Dephosphorylation of the Cdc2 kinase by the Cdc25 tyrosine phosphatase is the universally conserved trigger for mitotic entry. Cdc25 is also the point of convergence for checkpoint signaling pathways which monitor the genome for damaged DNA and incomplete replication. In addition, Cdc25 is inhibited by a MAP kinase cascade in the event of osmotic, oxidative and/or heat stress. These pathways inhibit cell cycle progression by phosphorylating Cdc25 resulting in its association with 14-3-3 and nuclear export. Although Cdc25 can be observed leaving the nucleus following inhibitory signals it is controversial whether phosphorylation, 14-3-3 binding or export itself is required for checkpoint proficiency. In fission yeast, Cdc25 is phosphorylated in vitro on 12 serine and threonine residues by the effector kinase of the DNA replication checkpoint, Cds1. Nine of these residues reside in the N-terminal regulatory region, while three are found in the extreme C-terminus of the protein. We show here that phosphorylation the nine N-terminal residues, nor any of the 12 in vitro sites, are required for enforcement of the DNA replication checkpoint. In lieu of Cdc25 phosphorylation the phosphatase is rapidly degraded and mitotic entry prevented by the action of the Mik1 kinase, targeting Cdc2. Thus, multiple mechanisms exist for preventing mitotic entry when S-phase progression is inhibited. The three C-terminal in vitro phosphorylation sites have not previously been examined in fission yeast. However, homology exists between the S. pombe protein and the Cdc25 orthologues in humans, Xenopus and Drosophila in this region. We report here that in S. pombe these sites are required to prevent mitotic entry following replication arrest in the absence of Mik1, and in the maintenance, but not establishment, of arrest following DNA damage. Our previous work showed that Cdc25 nuclear import requires the Sal3 importin-β but at the time we were unable to show a direct interaction between these two proteins. The final chapter of this thesis proves physical interaction by co-immunoprecipitation. Cdc25 mutants lacking all twelve putative Cds1 sites show nuclear localization during mitosis in a sal3- background, effectively reversing the cell cycle regulated pattern of accumulation of the phosphatase. / Thesis (Ph.D, Biology) -- Queen's University, 2011-06-20 12:16:15.71

Notre génome subit constamment les effets néfastes des agents endommageant de l'ADN. Afin de se protéger de ces effets délétères, les cellules disposent d’un système de détection des dommages à l’ADN (point de contrôle ou « checkpoint »). Certaines lésions peuvent persister quand les cellules entrent en phase S et inhiber ainsi la synthèse de l’ADN en interférant avec les ADN polymérases réplicatives. Ceci peut provoquer des arrêts prolongés des fourches de réplication ce qui fragilise l’ADN. Pour préserver l’intégrité de l’information génétique, les cellules ont développé une voie de tolérance qui implique des ADN polymérases spécialisées dans la réplication des lésions, appelées ADN Polymérases translésionnelles (Pols TLS). Dans ce processus, PCNA joue le rôle de facteur d’échafaudage pour de nombreuses protéines impliquées dans le métabolisme de l'ADN. Les mécanismes de régulation des échanges entre les différents partenaires de PCNA ne sont pas très bien compris. Parmi les protéines qui interagissent avec PCNA, CDT1, p21 ou encore PR-Set7/Set8 sont caractérisées par une forte affinité pour cette protéine. Ces dernières possèdent un motif d’interaction particulier avec PCNA, nommé « PIP degron », qui favorise leur protéolyse d'une manière dépendante de l’E3 ubiquitine ligase CRL4Cdt2. Après irradiation aux UV-C, le facteur d’initiation de la réplication CDT1 est rapidement détruit d’une manière dépendante de son PIP degron, Dans la première partie de mon travail, j’ai contribué à comprendre le rôle fonctionnel de cette dégradation. Les résultats obtenus ont fourni des évidences expérimentales qui montrent que l’inhibition de la dégradation de CDT1 par CRL4Cdt2 dans les cellules de mammifères compromet la relocalisation des TLS Pol eta et Pol kappaen foyers nucléaires induits par les irradiations UV-C. On a constaté que seules les protéines qui contiennent un PIP degron interfèrent avec la formation de foyers de Pol eta. La mutagenèse du PIP degron de CDT1 a révélé qu'un résidu de thréonine conservé parmi les PIP degrons est essentiel pour l'inhibition de la formation des foyers des TLS Polymérases. Les résultats obtenus suggèrent que l’élimination de protéines contenant des PIP degrons par la voie CRL4Cdt2 régule le recrutement de TLS Polymérases au niveau des sites des dommages induits par les UV-C.Dans un second temps, on s’est intéressé à l’étude du checkpoint des dommages à l’ADN au cours de l’embryogénèse. En effet, dans les embryons précoces, le checkpoint est silencieux jusqu'à la transition de mid-blastula (MBT), en raison de facteurs maternels limitants. Dans ce travail, nous avons montré, aussi bien in vitro qu’in vivo, que l’ubiquitine ligase de type E3 RAD18, un régulateur majeur de la translésion, est un facteur limitant pour l’activation du checkpoint dans les embryons de xénope. Nous avons montré que l'inactivation de la fonction de l’ubiquitine ligase RAD18 conduit à l'activation du checkpoint par un mécanisme qui implique l’arrêt des fourches de réplication en face des lésions produites par les UV-C. De plus, nous avons montré que l'abondance de RAD18 et de PCNA monoubiquitiné (PCNAmUb) est régulée au cours de l’embryogénèse. À l’approche de la MBT, l’abondance de l'ADN limite la disponibilité de RAD18, réduisant ainsi la quantité de PCNAmUb et induisant la dé-répression du checkpoint. En outre, nous avons montré que cette régulation embryonnaire peut être réactivée dans les cellules somatiques de mammifères par l'expression ectopique de RAD18, conférant une résistance aux agents qui causent des dommages à l'ADN. Enfin, nous avons trouvé que l'expression de RAD18 est élevée dans les cellules souches cancéreuses de glioblastome hautement résistantes aux dommages de l'ADN. En somme, ces données proposent RAD18 comme un facteur embryonnaire critique qui inhibe le point de contrôle des dommages de l’ADN et suggèrent que le dérèglement de l’expression de RAD18 peut avoir un potentiel oncogénique inattendu / Our genome is continuously exposed to DNA damaging agents. In order to preserve the integrity of their genome, cells have evolved a DNA damage signalling pathway known as checkpoint. Some lesions may persist when cells enter the S-phase and halt the progression of replicative DNA polymerases. This can cause prolonged replication forks stalling which threaten the stability of the genome. To preserve the integrity of genetic information, cells have developed a tolerance pathway which involves specialized DNA polymerases, called translesion DNA polymerases (TLS Pols). These polymerases have the unique ability to accommodate the damaged bases thanks to their catalytic site. In this process, PCNA acts as a scaffold for many proteins involved in DNA metabolism. The mechanisms governing the exchanges between different PCNA partners are not well understood. Among the proteins that interact with PCNA, CDT1, p21 and PR-Set7/set8 are characterized by a high binding affinity. These proteins have a particular interaction domain with PCNA, called "PIP degron", which promotes their proteasomal degradation via the E3 ubiquitin ligase CRL4Cdt2. After UV-C irradiation, the replication initiation factor CDT1 is rapidly degraded in a PIP degron-dependent manner. During the first part of my work, we wanted to understand the functional role of this degradation. Our results have shown that inhibition of CDT1 degradation by CRL4Cdt2 in mammalian cells, compromises the relocalisation of TLS Pol eta and Pol kappato nuclear foci after UV-C irradiation. We also found that only the proteins which contain a PIP degron interfere with the formation of Pol eta foci. Mutagenesis experiments on CDT1 PIP degron revealed that a threonine residue conserved among PIP degrons is essential for inhibiting foci formation of at least two TLS polymerases. This results suggest that CRL4Cdt2-dependent degradation of proteins containing PIP degrons regulates the recruitment of TLS polymerases at sites of UV-induced DNA damage.During the second part of my thesis, we studied DNA damage checkpoint regulation during embryogenesis. Indeed, in early embryos, the DNA damage checkpoint is silent until the mid-blastula transition (MBT) due to maternal inhibiting factors. In this work, we have shown, both in vitro and in vivo, that the E3 ubiquitin ligase RAD18, a major regulator of translesion DNA synthesis, is a limiting factor for the checkpoint activation in Xenopus embryos. We have also shown that RAD18 depletion leads to the activation of DNA damage checkpoints by inducing replication fork uncoupling in front of the lesions. Furthermore, we showed that the abundance of RAD18 and PCNA monoubiquitination (PCNAmUb) is regulated during embryonic development. Near the MBT, the increased abundance of DNA limits the availability of RAD18, thereby reducing the amount of PCNAmUb and inducing the de-repression of the checkpoint. Moreover, we have shown that this embryonic-like regulation can be reactivated in somatic mammalian cells by ectopic expression of RAD18, conferring resistance to DNA damaging. Finally, we found high RAD18 levels in glioblastoma cancer stem cells highly resistant to DNA damage. All together, these data propose RAD18 as a critical factor that inhibits DNA damage checkpoint in early embryos and suggests that dysregulation of RAD18 expression may have an unexpected oncogenic potential

This thesis investigates how pairwise combinatorial gene and stimulus perturbation experiments are conducted and interpreted. In particular, I investigate gene perturbation in the form of knockout, which can be achieved in a pairwise manner by SGA or CRISPR/Cas9 methods. In the present literature, I distinguish two approaches to interpretation: the calculation of stimulus and gene interactions, and the identification of equality among phenotypes measured for distinct perturbation conditions. I describe how each approach has been applied to derive hypotheses about gene regulatory networks. I identify conflicts and uncertainties in the assumptions allowing these derivations, and explore theoretically and experimentally approaches to improve the interpretation of genetic interaction data. I apply the approaches to a well-studied gene regulatory branch of the DNA damage checkpoint (DDC) pathway of Saccharomyces cerevisiae, and confirm the known order of genes within this pathway. I also describe observations that seem inconsistent with this pathway structure. I explore this inconsistency experimentally and discover that high concentrations of the DNA alkylating drug methyl methanesulfonate cause a cell division arrest program distinct from a G1 or G2/M checkpoint or from DNA damage adaptation, that resembles an endocycle.

Replication protein A (RPA) is a eukaryotic single-stranded DNA-binding protein consisting of three subunits of 70-, 32-, and 14-kDa (RPA70, RPA32, RPA14, respectively). It is a protein essential for most cellular DNA metabolic pathways. Checkpoint proteins Rad9, Rad1, and Hus1 form a clamp-like complex which plays a central role in the DNA damage-induced checkpoint response. In this report, we presented the evidence that Rad9-Rad1-Hus1 (9-1-1) complex directly interacted with RPA in human cells, and this interaction was mediated by the binding of Rad9 protein to both RPA70 and RPA32 subunits. In addition, the cellular interaction of 9-1-1 with RPA or hyperphosphorylated RPA was stimulated by UV irradiation or camptothecin treatment in a dose-dependent manner. Such treatments also resulted in the colocalization of the nuclear foci formed with the two complexes. Consistently, knockdown of the RPA expression in cells by the small interference RNA (siRNA) blocked the DNA damage-dependent chromatin association of 9-1-1, and also inhibited the 9-1-1 complex formation. Taken together, our results suggest that 9-1-1 and RPA complexes collaboratively function in DNA damage responses, and that the RPA may serve as a regulator for the activity of 9-1-1 complex in the cellular checkpoint network.

An intact cellular response to DNA damage is important for the maintenance of genomic stability and tumour prevention. EDD, the human orthologue of Drosophila melanogaster ???hyperplastic discs???, is over-expressed or mutated in a number of common human cancers. EDD is a progestin regulated gene that encodes an E3 ubiquitin ligase involved in cell communication and cell adhesion, and although it has also been implicated in the DNA damage response through its association with DNA damage proteins, a definitive role has yet to be demonstrated. The work presented herein shows that EDD is necessary for an adequate cellular response to double-strand DNA breaks. Cells depleted of EDD exhibit reduced survival, radio-resistant DNA synthesis and failure to maintain G2/M arrest following DNA damage induced by phleomycin exposure. Furthermore, EDD-depleted cells display impaired activating phosphorylation and kinase activity of the checkpoint kinase CHK2 after DNA damage. These effects appear to be largely modulated through a phospho-dependent interaction involving the CHK2 FHA domain and a region of EDD spanning a number of putative FHA-binding threonines. These results identify EDD as a novel mediator in DNA damage signal transduction via CHK2 and emphasise the potential importance of EDD in cancer.

An intact cellular response to DNA damage is important for the maintenance of genomic stability and tumour prevention. EDD, the human orthologue of Drosophila melanogaster ???hyperplastic discs???, is over-expressed or mutated in a number of common human cancers. EDD is a progestin regulated gene that encodes an E3 ubiquitin ligase involved in cell communication and cell adhesion, and although it has also been implicated in the DNA damage response through its association with DNA damage proteins, a definitive role has yet to be demonstrated. The work presented herein shows that EDD is necessary for an adequate cellular response to double-strand DNA breaks. Cells depleted of EDD exhibit reduced survival, radio-resistant DNA synthesis and failure to maintain G2/M arrest following DNA damage induced by phleomycin exposure. Furthermore, EDD-depleted cells display impaired activating phosphorylation and kinase activity of the checkpoint kinase CHK2 after DNA damage. These effects appear to be largely modulated through a phospho-dependent interaction involving the CHK2 FHA domain and a region of EDD spanning a number of putative FHA-binding threonines. These results identify EDD as a novel mediator in DNA damage signal transduction via CHK2 and emphasise the potential importance of EDD in cancer.

The MCM helicase plays an important role in eukaryotic DNA replication, unwinding double stranded DNA ahead of the replication fork. MCM is a hetero-hexamer consisting of the six related proteins, Mcm2-Mcm7. The distantly related MCM-binding protein (MCM-BP) was first identified in a screen for proteins interacting with MCM2-7 in human cells and was found to specifically interact with Mcm3-7 but not Mcm2. It is conserved in most eukaryotes and seems to play an important role in DNA replication but its exact function is not clear yet. This study contributes to the understanding of the fission yeast homologue of MCM-BP, named Mcb1, but also of MCM-BP in general. Results presented in this thesis document the initial biochemical characterisation of the complex Mcb1 forms with Mcm proteins, the MCMᴹᶜᵇ¹ complex. Interactions of Mcb1 with Mcm proteins, potential interaction sites between the proteins and the size of the complex were analysed using a variety of methods, including tandem affinity purification, co-immunoprecipitation, sucrose gradients and in vitro pull-down assays. Sequence analysis and structure prediction were utilised to gain some insight into Mcb1 and MCM-BP ancestry and structure. Results presented here indicate that fission yeast Mcb1 shares homology with Mcm proteins and forms a complex with Mcm3-Mcm7 but not Mcm2 and thus replaces the latter in an alternative high molecular weight complex that is likely to have an MCM-like appearance. Deletion of mcb1⁺ showed that Mcb1 is essential in fission yeast. To examine the cellular function of the protein, temperature-sensitive mutants were generated. Inactivation of Mcb1 leads to an increase in DNA damage and cell cycle arrest in G2-phase depending on the activation of the Chk1 dependent DNA damage checkpoint. Similar observations were made when Mcb1 was overexpressed, indicating that certain levels of the protein are important for accurate DNA replication. Construction of truncated versions of Mcb1 suggested that almost the full-length protein is needed for proper function.

Le virus de l’immunodéficience humaine de type 1 (VIH-1), l’agent étiologique du SIDA, est un rétrovirus complexe arborant plusieurs protéines accessoires : Nef, Vif, Vpr, et Vpu. Celles-ci sont impliquées dans la modulation de la réplication virale, dans l’évasion immunitaire et dans la progression de la pathogenèse du SIDA. Dans ce contexte, il a été démontré que la protéine virale R (Vpr) induit un arrêt de cycle cellulaire en phase G2. Le mécanisme par lequel Vpr exerce cette fonction est l’activation, ATR (Ataxia telangiectasia and Rad3 related)-dépendante, du point de contrôle de dommage à l’ADN, mais les facteurs et mécanismes moléculaires directement impliqués dans cette activité demeurent inconnus. Afin d’identifier de nouveaux facteurs cellulaires interagissant avec Vpr, nous avons utilisé une purification d’affinité en tandem (TAP) pour isoler des complexes protéiques natifs contenant Vpr. Nous avons découvert que Vpr s’associait avec CRL4A(VprBP), un complexe cellulaire d’E3 ubiquitine ligase, comprenant les protéines Cullin 4A, DDB1 (DNA damage-binding protein 1) et VprBP (Vpr-binding protein). Nos études ont mis en évidence que le recrutement de la E3 ligase par Vpr était nécessaire mais non suffisant pour l’induction de l’arrêt de cycle cellulaire en G2, suggérant ainsi que des événements additionnels seraient impliqués dans ce processus. À cet égard, nous apportons des preuves directes que Vpr détourne les fonctions de CRL4A(VprBP) pour induire la polyubiquitination de type K48 et la dégradation protéosomale de protéines cellulaires encore inconnues. Ces événements d’ubiquitination induits par Vpr ont été démontrés comme étant nécessaire à l’activation d’ATR. Finalement, nous montrons que Vpr forme des foyers ancrés à la chromatine co-localisant avec VprBP ainsi qu’avec des facteurs impliqués dans la réparation de l’ADN. La formation de ces foyers représente un événement essentiel et précoce dans l’induction de l’arrêt de cycle cellulaire en G2. Enfin, nous démontrons que Vpr est capable de recruter CRL4A(VprBP) au niveau de la chromatine et nous apportons des preuves indiquant que le substrat inconnu ciblé par Vpr est une protéine associée à la chromatine. Globalement, nos résultats révèlent certains des ménanismes par lesquels Vpr induit des perturbations du cycle cellulaire. En outre, cette étude contribue à notre compréhension de la modulation du système ubiquitine-protéasome par le VIH-1 et son implication fonctionnelle dans la manipulation de l’environnement cellulaire de l’hôte. / Human immunodeficiency virus 1 (HIV-1), the etiologic agent of AIDS, is a complex retrovirus with several accessory proteins. HIV-1 accessory proteins Nef, Vif, Vpr, and Vpu have been implicated in the modulation of viral replication, enhancement of viral fitness, immune evasion, and progression of AIDS pathogenesis. In that regard, viral protein R (Vpr) induces a cell cycle arrest in the G2 phase by activating the canonical ATR (Ataxia telangiectasia and Rad3 related)-mediated DNA damage checkpoint, but cellular factors and mechanisms directly engaged in this process remain unknown. To identify novel Vpr-interacting cellular factors, we used tandem affinity purification (TAP) to isolate native Vpr-containing complexes. We found that Vpr hijacks a cellular E3 ubiquitin ligase complex, CRL4A(VprBP), composed of Cullin 4A, DDB1 (DNA damage-binding protein 1) and VprBP (Vpr-binding protein). Moreover, we observed that recruitment of the E3 ligase by Vpr was necessary but not sufficient for the induction of G2 cell cycle arrest, suggesting that additional events are involved. In this context, we provide direct evidence that Vpr usurps the function of CRL4A(VprBP) to induce the K48-linked polyubiquitination and proteasomal degradation of as-yet-unknown cellular proteins. These ubiquitination events mediated by Vpr were necessary for the activation of ATR. Moreover, we show that Vpr forms chromatin-associated foci that co-localize with VprBP and DNA repair factors. Our data indicate that formation of these foci represent a critical early event in the induction of G2 arrest. Finally, we show that Vpr is able to recruit CRL4A(VprBP) on chromatin and we provide evidence that the unknown substrate targeted by Vpr is a chromatin-associated protein. Overall, our results reveal some of the mechanisms by which Vpr induces cell cycle perturbations. Furthermore, this study contributes to our understanding of the modulation of the ubiquitin-proteasome system by HIV-1 and its functional implication in the manipulation of the host cellular environment.

Le virus de l’immunodéficience humaine de type 1 (VIH-1), l’agent étiologique du SIDA, est un rétrovirus complexe arborant plusieurs protéines accessoires : Nef, Vif, Vpr, et Vpu. Celles-ci sont impliquées dans la modulation de la réplication virale, dans l’évasion immunitaire et dans la progression de la pathogenèse du SIDA. Dans ce contexte, il a été démontré que la protéine virale R (Vpr) induit un arrêt de cycle cellulaire en phase G2. Le mécanisme par lequel Vpr exerce cette fonction est l’activation, ATR (Ataxia telangiectasia and Rad3 related)-dépendante, du point de contrôle de dommage à l’ADN, mais les facteurs et mécanismes moléculaires directement impliqués dans cette activité demeurent inconnus. Afin d’identifier de nouveaux facteurs cellulaires interagissant avec Vpr, nous avons utilisé une purification d’affinité en tandem (TAP) pour isoler des complexes protéiques natifs contenant Vpr. Nous avons découvert que Vpr s’associait avec CRL4A(VprBP), un complexe cellulaire d’E3 ubiquitine ligase, comprenant les protéines Cullin 4A, DDB1 (DNA damage-binding protein 1) et VprBP (Vpr-binding protein). Nos études ont mis en évidence que le recrutement de la E3 ligase par Vpr était nécessaire mais non suffisant pour l’induction de l’arrêt de cycle cellulaire en G2, suggérant ainsi que des événements additionnels seraient impliqués dans ce processus. À cet égard, nous apportons des preuves directes que Vpr détourne les fonctions de CRL4A(VprBP) pour induire la polyubiquitination de type K48 et la dégradation protéosomale de protéines cellulaires encore inconnues. Ces événements d’ubiquitination induits par Vpr ont été démontrés comme étant nécessaire à l’activation d’ATR. Finalement, nous montrons que Vpr forme des foyers ancrés à la chromatine co-localisant avec VprBP ainsi qu’avec des facteurs impliqués dans la réparation de l’ADN. La formation de ces foyers représente un événement essentiel et précoce dans l’induction de l’arrêt de cycle cellulaire en G2. Enfin, nous démontrons que Vpr est capable de recruter CRL4A(VprBP) au niveau de la chromatine et nous apportons des preuves indiquant que le substrat inconnu ciblé par Vpr est une protéine associée à la chromatine. Globalement, nos résultats révèlent certains des ménanismes par lesquels Vpr induit des perturbations du cycle cellulaire. En outre, cette étude contribue à notre compréhension de la modulation du système ubiquitine-protéasome par le VIH-1 et son implication fonctionnelle dans la manipulation de l’environnement cellulaire de l’hôte. / Human immunodeficiency virus 1 (HIV-1), the etiologic agent of AIDS, is a complex retrovirus with several accessory proteins. HIV-1 accessory proteins Nef, Vif, Vpr, and Vpu have been implicated in the modulation of viral replication, enhancement of viral fitness, immune evasion, and progression of AIDS pathogenesis. In that regard, viral protein R (Vpr) induces a cell cycle arrest in the G2 phase by activating the canonical ATR (Ataxia telangiectasia and Rad3 related)-mediated DNA damage checkpoint, but cellular factors and mechanisms directly engaged in this process remain unknown. To identify novel Vpr-interacting cellular factors, we used tandem affinity purification (TAP) to isolate native Vpr-containing complexes. We found that Vpr hijacks a cellular E3 ubiquitin ligase complex, CRL4A(VprBP), composed of Cullin 4A, DDB1 (DNA damage-binding protein 1) and VprBP (Vpr-binding protein). Moreover, we observed that recruitment of the E3 ligase by Vpr was necessary but not sufficient for the induction of G2 cell cycle arrest, suggesting that additional events are involved. In this context, we provide direct evidence that Vpr usurps the function of CRL4A(VprBP) to induce the K48-linked polyubiquitination and proteasomal degradation of as-yet-unknown cellular proteins. These ubiquitination events mediated by Vpr were necessary for the activation of ATR. Moreover, we show that Vpr forms chromatin-associated foci that co-localize with VprBP and DNA repair factors. Our data indicate that formation of these foci represent a critical early event in the induction of G2 arrest. Finally, we show that Vpr is able to recruit CRL4A(VprBP) on chromatin and we provide evidence that the unknown substrate targeted by Vpr is a chromatin-associated protein. Overall, our results reveal some of the mechanisms by which Vpr induces cell cycle perturbations. Furthermore, this study contributes to our understanding of the modulation of the ubiquitin-proteasome system by HIV-1 and its functional implication in the manipulation of the host cellular environment.

No description available.