviluppano mielosoppressione, neuropatia e acidosi lattica in seguito a inibizione della sintesi proteica mitocondriale. L’attività di ricerca ha avuto come oggetto di studio il ruolo della variabilità del genoma mitocondriale (mtDNA) nella suscettibilità individuale alla tossicità da farmaci (Linezolid e Cisplatino) o da agenti tossici inquinanti ambientali (rotenone, MPP+, paraquat ed estratto di fumo) in un modello in vitro costituito, da ibridi transcitoplasmatici (cibridi) creati mediante fusione di fibroblasti da donatore, dopo enucleazione, con cellule di osteosarcoma private di mtDNA (rho0). Sono così stati originati cloni di cibridi appartenenti ai principali aplogruppi mitocondriali europei (N1b, H, J, T, U, K) o portatori di diverse mutazioni LHON, e caratterizzati mediante analisi di sequenza della regione non codificante di 1122 bp o“Displacement loop” (Dloop) e dell’intero genoma mitocondriale. Gli studi sono stati eseguiti valutando la vitalità cellulare, la funzionalità (sintesi di ATP, produzione di ROS) e la biogenesi mitocondriale (numero di copie di mtDNA). I risultati ottenuti da queste analisi hanno dimostrato che una variabilità genetica mitocondriale può influenzare la suscettibilità individuale alla tossicità da agenti tossici inquinanti ambientali e farmaci. In particolare sono state evidenziate alcune interessanti associazioni tra specifici aplogruppi mitocondriali, alterazioni mitocondriali e agente tossico: 1) l’aplogruppo K1 sembra svolgere un’azione protettiva rispetto al rotenone mentre l’aplogruppo J1 sembra più sensibile all’azione del pesticida e del MPP+ anche se quest’ultimo è meno specifico e affine al CI.; 2) l’aplogruppo T sembra più suscettibile all’azione del paraquat.; 3) gli aplogruppi H12 e T1 quando associati a mutazione LHON 3460/ND1 e gli aplogruppi J1c e J2a aumentano la suscettibilità al danno mitocondriale da fumo. Successive evidenze hanno dimostrato che l’aplogroup H1, caratterizzato dai polimorfismi 2706A e 3010A nel gene 16SrRNA, sembra essere il più sensibile all’azione tossica del Linezolid così come l’aplogruppo J è risultato più sensibile alla citotossicità da Cisplatino. Questi dati suggeriscono che procedere nello studio di associazioni tra aplogruppo mitocondriale e tossicità da farmaci e agenti chimici potrebbe consentire di individuare precocemente il rischio tossicologico individuale. Queste conoscenze potrebbero essere di particolare impatto per prevenire reazioni avverse a farmaci in alcuni casi causa di ritiro dal commercio o black box. Con questo scopo di recente le aziende farmaceutiche hanno introdotto nelle fasi iniziali dello sviluppo di un farmaco studi in vitro per valutare eventuali effetti sulla funzionalità mitocondriale (catena respiratoria, ROS, potenziale di membrana e mtDNA).

Repairing DNA damage is brought about by highly specific proteins that partake in a variety of DNA repair. Two of the most common types of damage are double-strand breaks (DSBs) and interstrand crosslinks. A single DSB or crosslink can potentially kill a cell if it is not repaired~ In human and other vertebrate cells, DSBs are repaired by two different mechanisms. The nonhomologous end-joining pathway can bring together the broken ends and join them, usually with the loss of some nucleotide sequence. A second pathway, homologous recombinational repair (HRR), is equally important. This repair process utilizes the information provided by another DNA molecule to restore damaged DNA. This molecule is usually a sister chromatid arising from DNA replication. This process is essentially error-free, unlike the end-joining process. Some HRR activity is required for proliferating cells to remain viable. The central protein player is RAD51, which with the help of other proteins such as XRCC2, XRCC3, RAD51B, RAD51C, and RAD51D, performs the critical initiating steps of homologous pairing and strand transfer. The proteins encoded by the familial breast cancer genes, brcal and brca2, also play an important role in HRR. My project is concerned with studying proteins that interact with Drosophila melanogaster (XRCC2). Proteins interacting with DmXRCC2 were identified by using a yeast two hybrid system. "Bait fusion protein" (DmXRCC2 linked to GAIA BD) was constructed by Dr. Wrischnik. Tanya Dimetrijevich, a graduate student, used this bait to fish for interacting or "target" proteins. About 50 such proteins were found. I began validating these target proteins with the intention of exploring novel interactions and functions of DmXRCC2. The process of validating proteins interacting with DmXRCC2 yielded two very interesting candidate proteins-CaBPl and FAF. · CaBPl, also called protein disulfide isomerase P5, is an endoplasmic-reticulum calciumbinding protein. FAF belongs to a large family of deubiquitinating enzymes that cleave ubiquitin-protein bonds and play diverse roles in the ubiquitin pathway. One of the implications of such discoveries could be to compare and contrast DmXRCC2 and human XRCC2 in terms of their interactions and functions.

Plants maintain organelle genomes that are descended from ancient microbes. Ages ago, these ancient microbes were engulfed by larger cells, beginning a process of co-evolution we now call the endo-symbiotic theory. Over time, DNA from the engulfed microbe was transferred to the genome of the larger engulfing cell, eventually losing the ability to be free-living, and establishing a permanent residency in the larger cell. Similarly, the larger cell came to rely so much on the microbe it had engulfed, that it too lost its ability to survive without it. Thus, mitochondria and plastids were born. Nearly all multicellular eukaryotes possess mitochondria; however, different evolutionary pressures have created drastically different genomes in plants versus animals. For one, animals have very compact, efficient mitochondrial genomes, with about 97% of the DNA coding for genes. These genomes are very consistent in size across different animal species. Plants, on the other hand, have mitochondrial genomes 10 to more than 100 times as large as animal mitochondrial genomes. Plants also use a variety of mechanisms to replicate and maintain their DNA. Central to these mechanisms are nuclear-encoded, organelle targeted replication proteins. To date, there are two DNA polymerases that have been identified in plant mitochondria and chloroplasts, Pol1A and Pol1B. There is also a DNA helicase-primase that localizes to mitochondria and chloroplasts called Twinkle, which has similarities to the gp4 protein from T7 phage. In this dissertation, we discuss the roles of the polymerases and the effects of mutating the Pol1A and Pol1B genes respectively. We show that organelle genome copy number decreases slightly and over time but with little effect on plant development. We also detail the interactions between Twinkle and Pol1A or Pol1B. Plants possess the same organellar proteins found in animal mitochondria, which are homologs to T7 phage DNA replication proteins. We show that similar to animals and some phage, plants utilize the same proteins in similar interactions to form the basis of a DNA replisome. However, we also show that plants mutated for Twinkle protein show no discernable growth defects, suggesting there are alternative replication mechanisms available to plant mitochondria that are not accessible in animals.

Dividing cells are constantly under threat from both endogenous and exogenous DNA damaging stresses that can lead to mutations and structural variations in DNA. One contributor to genome instability is three-stranded DNA:RNA hybrid structures called R-loops. Though R-loops are known to induce DNA damage and DNA replication stress, it is unclear whether they are recognized and processed by an established DNA repair pathway prior to inducing DNA breaks. Canonically, DNA repair proteins work downstream of R-loop-induced DNA damage to stimulate repair and suppress genome instability. Recently, the possibility that some DNA repair pathways actively destabilize R-loops, thus preventing unscheduled DNA damage has emerged. Here we identify the helicase SGS1 as a suppressor of R-loop stability. Our data reveals that SGS1 depleted cells accumulate R-loops. In addition, we define a role for transcription in genome instability of cells lacking SGS1, which is consistent with an R-loop based mechanism. Hyper-recombination in SGS1 mutants is dependent on transcript length, transcription rate, and active DNA replication. Also, rDNA instability in sgs1Δ can be suppressed by ectopic expression of RNaseH1, a protein that degrades DNA:RNA hybrids. Interestingly, R-loops are known to form at rDNA loci. We favour a model in which SGS1 contributes to the stabilization of stalled replication forks associated with transcription complexes, and unresolved DNA:RNA hybrids. Finally, we showed that knockdown of the human Sgs1 orthologue BLM in HCT116 cells also led to the accumulation of more R-loops than control HCT116 cells. In summary, our data supports the idea that some DNA repair proteins involved in replication fork stabilization might also prevent and process R-loops.
Science, Faculty of
Graduate

Un nombre croissant d’études soutiennent le fait que les protéines majeures du métabolisme des ARN, telles que les hélicases ARN, sont impliquées dans la réponse aux dommages à l’ADN. Cette activité est généralement accomplie par leur interaction avec des facteurs de réparation de l’ADN. BRCA2, une protéine suppressive de tumeurs, joue un rôle crucial dans la réparation des cassures double-brin (CDB) de l'ADN par recombinaison homologue (RH) et donc, est un facteur essentiel pour l’intégrité du génome. Les cellules déficientes pour BRCA2 accumulent des hybrides ADN-ARN ou R-loops, une source de dommage à l'ADN, suggérant ainsi l’importance de cette protéine dans la prévention ou la suppression de ces structures. Toutefois, le rôle spécifique de BRCA2 dans la résolution des hybrides ADN-ARN reste inconnu.Afin de connaître des potentiels partenaires de BRCA2, une analyse par spectrométrie de masse réalisée dans notre laboratoire a révélé un enrichissement en protéines impliquées dans le métabolisme de l'ARN, comme les hélicases ARN. Ces résultats nous ont menés à examiner la coopération entre BRCA2 et les hélicases ARN dans la séparation des structures ADN-ARN. Nous avons d’abord confirmé l'interaction entre l'hélicase ARN DDX5 et BRCA2, qui est améliorée dans les cellules exposées à γ-irradiation. Ensuite, nous avons réduit l’interaction aux premiers 250 aa de BRCA2 (BRCA2T1) et avons constaté que celle-ci est directe en utilisant des protéines purifiées. En collaboration avec le laboratoire du docteur A. Aguilera (Cabimer, SP), nous avons montré que la déplétion de DDX5 conduit à une accumulation des hybrides ADN-ARN dans l’entièreté du génome, particulièrement aux sites de dommages à l’ADN. De plus, nos résultats indiquent que DDX5 localise aussi aux hybrides ARN-ADN qui se forment à proximité de CDB.De manière intéressante, nous avons constaté que BRCA2 est important pour la rétention de DDX5 aux sites de dommage à l’ADN induit par l’irradiation laser. Notamment, des tests de déroulement de brins in vitro en utilisant les protéines purifiées DDX5 et BRCA2 ont révélé que BRCA2 stimule l’activité de déroulement des R-loops de DDX5.Un variant de signification inconnue (VSI) trouvé dans de patients atteints de cancer du sein situé dans la région BRCA2T1 (T207A) réduit l’interaction de BRCA2 avec DDX5 et conduit à l’accumulation des hybrides ADN-ARN. Les cellules exprimant stablement BRCA2-T207A montrent également une diminution de l’association de DDX5 avec les hybrides ARN-ADN, en particulier lors d’une exposition de cellules à l’irradiation. L’analyse de l’efficacité de la réparation des CDB par RH dans les cellules déficientes en DDX5 ou exprimant BRCA2-T207A, montre une cinétique retardée de l’apparition des foyers de réparation RAD51 lors de l’irradiation, ce qui suggère un rôle actif de l’interaction BRCA2-DDX5 pour assurer la réparation par RH efficacement. En accord avec cette hypothèse, la ribonucléase RNAseH1, qui dégrade spécifiquement la fraction d’ARN dans les structures d’ADN-ARN, restaure partiellement le phénotype de cinétique des foyers RAD51 dans les cellules BRCA2 T207A. De plus, les cellules portant le variant BRCA2-T207A ont également montré un nombre réduit de foyers RPA par rapport aux cellules qui expriment BRCA2 sauvage, témoins d’un défaut dans l’étape qui précède le chargement de RAD51 aux CDB.Ensemble, nos résultats suggèrent que les hybrides ADN-ARN représentent un obstacle à la réparation des CDB par RH et révèlent BRCA2 et DDX5 en tant que facteurs actifs dans leur suppression
Increasing evidence support the idea that proteins involved in RNA metabolism such as RNA binding proteins (RBPs) and RNA helicases are directly implicated in the DNA damage response (DDR). This activity is generally achieved through their interaction with DNA repair factors.BRCA2 is a tumor suppressor protein that plays an important role in the repair of DNA double-strand breaks (DSBs) by homologous recombination (HR) as well as protecting stalled replication forks from unscheduled degradation; therefore, it is essential to maintain genome integrity. Interestingly, BRCA2 deficient cells accumulate DNA-RNA hybrids or R-loops, a known source of DNA damage and genome instability, providing evidence for its role in either R-loop prevention or processing. However, the specific role of BRCA2 on these structures remains poorly understood.A mass spectrometry screen to identify partners of BRCA2 performed in our laboratory revealed an enrichment of proteins involved in RNA metabolism such as RNA helicases. These findings led us to investigate whether BRCA2 could cooperate with these candidate interacting RNA helicases in processing DNA-RNA structures. First, we confirmed the interaction of BRCA2 and the DEAD-box RNA helicase DDX5, which we found is enhanced in cells exposed to -irradiation. Then, we narrowed down the interaction to the first 250 aa of BRCA2 (BRCA2T1) and found that it is direct using purified proteins. In collaboration with A. Aguilera lab (Cabimer, SP), we could show that depletion of DDX5 leads to a genome-wide accumulation of DNA-RNA hybrids that is particularly enriched at DNA damage sites. DDX5 associates with DNA-RNA hybrids that form in the vicinity of DSBs. Interestingly, we found that BRCA2 is important for the retention of DDX5 at laser irradiation-induced DNA damage. Notably, in vitro R-loop unwinding assays using purified DDX5 and BRCA2 proteins revealed that BRCA2 stimulates the R-loop helicase activity of DDX5.A breast cancer variant of unknown clinical significance (VUS) located in BRCA2T1 (T207A) reduced the interaction between BRCA2 and DDX5 and led to the accumulation of DNA-RNA hybrids. Cells stably expressing BRCA2-T207A also showed a decreased association of DDX5 with DNA-RNA hybrids, especially upon irradiation. Notably, monitoring RAD51 foci to evaluate HR-mediated DSBs repair efficiency in either DDX5-depleted cells or in BRCA2-T207A cells resulted in a delayed kinetics of appearance of RAD51 foci upon irradiation suggesting an active role of BRCA2-DDX5 interaction in ensuring timely HR repair. In agreement with this, overexpression of the RNAseH1 ribonuclease, that specifically degrades the RNA moiety in DNA-RNA structures, partially restored RAD51 kinetics phenotype of BRCA2-T207A cells. Moreover, cells bearing BRCA2-T207A variant also showed a reduced number of RPA foci compared to BRCA2 WT expressing cells, a step that precedes RAD51 loading at DSBs.Taken together, our results are consistent with DNA-RNA hybrids being an impediment for the repair of DSBs by HR and reveal BRCA2 and DDX5 as active players in their removal

L'ADN est en permanence exposé à un grand nombre d'événements dommageables déclenchées par des agents endogènes et exogènes. De nombreux travaux expérimentaux ont fourni des informations cruciales sur les propriétés structurelles et la réparation de certains des lésions de l'ADN. Cependant, il manque une vision mécanistique ou énergétique sur leur formation. La biochimie computationnelle a émergé comme un outil puissant pour comprendre les réactions biochimiques et les propriétés électroniques de systèmes complexes.Dans cette thèse, nous étudions la formation de lésions complexes intra-brin et inter-brin. Ces lésions tandem constituent une puissant menace à l'intégrité du génome, en raison de leur haute fréquence mutagenique. Tout d'abord, nous discutons l'attaque d'une liaison covalente entre un radical pyrimidinique. En comparant avec les bases isolees, nos simulations hybrides Car-Parrinello demontrent que la reactivité de la thymine et de la cytosine radicalaires sont inversees dans l'environnement B-helical. De plus, nos resultats montrent egalement une deformation plus importante pour la lesion G[8-5]C.Nous rationalisons également la plus grande réactivité des cytosines par rapport aux purines vers la formation multi-etapes de lésions complexes inter-brins par condensation avec un site C4' abasique. Ces résultats bases sur des simulations avec solvatation explicite et combines a la théorie de la fonctionnelle de la densité sont en accord avec les données expérimentales
DNA is continuously exposed to a vast number of damaging events triggered by endogenous and exogenous agents. Numerous experimental studies have provided key information regarding structural properties of some of the DNA lesions and their repair. However, they lack in mechanistic or energetic information pertaining to their formation. Computational Biochemistry has emerged as a powerful tool to understand biochemical reactions and electronic properties of large systems.In this thesis we study the formation of inter- and intra-strand cross-links. These tandem lesions pose a potent threat to genome integrity, because of their high mutagenic frequency. First, we discuss the formation of complex defects which arise from the attack of a pyrimidine radical onto guanine. In comparison with the reactivity of isolated nucleobases, our hybrid Car-Parrinello Molecular Dynamics simulations reveal that the reactivity of hydrogen-abstracted thymine and cytosine is reversed within a B-helix environment. Further, our data also suggest a more severe distortion of the B-helix for G[8-5]C.Second, we rationalize the higher reactivity of cytosine vs. purines toward the multistep formation of inter-strand crosslinks with a C4' oxidized a basic site, which is in qualitative agreement with experiments on isolated nucleobases, using explicit solvent simulations combined to density functional theory

La plupart des études sur l’hybride naturel entre Schistosoma haematobium (S.) et S. guineensis sont réalisées sur les vers adultes et contrairement aux études expérimentales sur l’hybridation, on ne retrouve pas de vers adultes hybrides après analyse de leur ADN. Avec cette étude, nous souhaitons mettre en évidence la présence d’hybride naturel entre ces deux espèces au Gabon à partir du premier élément suspect : l’œuf. Nous avons suivi l’œuf de son observation morphologique, à sa coloration par la technique de Ziehl-Neelsen jusqu’à l’amplification par PCR de son ADN et on a pu montrer qu’un œuf de morphologie suspecte observé dans les urines est capable d’amplifier à la fois une région spécifique de S. haematobium et de S. guineensis
Most studies on the natural hybrid between Schistosoma haematobium (S.) and S.guineensis are performed on adult worms and contrary to experimental studies of hybridization, we do not find an adult hybrid worm after analysis of their DNA. With this study, we wish to highlight the presence of a natural hybrid between these two species in Gabon from the first suspect element: the egg. We followed the egg from its morphological observation to its staining using Ziehl-Neelsen technique until PCR amplification of its DNA and it has been shown that a suspected egg morphology seen in the urine is able to amplify both a specific region of S. haematobium and S. guineensis

The negative effects from rising carbon levels have created the need to find alternative energy sources that are more carbon neutral. One such alternative energy source is to use the biomass derived from forest trees to fulfill the need for a renewable alternative fuel. Through increased understanding and optimization of regulatory mechanisms that control wood development the potential exists to increase biomass yield. Transcription factors (TFs) are DNA-binding regulatory proteins capable of either activation or repression by binding to a specific region of DNA, normally located in the 5-prime upstream promoter region of the gene. In the first section of this work, six DNA promoters from wood formation-related genes were screened by the Yeast One-Hybrid (Y1H) assay in efforts to identify novel interacting TFs involved in wood formation. The promoters tested belong to genes involved in lignin biosynthesis, programmed cell death, and cambial zone associated TFs. The promoters were screened against a mini-library composed of TFs expressed 4-fold or higher in differentiating xylem vs phloem-cambium. The Y1H results identified PtrRAD1 with interactions involving several of the promoters screened. Further testing of PtrRAD1 by Yeast Two-Hybrid (Y2H) assay identified a protein-protein interaction (PPI) with poplar DIVARACATA RADIALIS INTERACTING FACTOR (DRIF1). PtrDRIF1 was then used in the Y2H assay and formed PPIs with MYB/SANT domain proteins, homeodomain family (HD) TFs, and cytoskeletal-related proteins. In the second section of this work, PPIs involving PtrDRIF1s' interaction partners were further characterized. PtrDRIF1 is composed of two separate domains, an N-terminal MYB/SANT domain that interacted with the MYB/SANT domain containing PtrRAD1 and PtrDIVARICATA-like proteins, and a C-terminal region containing a Domain of Unknown Function 3755 (DUF3755). The DUF3755 domain interacted with HD family members belonging to the ancient WOX clade and Class II KNOX domain TFs. In addition, PtrDRIF1 was able to form a complex between PtrRAD1 and PtrWOX13c in a Y2H bridge assay. PtrDRIF1 may function as a regulatory module linking cambial cell proliferation, lignification, and cell expansion during growth. Combined, these findings support a role for PtrDRIF1 in regulating aspects of wood formation that may contribute to altering biomass yield.
Ph. D.

Telomeres are repetitive sequences of DNA at the ends of chromosomes that become progressively shorter during cell division, acting as a form of “biological clock” causing cell death once they have reached a certain length. Almost 90% of cancer cells overexpress the enzyme telomerase which can lengthen telomeres and confer immortality to the tumour cells. Thus, telomerase has become an important target for drug discovery in the oncology area, and there is also interest from researchers investigating the aging process. During the catalytic cycle of telomerase, a unique DNA/RNA hybrid duplex (DRH) forms that is typically between 6-11 base pairs long and is key to extending the telomere. There is interest in discovering small drug-like molecules that can recognize and bind to this hybrid duplex to inhibit selectively telomerase, either by stabilizing the structure and thereby preventing telomerase dissociation (a key step in the catalytic cycle) or by sufficiently distorting the hybrid duplex to cause the misalignment of key catalytic groups. This project began by using oligonucleotides representing DNA/RNA hybrid duplex (DRH), telomeric G-quadruplex and control duplex DNA sequences to screen against the National Cancer Institute compound libraries (i.e., Diversity Set II, Mechanistic Set and Natural Product Set) using a high throughput Fluorescent Resonance Energy Transfer (FRET)-based DNA thermal denaturation assay to determine binding affinity and specificity. Thirteen novel chemical scaffold families were identified in the assay, compounds which showed a >5 °C selective stabilization of the DNA/RNA hybrid duplex at a 1 μM ligand concentration. Chemical modifications were then made to these scaffolds to generate focused libraries of analogues to improve selectivity, potency and drug-likeness, and to provide Structure-Activity Relationship (SAR) information. A total of 49 novel molecules were synthesized and then screened against an expanded range of four different nucleic acid constructs including telomeric and DNA/RNA hybrid duplex sequences. A number of compounds showed selective DNA/RNA hybrid stabilization potential with some compounds also showing notable telomeric G-quadruplex stabilization without significant affinity for promoter G-quadruplexes (i.e., c-Kit1, c-Kit-2 and c-Myc) and control duplex DNA sequences. The compounds from library-1 provided DNA/RNA hybrid duplex stabilization in the 0.5-7.2 C range and telomeric G-quadruplex stabilization in the 0.2-6.5 C range at a 1 μM ligand concentration. Molecular modelling and molecular dynamics studies confirmed that the methylene spacer between the benzimidazole and phenylene moieties of molecules within library-1 is perfectly shaped to fit within the DRH sequence. In addition, it was confirmed that minor-groove binding and simultaneous intercalation between the nucleobases of a DNA/RNA hybrid duplex requires a linker of specific length (i.e., an eight methylene spacer as in compound 3.3). Selected compounds were then studied further using a variety of biological techniques to confirm selective telomerase inhibition and cell-based assays to utilize their potential as antitumour agents.

During DNA replication, forks often stall upon encountering obstacles blocking their progression. Cells will act to speedily remove or overcome such barriers, thus allowing complete synthesis of chromosomes. This is the case for R-loops, DNA/RNA hybrids that arise during transcription. One mechanism to remove such R-loops involve DNA/RNA helicases. Here, I have shown that one such helicase, Sen1, associates with replisome components during S phase in the model organism S. cerevisiae. I demonstrate that the N-terminal domain of Sen1 is both sufficient and necessary for the interaction of the protein with the replisome. I also identified Ctf4 as one of at least two replisome interactors of Sen1. By mutational analysis, a mutant of Sen1 (Sen1-3) that cannot interact with the replisome was created. This mutant is healthy on its own but is lethal in the absence of both RNase H1 and H2. Overexpression of the sen1-3 allele from the constitutive ACT1 promoter is able to suppress this synthetic lethality, suggesting that Sen1 travels with replisomes in order to be quickly recruited at sites of R-loops that impair fork progression so as to remove those R-loops. In some cases, cells exploit fork stalling for biologically important processes. This is the case in Sz. pombe, where an imprint prevents complete DNA replication, triggering cell-type switching. This imprint is dependent on Pol1, a component of the replisome. Importantly, a single imprinting-defective allele of pol1 has been identified to date. Using in vitro assays, I have shown that this Pol1 mutant has reduced affinity for its substrates and is a correspondingly poor polymerase. By generating novel alleles of pol1, I have also demonstrated that switching-deficiency correlates with the affinity of Pol1 for its substrates in vivo. Finally, two interactors of Pol1 (Mcl1Ctf4 and Spp1Pri1 ) have been shown to have switching defects. S. cerevisiae and Sz. pombe have similar yet distinct genetic nomenclature conventions. Given that both model organisms were used in this study, it is important to highlight the conventions for both organisms to prevent confusion. In S. cerevisiae, wildtype gene names are expressed as a three letter, uppercase and italic name followed by a number (e.g. SEN1). The three letter name often corresponds to the screen through which the gene in question was originally identified. Mutants are generally designated with the same three letter but in lower case (unless the mutant is dominant) and with an allele designation (e.g. sen1∆, sen1-1 and sen1-2). Because of historical context, the allele designations vary in format (e.g. leu2-3,112 is a mutant of LEU2). Protein names are given as a three letter name with the first letter in uppercase (e.g. Sen1). This is also true for mutant proteins, with the added allele designation (e.g Sen1-1 and Sen1-2). In this study, I have generated constructs of the SEN1 gene and these constructs are referred to as SEN1 (X-Y), where X and Y refer to the first and last residues being encoded for. The corresponding proteins are referred to as Sen1 (X-Y). Different promoters have been used and, where appropriate, the promoters are expressed similarly to their wildtype gene names (e.g. GAL1, SEN1 and ACT1). In Sz. pombe, wildtype gene names are expressed as a three letter, lowercase and italic name followed by a number (e.g. pol1). Mutants are generally designated in the same format but with an allele designation. Like in S. cerevisiae, the allele designation varies widely (e.g. pol1-1, pol1-H4 and pol1-ts13). Additionally, because of the historical context, some (but not all) alleles of pol1 are referred to as swi7 to reflect the fact that they are defective for cell-type switching. Similar to the situation in S. cerevisiae, proteins names are given as a three letter name with the first letter in uppercase for both wildtype and mutants (e.g. Pol1 and Swi7-1). Sometimes, for the sake of comparison, genes or proteins are referred to their S. cerevisiae orthologues (e.g. swi1TOF1 and Swi1Tof1 , respectively). Several protein tags have been used in this study. When written in gene form, they were written in capital letters and italicized, irrespective of the host (e.g. 5FLAG) and when in protein form, they were written in capital, irrespective of the host (e.g. 5FLAG).

Kyoto University (京都大学)
0048
新制・論文博士
博士(農学)
乙第11497号
論農博第2532号
新制||農||897(附属図書館)
学位論文||H16||N3958(農学部図書室)
22629
UT51-2004-J769
(主査)教授 谷坂 隆俊, 教授 山田 利昭, 教授 遠藤 隆
学位規則第4条第2項該当

Faithful transmission of genetic material is challenged by the presence of natural impediments affecting replication fork progression that jeopardize genome integrity. Transcription, which competes with DNA replication for the same template, is a common barrier to replication in both prokaryotes and higher eukaryotes. Multiple mechanisms minimize the consequences of DNA replication and transcription collisions in order to prevent torsional stress accumulation that occurs when replication fork encounters the transcription machinery. Defects in resolving topological problems during chromosome replication lead to fork reversal, R loop formation and recombination-induced genome rearrangements. Our interest is focused on the processes that coordinate replication with transcription at TERs (termination sites) and on the molecular pathways involved in termination of DNA replication. We investigated the roles of Rrm3, a DNA helicase that assists replication fork progression, and of Sen1, a DNA/RNA helicase that resolves the conflicts between replication and transcription. We found that Rrm3 and Sen1 mediate replication termination at specific TERs, preventing aberrant events ultimately leading to chromosome fragility. Our results contribute to the elucidation of mechanisms coordinating replication and transcription at TER zones in eukaryotes.

Les dommages oxydatifs de l’ADN sont impliqués dans les processus pathologiques que sont le cancer, les maladies neurodégénératives ou le vieillissement. Ces dommages résultent en partie de l’action des espèces réactives de l’oxygène (ERO), qui proviennent du métabolisme cellulaire ou d’agents exogènes (physiques ou chimiques), et qui conduisent à différents types de lésions parmi lesquelles l’oxydation des bases de l’ADN (8-oxoguanine, 8-oxoG) ou la formation de sites abasiques AP (apurique/apyrimidique). Ces lésions, qui si elles ne sont pas éliminées conduisent à des processus de mutagenèse ou de mort cellulaire, sont prises en charge spécifiquement par le système de réparation de l’ADN par excision de base ou BER. Le BER est initié par l’action d’une ADN glycosylase, telles que la 8-oxoG-ADN glycosylase (Ogg1) chargée d’éliminer la 8-oxoG, une lésion très abondante. Une étude par « double-hybride » initiatrice de ce projet a révélé l’existence d’une interaction in vivo chez S. cerevisiae entre la protéine yOgg1 et la sous-unité catalytique de l’ADN polymérase réplicative Polε (yPol2), également impliquée dans la voie BER chez la levure. Nos travaux démontrent que yOgg1 et yPol2 interagissent bien physiquement entre elles et de façon spécifique. Une étude par troncations et mutagenèse dirigée nous a permis d’identifier le domaine 3’→5’ exonucléase de yPol2 comme faisant partie de la forme tronquée minimale de yPol2 capable d’interagir avec yOgg1. La poche du site actif de yOgg1 et/ou son voisinage immédiat pourrait contenir pour partie le site d’interaction pour yPol2. Nous observons d’ailleurs une corrélation nette entre l’activité de yOgg1 et sa capacité à interagir avec yPol2 dans la levure. De même, l’activité 3’→5’ exonucléase de yPol2 pourrait être liée à son interaction avec yOgg1. D’un point de vue fonctionnel, yPol2 stimulerait l’activité AP lyase de yOgg1 et le couplage entre l’activité ADN glycosylase et AP lyase de l’enzyme, permettant ainsi une meilleure coordination de l’étape d’excision du nucléoside endommagé et l’étape de resynthèse de l’ADN dans la voie BER
Oxidative DNA damages are involved in pathological processes such as cancer, neurodegenerative diseases and aging. Part of these damages results from the action of reactive oxygen species (ROS), which are produced by cellular metabolism or (physical or chemical) exogenous agents. They lead to different types of DNA lesions including DNA base oxidation (8-oxoguanine, 8-oxoG) and abasic site formation (AP, apuric/apyrimidic). If not removed, these lesions lead to mutagenesis or cell death. Most of base lesions are dealt specifically by the base excision repair (BER) pathway. BER is initiated by a DNA glycosylase, such as 8-oxoG-DNA glycosylase (Ogg1) which is responsible for the removal of 8-oxoG. In previous unpublished work, a yeast two-hybrid study revealed the existence in S. cerevisiae of an interaction between yOgg1 and the catalytic subunit of the replicative DNA polymerase Polε (yPol2), also involved in the BER pathway in eukaryotes. Our work shows that yOgg1 and yPol2 physically and specifically interact with each other. Truncation and site-directed mutagenesis studies allowed us to identify the 3 ' → 5' exonuclease activity domain of yPol2 as part of the minimal form of yPol2 still able to interact with yOgg1. The active site of yOgg1 and/or its immediate vicinity may contain part of its interaction domain with yPol2. Besides, we observe a clear correlation between yOgg1 catalytic activity and its ability to interact with yPol2 in vivo. Similarly, the 3'→5' exonuclease activity of yPol2 could be useful to its interaction with yOgg1. From a functional point of view, yPol2 stimulates in vitro the AP lyase activity of yOgg1 and the coupling of both DNA glycosylase and AP lyase enzyme activity. The interaction yOgg1/yPol2 could allow a better coordination of damaged nucleoside excision and DNA re-synthesis steps in BER

The elucidation of the composition and organization of genomes of higher plants is a fundamental problem of modern molecular biology. The genus Beta containing 14 species assigned to the sections Beta, Corollinae, Nanae and Procumbentes provides a suitable system for the comparative study of the nuclear genomes. Sugar beet Beta vulgaris has a genome size of 758 Mbp DNA with estimated 63 % repetitive sequences and the number of chromosomes n=9. The wild beet Beta procumbens is an important natural pool of resistance against pests and tolerance to unfavorable growth conditions. The subject of this research was the isolation and description of new repetitive DNA families from genomes of this Beta species. This work presents the molecular investigation and cytogenetic characterization by high-resolution multicolor fluorescent in situ hybridization (FISH) of the satellite and dispersed repetitive sequences in wild and cultivated beet species and in their hybrids. New repetitive sequences were isolated from the B. procumbens genome. The AluI restriction satellite repeats pAp11 are 229-246 bp long and form subfamilies. The satellite is amplified in the section Procumbentes, but also found in distantly related section Beta. Thus, pAp11 is probably an ancient component of Beta genomes. It could be the ancestor of the satellite subfamily pEV4 in B. vulgaris based on sequence analysis, Southern hybridization and comparative FISH. pAp11 was found at centromeric and a few intercalary sites in B. procumbens and formed intercalary blocks on B. vulgaris chromosomes where it co-localized with pEV4. These remarkable differences in the chromosomal position of pAp11 between Procumbentes and Beta species indicate that both satellites were likely involved in the expansion or rearrangement of the intercalary heterochromatin of B. vulgaris. Other two sequence families characterized on molecular, genomic and chromosomal levels are the non-homologous repeats pAp4 and pAp22, 1354 and 582 bp long. They have a dispersed organization in the genome and are widely scattered along B. procumbens chromosomes. pAp4 and pAp22 are specific for the section Procumbentes and can be used as DNA probes to discriminate parental genomes in interspecific hybrids. High-resolution FISH on meiotic chromosomes showed that the both sequences mostly co-localize. The PCR analysis of their flanking regions revealed that pAp22 is a part of a Long Terminal Repeat (LTR) of an Athila-like env-class retrotransposon. This is the first indication that the retrovirus-like DNA elements exist in Beta. An ancient family of subtelomeric satellite DNA pAv34 was isolated from all four sections of the genus Beta and from spinach, a related Chenopodiaceae. Five clones were analyzed from each of the five species. The genomic organization and species distribution of the satellites were studied by sequencing and Southern hybridization. The repeating units in all families are 344-362 bp long and share 46.2-98.8 % similarity. Each monomer consists of two subunits SU1 and SU2 of 165-184 bp. The maximum likelihood and neighbor joining analyses of the 25 subtelomeric satellite monomers and their subunits indicated, that the duplication leading to the emergence of the 360 bp satellite should have occurred early in the phylogeny. The two directions of diversification are the clustering of satellites in two groups of subunits SU1 and SU2 and the arrangement of satellite repeats in section-specific groups. The comparative chromosomal localization of the telomeric repeat, pAv34 and rDNA was investigated by multicolor FISH. B. vulgaris chromosome termini showed unique physical organization of telomeric repeat and the subtelomeric satellite, as studied by high-resolution FISH on extended DNA fibers. The estimated length of the telomeric array was 0.55 - 62.65 kb, the length of pAv34 was 5.0-125.25 kb, the spacer between these sequences spanned 1.0-16.60 kb. Eight various classes of repeats were used to characterize the minichromosomes of the sugar beet fragment addition lines PRO1 and PAT2 by comparative multi-color FISH. The study allowed to propose a schematic pattern of repetitive DNA organization on the PRO1 and PAT2 minichromosomes. PRO1 has an acrocentric minichromosome, while PAT2 possesses a metacentric or submetacentric chromosome fragment. The functional integrity of the fragment addition line centromeres was confirmed by an immunostaining localization of the proteins specific to the active kinetochore. The serine 10-phosphorylated histone H3 was detected in pericentromeric regions of the PRO1 chromosomes. The microtubuli attachment sites were visualized as parts of kinetochore complexes.