Ribonucleotide reductases (RNR) are essential enzymes that catalyze the reduction of ribonucleotides to 2'-deoxyribonucleotides, which is a critical step that produces precursors for DNA replication and repair. The inactivation of RNR, logically, would discontinue producing the precursors of the DNA of viral or cancer cells, which then would consequently end the cycle of DNA replication. Among different compounds that were found to be inhibitors of RNR, 2'-azido-2'-deoxynucleotide diphosphates (N3NDPs) have been investigated in depth as potent inhibitors of RNR. Decades of investigation has suggested that the inactivation of RNR by N3NDPs is a result of the formation of a nitrogen-centered radical (N•) that is covalently attached to the nucleotide at C3' and cysteine molecule C225 [3'-C(R-S-N•-C-OH)]. Biomimetic simulation reactions for the generation of the nitrogen-centered radicals similar to the one observed during the inactivation of the RNR by azionuclotides was investigated. The study included several modes: (i) theoretical calculation that showed the feasibility of the ring closure reaction between thiyl radicals and azido group; (ii) synthesis of the model azido nucleosides with a linker attached to C3' or C5' having a thiol or vicinal dithiol functionality; (iii) generation of the thiyl radical under both physiological and radiolysis conditions whose role is important in the initiation on RNR cascades; and (iv) analysis of the nitrogen-centered radical species formed during interaction between the thiyl radical and azido group by electron paramagnetic resonance spectroscopy (EPR). Characterization of the aminyl radical species formed during one electron attachment to the azido group of 2'-azido-2'-deoxyuridine and its stereospecifically labelled 1'-, 2'-, 3'-, 4'- or 5,6-[2H2]-analogues was also examined. This dissertation gave insight toward understanding the mechanism of the formation of the nitrogen-centered radical during the inactivation of RNRs by azidonucleotides as well as the mechanism of action of RNRs that might provide key information necessary for the development of the next generation of antiviral and anticancer drugs.

No description available.

Face aux agressions constantes que subit l’ADN, les cellules ont développé des mécanismes de protection, nommés checkpoints pour maintenir l’intégrité de leur génome. Chez Saccharomyces cerevisiae, la kinase Rad53 joue un rôle central dans ces voies et son activation conduit à de nombreux effets cellulaires tels que le ralentissement du cycle cellulaire, le ralentissement de la réplication, l’activation de la transcription de certains gènes, l’activation de la réparation… Lors d’un crible transcriptomique, utilisant une souche exprimant une forme hyperactive de Rad53, nous avons identifié le gène HUG1 comme l’un des gènes les plus transcrits suite à l’activation de la voie RAD53. Cependant les fonctions de Hug1 demeurent énigmatiques.Pour mieux comprendre les fonctions de Hug1 dans la réponse aux dommages de l’ADN, nous avons recherché ses partenaires physiques et avons identifié les protéines Rnr2 et Rnr4, les deux composants de la petite sous-unité de la Ribonucléotide Réductase (RNR). La RNR est un complexe enzymatique qui catalyse l’étape limitante de synthèse des nucléotides. Nous avons alors cherché à caractériser cette interaction par diverses méthodes. Nous avons ainsi montré que Hug1 est une protéine intrinsèquement désordonnée capable d’interagir physiquement avec la petite sous-unité de la RNR et qu’au moins onze acides aminés de Hug1 sont impliqués dans son interaction avec la RNR. Lors de nos investigations, nous avons observé que le fait d’étiqueter Rnr2 en position C-terminale sensibilisait les souches aux stress génotoxiques et que cette sensibilité était supprimée si on abrogeait la fonction de HUG1, faisant de Hug1 un nouvel inhibiteur de la RNR. Ainsi nous sommes parvenus à proposer un modèle de régulation de la RNR par Hug1. / To maintain genome integrity, cells have developed protection mechanisms, called checkpoints, in response to DNA damage insults. In Saccharomyces cerevisiae, Rad53 protein kinase is one of the major actors in these mechanisms, and its activation triggers several cellular responses such as cell cycle delay, replication delay, transcription modifications, activation of DNA repair pathways… Using an hyperactivative allele of RAD53, we identified HUG1, as one of the most induced gene in a transcriptomic analysis upon RAD53 pathway activation. However Hug1’s functions remains elusive.To better understand Hug1’s functions in DNA damage response, we searched for physical partners and identified Rnr2 and Rnr4 proteins, which are the two small subunits of Ribonucleotide Reductase (RNR). The RNR is an enzymatic complex that catalyses nucleotide reduction, a step limiting for dNTPs synthesis. We next experimentally tackled the Hug1-RNR interaction using various methods. We showed so that Hug1 is a small intrinsically disordered protein able to interact physically with the small RNR subunit and that at least eleven amino acids in Hug1 are involved in this interaction. During our investigations, we observed that C-terminal tagging of Rnr2 sensitizes strains to genotoxics stress and that this sensitivity was suppressed when HUG1’s function is abrogated. Hence, we showed that Hug1 is a negative RNR regulator and propose a model for Hug1’s function.

<p>Hybrid density functional theory (DFT) B3LYP method is applied to study the four diverse enzyme systems: <i>zinc-containing peptidases</i> (thermolysin and stromelysin),<i> methyl-coenzyme M reductase</i>, <i>ribonucleotide reductases</i> (classes I and III), and <i>superoxide dismutases</i> (Cu,Zn- and Ni-dependent enzymes). Powerfull tools of modern quantum chemistry are used to address the questions of biological pathways at their molecular level, proposing a novel mechanism for methane production by methyl-coenzyme M reductase and providing additional insights into hydrolysis by zinc peptidases, substrate conversion by ribonucleotide reductases, and biological superoxide dismutation. Catalysis by these enzymes, with the exception of zinc peptidases, involves radical chemistry.</p>

Hybrid density functional theory (DFT) B3LYP method is applied to study the four diverse enzyme systems: zinc-containing peptidases (thermolysin and stromelysin), methyl-coenzyme M reductase, ribonucleotide reductases (classes I and III), and superoxide dismutases (Cu,Zn- and Ni-dependent enzymes). Powerfull tools of modern quantum chemistry are used to address the questions of biological pathways at their molecular level, proposing a novel mechanism for methane production by methyl-coenzyme M reductase and providing additional insights into hydrolysis by zinc peptidases, substrate conversion by ribonucleotide reductases, and biological superoxide dismutation. Catalysis by these enzymes, with the exception of zinc peptidases, involves radical chemistry.

Diss. (sammanfattning) Umeå : Univ., 2008. / Härtill 4 uppsatser.

No description available.

La division cellulaire est un processus fondamental des êtres vivants. À chaque division cellulaire, le matériel génétique d'une cellule mère est dupliqué et ségrégé pour produire deux cellules filles identiques; un processus nommé la mitose. Tout d'abord, la cellule doit condenser le matériel génétique pour être en mesure de séparer mécaniquement et également le matériel génétique. Une erreur dans le niveau de compaction ou dans la dynamique de la mitose occasionne une transmission inégale du matériel génétique. Il est suggéré dans la littérature que ces phénomènes pourraient causé la transformation des cellules cancéreuses. Par contre, le mécanisme moléculaire générant la coordination des changements de haut niveau de la condensation des chromosomes est encore incompris. Dans les dernières décennies, plusieurs approches expérimentales ont identifié quelques protéines conservées dans ce processus. Pour déterminer le rôle de ces facteurs dans la compaction des chromosomes, j'ai effectué un criblage par ARNi couplé à de l'imagerie à haute-résolution en temps réel chez l'embryon de C. elegans. Grâce à cette technique, j'ai découvert sept nouvelles protéines requises pour l'assemblage des chromosomes mitotiques, incluant la Ribonucléotide réductase (RNR) et Topoisomérase II (topo-II). Dans cette thèse, je décrirai le rôle structural de topo-II dans l'assemblage des chromosomes mitotiques et ces mécanismes moléculaires. Lors de la condensation des chromosomes, topo-II agit indépendamment comme un facteur d'assemblage local menant par la suite à la formation d'un axe de condensation tout au long du chromosome. Cette localisation est à l'opposé de la position des autres facteurs connus qui sont impliqués dans la condensation des chromosomes. Ceci représente un nouveau mécanisme pour l'assemblage des chromosomes chez C. elegans. De plus, j'ai découvert un rôle non-enzymatique à la protéine RNR lors de l'assemblage des chromosomes. Lors de ce processus, RNR est impliqué dans la stabilité des nucléosomes et alors, permet la compaction de haut niveau de la chromatine. Dans cette thèse, je rapporte également des résultats préliminaires concernant d'autres nouveaux facteurs découverts lors du criblage ARNi. Le plus important est que mon analyse révèle que la déplétion des nouvelles protéines montre des phénotypes distincts, indiquant la fonction de celles-ci lors de l'assemblage des chromosomes. Somme toute, je conclus que les chromosomes en métaphase sont assemblés par trois protéines ayant des activités différentes d'échafaudage: topoisomérase II, les complexes condensines et les protéines centromériques. En conclusion, ces études prouvent le mécanisme moléculaire de certaines protéines qui contribuent à la formation des chromosomes mitotiques. / Cell division is a fundamental process that continuously happens in all living organisms. In each cell division, genetic material of the parent cell duplicates and segregates to produce genetically identical daughter cells in a process called mitosis. Cells need to condense their genetic material to be able to partition them equally. Any subtle defects, either timing or compaction level, could lead to the unequal inheritance of genetic material, a phenomenon that is believed to be the leading cause of cancerous transformation. However, the precise molecular mechanisms underlying the coordinated changes of higher-order chromosome structure are poorly understood. In the last two decades, various approaches have identified several conserved factors required for chromosome condensation. To define the roles of known and novel factors in this process, I performed an RNAi based screen using high-resolution live imaging of the C. elegans one-cell embryo. Importantly, using an in vivo approach, I discovered seven novel factors required for mitotic chromosome assembly, including Ribonulceotide reducatase (RNR) and DNA topoisomerase II (topo-II). In this thesis, I report a structural role for topo-II in mitotic chromosome assembly and underlying molecular mechanisms. During chromosome condensation process, topo-II acts independently as a local assembly factor leading to global chromosome axis formation, contradicting models that chromosomes organize around preassembled scaffolds, thus representing a novel pathway for chromosome assembly in C. elegans. Furthermore, I also discovered a non-enzymatic role of RNR in the mitotic chromosome assembly process. During this process, RNR is involved in nucleosome stability, and thereby, it allows higher-order chromatin assembly. In this thesis, I also report preliminary data for other novel factors that I discovered in the RNAi based screen for factors involved in chromosome condensation. Importantly, my analyses revealed that the depletion of several proteins results in distinct chromosome condensation phenotypes, indicating that they function in discrete events during mitotic chromosome assembly. In sum, I conclude that metaphase chromosomes are built by the distinct scaffolding activities of three proteins: DNA topoisomerase II, condensin complexes and centromere proteins. Taken together, these studies provide underlying molecular mechanisms contributing to the mitotic chromosome formation.

Understanding the functional impact of physical interactions between proteins and DNA on gene expression is important for developing approaches to correct disease-associated gene dysregulation. I conducted a systematic, functional genetic analysis of protein-DNA interactions in the promoter region of the yeast ribonucleotide reductase subunit gene RNR3. I measured the transcriptional impact of systematically perturbing the major transcriptional regulator, Crt1, and three X-box sites on the DNA known to physically bind Crt1. This analysis revealed interactions between two of the three X-boxes in the presence of Crt1, and unexpectedly, a significant functional role of the X-boxes in the absence of Crt1. Further analysis revealed Crt1- independent regulators of RNR3 that were impacted by X-box perturbation. Taken together, these results support the notion that higher-order X-box-mediated interactions are important for RNR3 transcription, and that the X-boxes have unexpected roles in the regulation of RNR3 transcription that extend beyond their interaction with Crt1.

Understanding the functional impact of physical interactions between proteins and DNA on gene expression is important for developing approaches to correct disease-associated gene dysregulation. I conducted a systematic, functional genetic analysis of protein-DNA interactions in the promoter region of the yeast ribonucleotide reductase subunit gene RNR3. I measured the transcriptional impact of systematically perturbing the major transcriptional regulator, Crt1, and three X-box sites on the DNA known to physically bind Crt1. This analysis revealed interactions between two of the three X-boxes in the presence of Crt1, and unexpectedly, a significant functional role of the X-boxes in the absence of Crt1. Further analysis revealed Crt1- independent regulators of RNR3 that were impacted by X-box perturbation. Taken together, these results support the notion that higher-order X-box-mediated interactions are important for RNR3 transcription, and that the X-boxes have unexpected roles in the regulation of RNR3 transcription that extend beyond their interaction with Crt1.