Em eucariotos, a origem de replicação é reconhecida por um complexo ORC, a proteína Cdc6 e outras proteínas. Nos tripanossomas, encontramos somente uma proteína similar a Orc1 e Cdc6, que chamaremos de Orc1/Cdc6. Nesta tese serão descritos os estudos realizados sobre Orc1/Cdc6 do Trypanosoma cruzi e do Trypanosoma brucei. As proteínas recombinantes dos tripanossomas apresentam atividade de ATPase e são capazes de substituir Cdc6 em ensaio de complementação em leveduras. A indução do silenciamento do gene de Orc1/Cdc6 por RNAi resulta em células anucleadas. Orc1/Cdc6 é expressa durante todo o ciclo celular das formas replicativas, permanecendo associada à cromatina. No caso do T.cruzi, Orc1/Cdc6 é expressa não só nas formas replicativas, mas também nas formas não replicativas. Nestas últimas, a proteína expressa não interage com o DNA, este resultado sugere que a ausência desta interação deve contribuir para ausência da duplicação do DNA nas formas infectivas do T. cruzi. / In eukaryotes, the replication origin is recognized by a complex ORC, Cdc6 and other proteins. The trypanosomes contain only one protein, we named it Orc1/Cdc6. Here we show that the recombinant Orc1/Cdc6 from T.cruzi (TcOrc1/Cdc6) and from T.brucei (TbOrc1/Cdc6) present ATPase activity, replaced yeast Cdc6 in a phenotypic complementation assay. The induction of Orc1/Cdc6 silencing by RNA interference in T.brucei resulted in enucleated cells. Orc1/Cdc6 is expressed during the entire cell cycle and in all stages of the life cycle of trypanosomes, remaining associated with chromatin in all stages of the cell cycle. This association is different among the stages from T. cruzi life cycle. In the non replicative ones, Orc1/Cdc6 does not interact with DNA. The lack of pre-replication machinery-DNA interaction in T. cruzi non-replicative stages might contribute to the absence of DNA replication in these stages.

Em eucariotos, a origem de replicação é reconhecida por um complexo ORC, a proteína Cdc6 e outras proteínas. Nos tripanossomas, encontramos somente uma proteína similar a Orc1 e Cdc6, que chamaremos de Orc1/Cdc6. Nesta tese serão descritos os estudos realizados sobre Orc1/Cdc6 do Trypanosoma cruzi e do Trypanosoma brucei. As proteínas recombinantes dos tripanossomas apresentam atividade de ATPase e são capazes de substituir Cdc6 em ensaio de complementação em leveduras. A indução do silenciamento do gene de Orc1/Cdc6 por RNAi resulta em células anucleadas. Orc1/Cdc6 é expressa durante todo o ciclo celular das formas replicativas, permanecendo associada à cromatina. No caso do T.cruzi, Orc1/Cdc6 é expressa não só nas formas replicativas, mas também nas formas não replicativas. Nestas últimas, a proteína expressa não interage com o DNA, este resultado sugere que a ausência desta interação deve contribuir para ausência da duplicação do DNA nas formas infectivas do T. cruzi. / In eukaryotes, the replication origin is recognized by a complex ORC, Cdc6 and other proteins. The trypanosomes contain only one protein, we named it Orc1/Cdc6. Here we show that the recombinant Orc1/Cdc6 from T.cruzi (TcOrc1/Cdc6) and from T.brucei (TbOrc1/Cdc6) present ATPase activity, replaced yeast Cdc6 in a phenotypic complementation assay. The induction of Orc1/Cdc6 silencing by RNA interference in T.brucei resulted in enucleated cells. Orc1/Cdc6 is expressed during the entire cell cycle and in all stages of the life cycle of trypanosomes, remaining associated with chromatin in all stages of the cell cycle. This association is different among the stages from T. cruzi life cycle. In the non replicative ones, Orc1/Cdc6 does not interact with DNA. The lack of pre-replication machinery-DNA interaction in T. cruzi non-replicative stages might contribute to the absence of DNA replication in these stages.

No description available.

Tumorigenesis is a multifaceted set of events consisting of the deregulation of several cell-autonomous and tissue microenvironmental processes that ultimately leads to the acquisition of malignant disease. Transforming growth factor beta (TGFß) and its family members are regulatory cytokines that function to ensure proper organismal development and the maintenance of homeostasis by controlling cellular differentiation, proliferation, adhesion, and survival, as well as by modulating components of the cellular microenvironment and immune system. The pleiotropic control by TGFß of these cell intrinsic and extrinsic factors is intimately linked to the prevention of tumor formation, the specifics of which are dependent on the various cellular and/or molecular signaling contexts that exist for TGFß. The diverse roles and the various levels of signal control for TGFß lend themselves to certain characteristics that are more advantageous for cancers to usurp in order to promote tumorigenesis, while other anti-tumorigenic roles for TGFß are more beneficial to tumor development if they are circumvented or disabled. Transforming growth factor ß1 (TGF-ß1) exerts its anti-tumor effects in large part by potently inhibiting cell cycle progression at any point in G1 phase to control the proliferation of a variety of cell lineages. Loss of sensitivity to TGF-ß1-induced cell cycle arrest is a crucial event during early tumorigenesis. Indeed, cancer cells of almost all tumor types display insensitivity to TGF-ß1 inhibition. As such, the pursuit of the molecular details underlying the TGF-ß1 growth arrest pathway is important for our understanding of cell cycle regulation, and significantly, how disruption of these mechanisms contributes to TGF-ß1 insensitivity and tumorigenesis. TGF-ß1 inhibition of the cell cycle in G1 phase has been shown to involve two main transcriptionally based molecular events, including the induction of cyclin-dependent kinase (CDK) inhibitors and the suppression of the c-Myc protein. Both mechanisms contribute to the maintenance of the retinoblastoma (Rb) protein in its hypophosphorylated and antiproliferative form, thus preventing progression through the cell cycle. However, this type of regulation does not offer answers to all of the questions regarding TGF-ß1 arrest. While these transcriptional mechanisms provide explanations for TGF-ß1 arrest throughout most of G1, inhibition late in G1 by TGF-ß1 however, does not require any acute regulation of transcription. In addition, the chance to utilize canonical TGF-ß1 arrest mechanisms at this time has already passed (i.e. Rb is already hyperphosphorylated by late-G1). Previous work from our group shows instead that late-G1 TGF-ß1 cell cycle arrest requires an intact direct interaction between the N-terminus of Rb (RbN) and the C terminus of Mcm7, a subunit of the Cdc45-MCM-GINS (CMG) replicative helicase. Our studies show that TGF-ß1 exposure in late-G1 prevents the disassociation of Rb with fully assembled helicases, which remain inactive. In addition, it was found that early-G1 treatment with TGF-ß1 also targets CMG components, namely MCM protein accumulation (and therefore hexamer formation) in G1 is blocked. However, the residue(s) of RbN involved as well as the molecular mechanisms Rb utilizes for late-G1 TGF-ß1 arrest are not described, nor is it evident from this work if TGF-ß1 affects other genes involved in CMG assembly and/or activation. In the following study we explore these unanswered questions for TGF-ß1 growth arrest as a means to understand novel aspects of cell cycle regulation that must be abrogated during tumorigenesis. Our hypothesis is that CMG helicase control on some level is critical for all TGF-ß1-induced inhibition of cell cycle progression throughout the entire G1 phase. In Chapter 2 herein we have investigated the details and mechanistic implications of the Rb/RbN inhibitory-interaction with the CMG helicase that is required for late-G1 TGF-ß1 arrest. We show that N-terminal exons of Rb that are lost in partially penetrant hereditary retinoblastomas inhibit DNA replication and elongation using a bipartite mechanism. Specifically, Rb exon 7 is necessary and sufficient to inhibit CMG helicase activation, while an independent loop domain within RbN that forms a projection blocks DNA polymerase α (Pol-α) and Ctf4 recruitment without affecting polymerases δ and ε or the CMG helicase. Individual disruption of exon 7 or the projection in RbN or Rb, as occurs in inherited cancers, partially impairs the ability of Rb/RbN to inhibit DNA replication and block G1-S cell cycle transit. Importantly, their combined loss abolishes these functions of Rb. Thus, TGF-ß1 cell cycle arrest in late-G1 requires the growth suppressive role of Rb in which replicative complexes are blocked directly via independent and additive N-terminal domains. TGF-ß1-induced arrest in late-G1 also requires the presence of Smad3 and Smad4, suggesting that a novel transcription-independent role may exist for Smad signaling proteins in blocking cell cycle transit directly in Rb-CMG inhibitory complexes. TGF-ß1 is thought to require a functional Rb protein to inhibit the cell cycle at any point in G1 phase. Intriguingly, while cells lacking Rb (and the inhibitory N-terminal domains) lose sensitivity to TGF-ß1 arrest in late-G1, these same cells remain sensitive to TGF-ß1 inhibition in early-G1. This Rb-independent TGF-ß1 growth arrest also occurs in the absence of c-Myc and MCM suppression, as well as without CyclinE-Cdk2 inhibition, but requires Smad3 and Smad4 respectively. Here (Chapter 3) we have identified the mechanism by which TGF-ß1 achieves Smad-dependent G1 arrest in the absence of these common mediators. TGF-ß1 inhibits the assembly of CMG replicative helicases by suppressing the recruitment of the MCM complex to chromatin. Accordingly, the entire heterohexamer fails to load onto DNA. Cdc6 phosphorylation in its amino terminus is known to be required for Cdt1-dependent loading of the MCM complex. We show that in Rb-lacking cells early-G1 TGF-ß1 treatment blocks the phosphorylation of Cdc6 at serine 54, without affecting total Cdc6 protein levels, to prevent MCM heterohexamer formation on DNA. Consistent with TGF-ß1 signals targeting this recruitment and loading step, Cdt1 overexpression promotes S-phase entry in the presence of TGF-ß1, circumventing the need for Cdc6 phosphorylation. Importantly, Cdt1 requires an intact C-terminal MCM-binding domain in order to overcome this TGF-ß1-induced cell cycle arrest mechanism. These data indicate that early-G1 TGF-ß1 arrest can occur by perturbing Cdc6 phosphorylation to block Cdt1-mediated MCM recruitment and loading, leading to inhibition of CMG assembly and S-phase entry despite the lack of Rb and normal c-Myc and CyclinE-Cdk2 activities. We conclude that the main event governing TGF-ß1-induced cell cycle arrest at any point in G1 is the inhibition of the assembly and/or activation of the replicative CMG helicase. However, TGF-ß1 growth arrest has a temporal dependence on the presence of the Rb protein. In normal cells containing Rb, the accumulation of MCM subunit proteins is blocked by TGF-ß1 in early-G1 and accordingly MCM heterohexamers are unable to form. However, if cells are allowed to transit to late-G1 when MCM complexes have already assembled on origins, but before functional CMG helicases have formed at G1-S, exposure to TGF-ß1 signaling prevents CMG activation via interactions with critical inhibitory domains within RbN. Cells lacking Rb (and these residues) are not sensitive to TGF-ß1 in late-G1. Surprisingly, these cells remain sensitive to TGF-ß1 early in G1 phase despite a lack of c-Myc/MCM protein suppression and CyclinE-Cdk2 inhibition. In these cells the recruitment and loading of the MCM complex is blocked to facilitate a TGF-ß1-mediated G1 arrest. It is only when this mechanism is overcome by Cdt1 overexpression that TGF-ß1 is unable to elicit cell cycle arrest in these cells. These data provide molecular explanations for studies reporting instances of TGF-ß1 arrest without canonical effectors, such as Rb, c-Myc loss, or CDK inhibitors. Additionally, this work argues for the development of novel cancer therapeutics targeting CMG helicase assembly or activation, the regulation of which is likely lost in a variety of TGF-ß1-insensitive and/or Rb-deficient malignancies. Indeed, reintroduction of these tumor suppressive pathways has shown efficacy in blocking growth of tumors or cancer cells lacking the same mechanisms. Our studies of Rb/RbN inhibition of DNA replication also provide proof of principle for this type of therapy, as well as the framework for how the CMG might be targeted by exploring further and perhaps mimicking Rb exon7-mediated CMG inhibition biochemically.

L’objectif de cette thèse a été de comprendre deux caractéristiques majeures des divisions méiotiques chez la femelle: le blocage en prophase de 1ère division méiotique qui permet à l’ovocyte d’accumuler des réserves énergétiques et des déterminants nécessaires au développement embryonnaire ; et l’absence de phase-S entre les deux divisions méiotiques ce qui permet de former des cellules haploïdes aptes à la fécondation. Pour cela, j’ai choisi comme modèle d’étude l’ovocyte de Xénope qui permet de suivre ces processus in vitro en réponse à la progestérone. L’ovocyte subit les deux divisions méiotiques grâce à l’activation du facteur universel de la division cellulaire, le MPF, et se bloque en métaphase de 2ème division méiotique dans l’attente d’être fécondé. Chez tous les vertébrés, le 1er arrêt en prophase dépend de l’activité de la protéine kinase dépendante de l’AMPc, PKA, dont l’inactivation est nécessaire pour la reprise de la méiose. Le substrat de PKA dans l’ovocyte était resté inconnu. Nous avons découvert que la protéine Arpp19, jusqu’alors connue pour son rôle positif dans l’activation du MPF, est phosphorylée par PKA de cette phosphorylation bloque l’activation du MPF nécessaire pour la levée du blocage en prophase. ARPP19 possède donc un double rôle, le 1er exercé comme substrat de PKA et responsable de l’arrêt en prophase, le second dans l’activation du MPF suite à un changement dans sa phosphorylation. Dans un second temps, nous avons étudié la protéine Cdc6, un acteur majeur de la réplication de l’ADN. Absente en prophase, Cdc6 s’accumule entre les deux divisions méiotiques ce qui permet à l’ovocyte d’acquérir la compétence à répliquer l’ADN. Cette compétence ne s’exprime pas ce qui permet de réduire de moitié la ploïdie. Nous avons montré que Cdc6 est un inhibiteur puissant du MPF capable de bloquer les divisions méiotiques et d’induire la réplication de l’ADN. Pour éviter ces effets délétères l’accumulation de Cdc6 est strictement régulée lors des deux divisions méiotiques, ce qui est absolument requis pour assurer l’enchainement des deux divisions cellulaires sans phase-S intercalaire. / The goal of my PhD project was to understand two main features of the female meiotic division: the arrest in prophase of the 1st meiotic division that allows the accumulation of nutrients and determinants necessary for the embryonic cell cycles; and the absence of S-phase between the two meiotic divisions in order to produce haploid gametes. For this purpose, I studied Xenopus oocytes, a powerful model system that allows the biochemical analysis of these two processes in vitro. In ovary, oocytes are arrested in prophase I and resume meiosis in response to progesterone. The oocytes then proceed through the 1st and the 2nd meiotic divisions and halt at metaphase II, awaiting for fertilization. These two consecutive divisions are controlled by two waves of Cdk1 activation, the universal factor responsible for the entry into mitosis. I analysed the mechanisms responsible for arresting the oocyte in prophase I. In all vertebrates, this arrest depends on a high activity of the cAMP-dependent protein kinase, PKA, whose downregulation is required for the release of the prophase block. The substrate of PKA had never been identified up to date. I discovered that the small protein Arpp19, already known for positively regulating entry into M-phase, is phosphorylated by PKA in prophase I and is dephosphorylated upon progesterone addition, an event required for Cdk1 activation. Hence, Arpp19 has a dual function, responsible of the prophase arrest as a PKA substrate, and then converted into an activator of Cdk1 by changes of its phosphorylation pattern. The second part of my thesis has been dedicated to understanding the role and the regulation of the Cdc6 protein during meiotic divisions. This protein is essential for DNA replication in somatic cells. It is accumulated between the two oocyte meiotic divisions and restores the competence to replicate DNA in oocyte. However, this competence is repressed before fertilization, allowing formation of haploid cells. I found that the accumulation of Cdc6 is tightly controlled during meiotic maturation by the Cyclin B accumulation and the Mos/MAPK pathway. I further demonstrated that Cdc6 is a strong inhibitor of Cdk1 in Xenopus oocytes and that the timely accumulation of Cdc6 is required to coordinate the two meiotic divisions with no intercaling S-phase.

Les organismes cellulaires appartiennent à l'un des trois domaines du vivant : Archaea, Bacteria, Eucarya. Les Archaea sont des organismes unicellulaires avec un phénotype bactérien mais qui possèdent de nombreux caractères moléculaires eucaryotes. En particulier, la machinerie de réplication archéenne est une version homologue et simplifiée de celle des eucaryotes. Au cours de cette thèse, j'ai étudié la réplication de l'ADN chez les Archaea en combinant des approches in vitro et in silico.<br />Premièrement, j'ai essayé de purifier la protéine initiatrice de la réplication Cdc6/Orc1, sous une forme native, dans l'espoir de mettre au point le premier système de réplication de l'ADN in vitro chez les Archaea. Malheureusement, cette approche a été infructueuse en raison de l'instabilité et des propriétés d'agrégation de la protéine.<br />Deuxièmement, j'ai réalisé une analyse comparative du contexte génomique des gènes de réplication dans les génomes d'Archaea. Cette analyse nous a permis d'identifier une association très conservée entre des gènes de la réplication et des gènes liés au ribosome. Cette organisation suggère l'existence d'un mécanisme de couplage entre la réplication de l'ADN et la traduction. De manière remarquable, des données expérimentales obtenues chez des modèles bactériens et eucaryotes appuient cette idée. J'ai ensuite mis au point des outils expérimentaux qui permettront d'éprouver la pertinence biologique de certaines des prédictions effectuées.<br />Finalement, j'ai examiné la distribution taxonomique des gènes de la réplication dans les génomes d'Archaea afin de prédire la composition probable de la machinerie de réplication de l'ADN chez le dernier ancêtre commun des Archaea. Dans leur ensemble, les profils phylétiques des gènes de la réplication suggèrent que la machinerie ancestrale était plus complexe que celle des organismes archéens contemporains.

La maturation méiotique des vertébrés diffère de la mitose par plusieurs aspects. J'ai étudié deux de ces particularités. 1) En méiose I, les chromosomes homologues sont ségrégés, en mitose, les chromatides sœurs sont séparées. En mitose, un mécanisme de contrôle bloque la cellule en métaphase en inhibant l'APC/C tant que tous les chromosomes ne sont pas correctement alignés sur le fuseau. En méiose I, des résultats contradictoires existent selon les espèces quant à l'existence d'un mécanisme de contrôle de ce type. J'ai montré que l'activité séparase (activité indirectement régulée par l'APC/C) est requise pour effectuer la transition métaphase/anaphase en méiose I, suggérant qu'un mécanisme de contrôle de ce type est requis chez la souris, organisme proche de l'homme. 2) A l'issue de la maturation méiotique, l'ovocyte reste bloqué en métaphase de méiose II en attendant la fécondation, alors que la mitose s'achève toujours. Ce blocage est dû à l'activité CSF et requiert la voie Mos/.../MAPK. J'ai montré que DOC1R, un nouveau substrat des MAPK, contrôle l'organisation des microtubules au cours de l'arrêt CSF. Ces résultats font évoluer la vision de l'arrêt CSF qui était considéré comme une voie linéaire aboutissant à la stabilisation du MPF. L'arrêt CSF est une voie non linéaire contrôlant aussi la morphologie de l'ovocyte.