Thèses sur le sujet « Transcription and repair »
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Chambers, Anna Louise. « Transcription termination by a transcription-repair coupling factor ». Thesis, University of Bristol, 2005. http://hdl.handle.net/1983/b95a2024-73ae-460d-89bf-3c064a780c78.
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Lainé, Jean-Philippe. « TFIIH and transcription coupled repair ». Université Louis Pasteur (Strasbourg) (1971-2008), 2005. http://www.theses.fr/2005STR13195.
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La bonne coordination des différents évènements qui participent au maintien de l’intégrité du génome et régulent son expression est un pré-requis pour la différentiation, la prolifération et le vie de la cellule. L’interconnection de ces divers phénomènes est illustrée par l’existence d’un complex aux multiples fonctions, le facteur TFIIH. Identifié à l’origine comme un facteur de transcription associé à l’ARN polymérase II, TFIIH participe également à la réparation de l’AND soumis à diverses attaques génotoxiques. Je me suis interessé à le contribution fonctionelle et structurale de la sous-unité p52, une des dix sous-unités de TFIIH, au sein du complex, ainsi qu’au lien reliant les mecanismes de transcription et de réparation, dans lesquelles TFIIH joue un role prépondérent. Premièrement, j’ai démontré que l’extrémité carboxyl terminale de p52 était importante pour stabiliser l’ancrage de XPB, une des sous-unité de TFIIH, au sein du complex. Cette interaction est primordiale pour permettre à XPB d’exercer son role dans l’ouverture de l’ADN au cours de l’initiation de la transcription. Puis, je me suis attaché aux mécanismes couplant la réparation à la transcription. J’ai montré qu’une ARN polymérase arretée sur une lésion est capable de recruter les différents facteurs de réparation et d’induire le relarguage d’un fragment d’ADN contenant le dommageAccurate coordination of the various events that maintain the integrity of the genome and regulate its expression is a prerequisite for differentiation, proliferation and cell life. The interconnection of such cellular processes is highlighted by the multi-functional complex TFIIH. Originally identified as a RNA polymerase II transcription factor, TFIIH also participates in the DNA nucleotide excision repair (NER) reaction. Ve focused my work on the functional/structural contribution within the complex of p52, one of the ten subunits of TFIIH, the link between transcription and NER, and the role of TFIIH in both. I first demonstrated that the carboxy-terminal of p52 is important for stabilizing the anchoring of XPB, another subunit of TFIIH, within the complex. This interaction is important for the role of XPB in the DNA opening step during transcription initiation. Then I focused my attention on the mechanism linking transcription to NER. I was able to show that a stalled elongating RNA polymerase II is able to recruit the repair factors at the site of the lesion and promote the removal of the DNA patch containing the lesion
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Kim, Young-In Timothy. « Determinants of bacterial transcription-coupled repair ». Thesis, University of Bristol, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.521101.
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Trautinger, Brigitte W. « Interplay between DNA replication, transcription and repair ». Thesis, University of Nottingham, 2002. http://eprints.nottingham.ac.uk/14281/.
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The Ruv ABC and RecBCD protein complexes together can collapse and repair arrested replication forks. With their help a fork structure can be re-established on which replication can be restarted. ruv and recB mutants are therefore quite sensitive to UV light. Their survival is greatly decreased in the absence of the signalling molecules (p)ppGpp and increased when excess (p)ppGpp is present. (p)ppGpp are the effector molecules of the stringent response, regulating adaptation to starvation and other stressful environmental changes. Absence of (p)ppGpp can be compensated for by mutations in RNA polymerase that are called stringent mutations. Some of those, called rpo *, also - like excess (p)ppGpp - increase the survival of UV irradiated ruv and recB cells. A model proposed by McGlynn and Lloyd (Cell, Vol. 101, pp35-45, March 31, 2000) suggests that this is achieved by modulation of RNA polymerase, which decreases the incidence of replication fork blocks. In this work twenty-seven rpo * mutants were isolated, sequenced and mapped on the 3D structure of Thermus aquatic us RNA polymerase. I have found mutants in the ~ and ~' subunits of RNA polymerase. They lie mostly on the inner surface of the protein, well placed to make contact with the DNA substrate or the RNA product. A large number of rifampicin resistant mutations among rpo* mutations is explained by an overlap between the so-called Rif pocket and the "rpo* pocket". rpo * mutations, like stringent mutations, lead to a decrease in cell size, suppress filamentation and increase viability. For in vitro studies I purified wild type and two mutant RNA polymerases with help of a his-tagged a subunit. The experiments confirmed that rpo* mutant RNA polymerases form less stable open complexes than wild type, just like previously investigated stringent RNA polymerases. In addition I have shown here that (p)ppGpp leads to the destabilisation of RNA polymerase complexes stalled by nucleotide starvation or UV-induced lesions, though there is as yet no indication that rpo * mutations act in the same way.
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Fan, Jun. « Single-molecule basis of transcription-coupled DNA repair ». Sorbonne Paris Cité, 2015. http://www.theses.fr/2015USPCC213.
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Les cellules vivantes sont constamment soumises à des agents, endogènes et exogènes, qui peuvent induire des lésions sur l'ADN. Ces agents peuvent menacer l'intégrité du génome, bloquer les processus de réplication, transcription et traduction, ou encore avoir des effets génotoxiques. Les organismes ont alors développé des systèmes de surveillance qui coordonnent la réparation de l'ADN et la progression du cycle cellulaire afin de lutter contre l'accumulation de dommages sur leur ADN. Les mécanismes de réparation de l'ADN, découverts à la fois dans les organismes procaryotes et eucaryotes, ont pour rôle le maintien de l'intégrité du matériel génétique. Ces mécanismes comprennent la réparation par excision de nucléotides (NER), la réparation par excision de bases (BER), la réparation des mésappariements (MMR) et la réparation des cassures double-brin (DSBR). La réparation couplée à la transcription (RCT) est une sous-voie de la réparation par excision de nucléotides qui permet une réparation rapide des lésions uniquement localisées sur le brin transcrit et affectant des gènes en cours de transcription. Elle se différencie de la sous-voie de la réparation du génome global (GGR) qui opère sur l'ensemble du génome sans distinction entre les brins transcrits et non transcrits. L'implication dans la RCT de l'ARN Polymérase (ARNP) et de la protéine Mfd (Mutation Frequency Decline), aussi connue sous le nom TRCF (Transcription Repair Coupling Factor), est ce qui permet la réparation préférentielle du brin transcrit. Le blocage de l'ARNP par une lésion sur le brin transcrit agit comme un senseur de dommage et déclenche la RCT. En effet, l'ARNP bloquée doit être déplacée afin de rendre la lésion accessible aux facteurs de réparation avals. Chez E. Coli c'est la translocase Mfd qui joue ce rôle : elle déplace l'ARNP bloquée et coordonne l'assemblage des facteurs UvrAB(C) au site de la lésion. Des études récentes ont montré que, après s'être lié à l'ARNP et l'avoir déplacée, Mfd reste sur l'ADN sous la forme d'un complexe de translocation stable impliquant l'ARNP même si cette dernière n'est plus directement liée à l'ADN. La technique de nanomanipulation par pince magnétique de molécules uniques d'ADN portant une lésion a été utilisée afin de comprendre comment UvrAB(C) sont recrutés via le complexe Mfd-ARNP. Cette technique a permis d'observer en temps réel jusqu'à l'incision par UvrC la RCT, qui comporte un grand nombre d'étapes et implique de nombreuses protéines. Il a été observé que le recrutement d'UvrA et d'UvrAB par le complexe Mfd-ARNP stoppe la translocation du complexe sur l'ADN et entraine la dissolution du complexe avec un passage de relais moléculaire dans une cinétique lente. La combinaison de la nanomanipulation de molécule-unique avec la fluorescence montre en outre que la dissolution du complexe entraine non seulement la perte de l'ARNP mais aussi celle de Mfd. Ce passage de témoin moléculaire permet d'avoir une réaction d'incision du brin endommagé par UvrC plus rapide par rapport à ce qui se produit lors de la réaction sur une molécule unique dans le cadre de la réparation globale du génome. Un modèle global intégrant les protéines impliquées dans la RCT et la GGR et donnant les caractéristiques cinétiques des différentes réactions se produisant en parallèle dans les deux sous-voies a été proposéThe DNA in living cells is constantly threatened by damages from both endogenous and exogenous agents, which can threaten genomic integrity, block processes of replication, transcription and translation and have also genotoxic effects. In response to the DNA damage challenge, organisms have evolved diverse surveillance mechanisms to coordinate DNA repair and cell-cycle progression. Multiple DNA repair mechanisms, discovered in both prokaryotic and eukaryotic organisms, bear the responsibility of maintaining genomic integrity; these mechanisms include nucleotide excision repair (NER), base excision repair (BER), mismatch repair (MMR) and double strand break repair (DSBR). Transcription-coupled DNA repair (TCR) is a specialized NER subpathway characterized by enhanced repair of the template strand of actively transcribed genes as compared to the classical global genome repair (GGR) subpathway of NER which does not distinguish between template and non-template strands. TCR achieves specialization via the involvement of RNA polymerase (RNAP) and the Mfd (Mutation Frequency Decline) protein, also known as TRCF (transcription repair coupling factor). TCR repair initiates when RNAP stalls at a DNA lesion on the transcribed strand and serves as the da mage sensor. The stalled RNAP must be displaced so as to make the lesion accessible to downstream repair components. E. Coli Mfd translocase participates in this process by displacing stalled RNAP from the lesion and then coordinating assembly of the UvrAB(C) components at th( damage site. Recent studies have shown that after binding to and displacing stalled RNAP, Mfd remains on the DNA in the form of a stable, translocating complex with evicted RNAP. So as to understand how UvrAB(C) are recruited via the Mfd-RNAP complex, magnetic trapping of individual, damaged DNA molecules was employed to observe-in real-time this multi¬component, multi-step reaction, up to and including the DNA incision reaction by UvrC. It was found that the recruitment of UvrA and UvrAB to the Mfd-RNAP complex halts the translocating complex and then causes dissolution of the complex in a molecular "hand-off" with slow kinetics Correlative single-molecule nanomanipulation and fluorescence further show that dissolution of the complex leads to loss of not only RNAP but also Mfd. Hand-off then allows for enhanced incision of damaged DNA by the UvrC component as compared to the equivalent single-moleculE GGR incision reaction. A global model integrating TCR and GGR components in repair was proposed, with the overall timescales for the parallel reactions provided
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Malik, Shivani. « REGULATORY MECHANISMS OF TRANSCRIPTION AND ASSOCIATED DNA REPAIR ». OpenSIUC, 2012. https://opensiuc.lib.siu.edu/dissertations/626.
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Transcription is a crucial regulatory step in gene regulation modulated by several proteins. Any misregulation during transcription can lead to many diseases including cancer, neurodegenerative disorders and aging making it imperative to have a detailed mechanistic view of the process. Over the recent years, 26S proteasome has been implicated in transcriptional regulation through its proteolytic and non-proteolytic activities. While, the proteolytic role of proteasome in transcription has been extensively studied, its non-proteolytic function is poorly understood. Thus, one of my thesis aims had been to analyze the non-proteolytic role of proteasome in transcription. My results have revealed the non-proteolytic role of 26S proteasome in establishing a specific protein interaction network at the promoter for stimulated transcriptional initiation in vivo . In addition to its roles in transcription, 26S proteasome also plays an important role in the degradation of RNA polymerase II stalled at DNA lesion facilitating the rapid repair of transcriptionally active genes through a process of transcription coupled repair (TCR). My studies have addressed the key question of the fate of RNA polymerase II stalled at a lesion. My findings show that RNA polymerase II interacts with an elongation and TCR-specific factor, Rad26p. Upon encountering a lesion, RNA polymerase II stalls and unloads Rad26p on the site of DNA damage. Subsequently, the elongating RNA polymerase II is disassembled through the degradation of its largest subunit, Rpb1p. Further; our studies have also uncovered a novel role of Rad26p in chromatin disassembly, which facilitates transcriptional elongation and hence TCR. This work provides valuable insights into interplay of chromatin structure, transcriptional elongation and TCR. Finally, extending the regulatory knowledge of sense transcriptional initiation to antisense, my work has revealed the extensive participation of GTFs in the process. Collectively, results of above studies provide a comprehensive view of transcription and associated process of active genome repair.
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Cerutti, Elena. « Nucleotide Excision Repair at the crossroad with transcription ». Thesis, Lyon, 2019. http://www.theses.fr/2019LYSE1057.
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L’intégrité de l’ADN est continuellement remise en question par divers agents endogènes et exogènes (p. ex., la lumière ultraviolette, la fumée de cigarette, la pollution de l’environnement, les dommages oxydatifs, etc.) qui causent des lésions de l’ADN qui interfèrent avec les fonctions cellulaires correctes. Le mécanisme de réparation par excision de nucléotides (NER) supprime les adduits d’ADN déformantes l’hélice tels que les lésions induites par les UV et il existe dans deux sous voies distinctes selon l’endroit où les lésions de l’ADN sont situées dans le génome. L’une de ces sous voies est directement liée à la transcription de l’ADN (TCR) par l’ARN Polymérase 2 (ARNP2). Dans la première partie de ce travail, nous avons démontré qu’un mécanisme NER entièrement compétent est également nécessaire pour la réparation de l’ADN ribosomique (ADNr), transcrite par ARN Polymérase 1 (ARNP1) et représentant 60 % de la transcription cellulaire totale. De plus, nous avons identifié et clarifié le mécanisme de deux protéines responsables du repositionnement nucléolaire dépendant des UV de l’ARNP1 et de l’ADNr observé pendant la réparation. Dans la deuxième partie de ce travail, nous avons étudié la fonctionne moléculaire de la protéine XAB2 lors de la réparation NER et nous avons démontré son implication dans le processus TCR. De plus, nous avons également montré la présence de XAB2 dans un complexe d’épissage du pré-ARNm. Enfin, nous avons décrit l’impact de XAB2 sur la mobilité de l’ARNP2 lors des premières étapes de la réparation TCR, suggérant ainsi un rôle de XAB2 dans le processus de reconnaissance des lésionsThe integrity of DNA is continuously challenged by a variety of endogenous and exogenous agents (e.g. ultraviolet light, cigarette smoke, environmental pollution, oxidative damage, etc.) that cause DNA lesions which interfere with proper cellular functions. Nucleotide Excision Repair (NER) mechanism removes helix-distorting DNA adducts such as UV-induced lesions and it exists in two distinct sub-pathways depending where DNA lesions are located within the genome. One of these sub pathways is directly linked to the DNA transcription by RNA Polymerase 2 (TCR). In the first part of this work, we demonstrated that a fully proficient NER mechanism is also necessary for repair of ribosomal DNA, transcribed by RNA polymerase 1 and accounting for the 60 % of the total cellular transcription. Furthermore, we identified and clarified the mechanism of two proteins responsible for the UV-dependent nucleolar repositioning of RNAP1 and rDNA observed during repair. In the second part of this work, we studied the molecular function of the XAB2 protein during NER repair and we demonstrated its involvement in the TCR process. In addition, we also shown the presence of XAB2 in a pre-mRNA splicing complex. Finally, we described the impact of XAB2 on RNAP2 mobility during the first steps of TCR repair, thus suggesting a role of XAB2 in the lesion recognition process
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MacKinnon-Roy, Christine. « The role of transcription elongation factor IIS in transcription-coupled nucleotide excision repair ». Thesis, University of Ottawa (Canada), 2010. http://hdl.handle.net/10393/28454.
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Transcription-coupled nucleotide excision repair (TC-NER) removes bulky DNA lesions from the template strand at actively transcribed genes. The RNA polymerase II (RNAPII) holoenzyme complex forms a stable ternary complex at the site of DNA damage which may block access of DNA repair proteins to the site of DNA lesions. Therefore, there is considerable interest in understanding how repair is coupled to transcription. Based on elegant in vitro studies, it has been hypothesized that transcription elongation factor IIS (TFIIS), by catalyzing the reverse translocation of RNAPII, may allow access of DNA repair proteins to sites of DNA damage. Here, we tested this hypothesis by assessing TC-NER capacity in cells in which TFIIS expression has been reduced by RNA interference. Surprisingly, we found that decreased TFIIS levels did not affect the repair of transcription-blocking DNA lesions and did not affect the sensitivity of targeted cells to UV light or cisplatin. These results do not support a role for TFIIS in TC-NER. We conclude conservatively that TFIIS levels are not limiting for TC-NER.
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Abdullah, Mohamad Faiz Foong. « Transcription factors and mismatch repair proteins in meiotic recombination ». Thesis, University of Oxford, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.249637.
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Riedl, Thilo. « Tfiih : A factor between DNA repair and transcriptional activation ». Université Louis Pasteur (Strasbourg) (1971-2008), 2003. http://www.theses.fr/2003STR13059.
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Haines, Nia. « Damage detection during transcription coupled DNA repair in Escherichia coli ». Thesis, University of Bristol, 2015. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.690774.
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Transcription coupled DNA repair (TCR) is a sub-pathway of nucleotide excision repair (NER), which enhances the repair of damage on the template strand of active genes. In E. coli, this is initiated by stalling of the transcribing RNA polymerase (RNAP) at the lesion. This results in recruitment of the Mfd helicase which displaces RNAP, allowing repair to be carried out by Uvr proteins through the NER pathway. In this work, in order to better understand damage recognition in this pathway, single lesion-containing plasmid templates were created and analysed for repair. In these experiments the RNAP stalling step was separated from damage recognition by artificially stalling the RNAP upstream of the lesion. The results demonstrated that RNAP does not need to be stalled directly at a lesion in order to stimulate enhanced repair of a downstream lesion on the transcribed strand. This occurs both for the classical cyclopyrimidine dimer TCR substrate and the non-RNAP-stalling biotin and abasic site lesions. The TCR pathway was also shown to be inherently strand specific, as even lesions far downstream of the stalled RNAP were only repaired by TCR if they were on the transcribed strand. Introduction of a backtracking OpS transcriptional pause instead of a complete RNAP stall was also shown to induce an increase in downstream repair ofnon-RNAPstalling lesions. This downstream repair ability is likely facilitated by the observed translocation ability of the Mfd protein after RNAP displacement, and suggests that Mfd is part of a translocating damage search complex which is able to detect damage away from the initial RNAP stall site. The possible role of UvrD in backtracking RNAP to facilitate an Mfd-independent TCR pathway was investigated, however no evidence for this proposed ability of UvrD was observed. To try and understand the complexity of TCR and the possibly overlapping pathways, the initial steps in developing a high throughput genome wide in vivo assay for TCR were carried out.
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Sen, Rwik. « REGULATION OF EUKARYOTIC TRANSCRIPTIONAL ELONGATION AND ASSOCIATED DNA REPAIR ». OpenSIUC, 2016. https://opensiuc.lib.siu.edu/dissertations/1205.
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Transcriptional elongation is a crucial step in eukaryotic gene regulation whose mis-regulation leads to cellular pathologies. This makes it quite imperative to aim for a better understanding of the processes regulating transcriptional elongation. An important process promoting the association of RNA Polymerase II (RNAPII) with the coding region of the active gene and hence transcriptional elongation is the monoubiquitination of histone H2B at lysine 123. A complex of an E2 conjugase, Rad6p, and an E3 ligase, Bre1p, is essential for this process. Consistent with the role of histone H2B monoubiquitination in promoting the association of RNAPII with the active gene, this process was found to be impaired in the absence of Rad6p or point mutation of lysine 123 to arginine (H2B-K123R). Intriguingly, the association of RNAPII with the coding region of the active gene was not impaired in the absence of Bre1p, even though Bre1p is essential for histone H2B monoubiquitination. However, deletion of Bre1p’s RING domain that is essential for histone H2B monoubiquitination led to an impaired RNAPII association with the active gene. This observation indicates a role of the non-RING domain of Bre1p in repressing the association of RNAPII with the active gene, resulting in no net decrease in RNAPII occupancy in the absence of Bre1p. Taken together, my results implicated both the stimulatory and repressive roles of the histone H2B ubiquitin ligase Bre1p in regulation of RNAPII association with the coding regions of active genes and hence transcriptional elongation. Interestingly, my work also revealed that for efficient transcriptional elongation by histone H2B monoubiquitination, its optimum level needs to be maintained by a proper balance between Rad6p-Bre1p-mediated ubiquitination and de-ubiquitination (DUB) by the DUB module of SAGA. It was found that Sus1p, a subunit of the DUB module, promotes transcriptional elongation, DNA repair and replication via regulation of histone H2B DUB. In addition to Rad6p- Bre1p and the DUB module, global level of histone H2B monoubiquitination is also critically regulated by Cdk9, a kinase essential for phosphorylation of the serine 2 residue in the C-terminal domain (CTD) of RNAPII, which promotes transcriptional elongation. Apart from serine phosphorylation, proline residues at RNAPII-CTD undergo isomerization by proline isomerases, which also regulate transcription. One of the proline isomerases, Rrd1p, has been previously implicated in transcription in response to rapamycin treatment. Based on this fact and Rrd1p’s known interaction with RNAPII-CTD, we predicted that Rrd1p might regulate transcription independently of rapamycin treatment. In agreement with this hypothesis, our work revealed Rrd1p’s role in facilitating transcription of both rapamycin responsive and non-responsive genes in the absence of rapamycin treatment. Consistently, the absence of Rrd1p led to an impaired nucleosomal disassembly at the active gene, which correlates with the role of Rrd1p in promoting transcription. This is because maintenance of proper nucleosomal dynamics is essential for efficient transcription. It is known that transcriptional elongation is facilitated by the regulation of nucleosomal dynamics via the histone chaperone, FACT. Efficient chromatin reassembly in the wake of elongating RNAPII contributing to the fidelity of transcription is promoted by FACT. Being evolutionarily conserved among eukaryotes, FACT is also known to regulate DNA replication and repair, apart from transcription. Intriguingly, FACT has been found to be upregulated in cancers while its downregulation leads to tumor cell death. However, the mechanism which fine-tunes FACT for normal cellular functions remained unknown. My studies revealed a novel mechanism of regulation of FACT by the ubiquitin-proteasome system in yeast. San1p, an E3 ligase involved in nuclear protein quality control, was found to associate with the active gene and regulate transcriptional elongation through its E3 ligase activity- mediated turnover of Spt16p component of FACT. This regulation was found to maintain optimum level of Spt16p/FACT to engage with the active gene for proper transcriptional elongation, DNA repair and replication. In spite of playing such crucial roles in gene regulation, it was not known how FACT is targeted to the active gene. We discovered that a direct physical interaction between FACT and Cet1p, the mRNA capping enzyme, targets FACT to the active gene independently of Cet1p’s mRNA capping activity. Such targeting of FACT to the active gene leads to the release of promoter proximally paused-RNAPII into transcriptional elongation. However, the progress of RNAPII along the active gene during transcriptional elongation is frequently impeded by various kinds of damages along the underlying template DNA. Even though some of these lesions are co-transcriptionally repaired, it was not known whether the repair of extremely toxic DNA double-strand breaks (DSBs) was coupled to transcription. My results showed that DSBs at the transcriptionally active state of a gene are repaired faster than at the inactive state but such repair was not mediated by a co-transcriptional recruitment of DSB repair factors. This observation is in contrast to other DNA repair pathways such as nucleotide excision repair (NER) where repair factors are co-transcriptionally recruited to the lesion containing DNA. In this regard, we found that an NER factor, Rad14p, co-transcriptionally associates with the active gene in the absence of DNA damage to promote transcription, which unraveled a new role of Rad14p in transcription in addition its established role in NER. In summary, my results provide significant novel insights into the regulation of transcriptional elongation and associated processes leading to better understanding of eukaryotic gene expression.
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López, de Heredia Luis. « Analysis of the C/EBP family of transcription factors in neuronal repair ». Thesis, Queen Mary, University of London, 2009. http://qmro.qmul.ac.uk/xmlui/handle/123456789/545.
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Neurons within the peripheral nervous system (PNS) have a remarkable ability to repair themselves after injury; however, neurons within the central nervous system (CNS) do not spontaneously regenerate. Therefore, understanding the molecular elements responsible for successful regenerative response in the PNS can help us to establish basic principles and strategies for promoting regeneration in CNS structures such as the spinal cord. Nerve repair in the PNS has been suggested to be in part due to the involvement of intrinsic molecules such as transcription factors. In this thesis, I am focusing on the C/EBP family of transcription factors and their potential role in axonal regeneration after PNS injury. I examined the expression of different C/EBP members in PNS after injury after, using the sciatic nerve crush injury model I found that C/EBPδ mRNA is upregulated 4, 24 and 72 hours in dorsal root ganglia (DRG) following injury, whereas C/EBPβ and C/EBPγ expression is transiently upregulated by 4 hours resuming background levels after 72 hours. Conversely, C/EBPα and C/EBPε did not show upregulation following injury. In order to determine the function of C/EBPs in axonal growth in an in vitro system I used the ND7/23 cell line where I found that upon neurite growth induced by cyclic adenosine monophosphate (cAMP), the mRNA levels of C/EBPβ and C/EBPδ were upregulated. Furthermore, the conditional expression of a C/EBP total inhibitor or a C/EBPδ antisense construct decreases neurite elongation in vitro. Additionally, I found that 24 hours after treatment of ND7/23 cells with trichostatin A (TSA) C/EBPδ expression is elevated. Subsequently, I found that in DRG cultures from C/EBPδ knock-out animals, the lack of C/EBPδ affects the intrinsic growth capacity of dorsal root ganglion neurons which show a 3 drastically reduced axonal growth in vitro. To address the role of C/EBPδ in vivo, peripheral nerve repair was assessed in transgenic C/EBPδ knock-out animals following sciatic nerve crush. C/EBPδ knock-outs show, by immunostaining, impaired nerve regeneration 3 days and 14 days after sciatic nerve injury. Furthermore, functional recovery and morphometric analysis indicate that nerve regeneration is delayed in C/EBPδ deficient animals. These data demonstrate that the C/EBPδ gene is involved in neuronal repair after peripheral nerve injury.
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Hill, Sarah J. « Familial ALS Proteins Function in Prevention/repair of Transcription-Associated DNA Damage ». Thesis, Harvard University, 2016. http://nrs.harvard.edu/urn-3:HUL.InstRepos:27007760.
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Amyotrophic lateral sclerosis (ALS) is a progressive motor neuron dysfunction disease that leads to paralysis and death. There is currently no defined molecular pathogenesis pathway. Multiple proteins involved in RNA processing are linked to ALS, including FUS and TDP43; and we propose a disease mechanism in which loss of function of one of these proteins leads to an accumulation of transcription-associated DNA damage contributing to motor neuron cell death and progressive neurological symptoms.
In support of this hypothesis, we found that depletion of FUS and TDP43 leads to increased sensitivity to a transcription-arresting agent due to increased DNA damage. This indicates that these proteins normally contribute to the prevention or repair of transcription-associated DNA damage. In addition, we observed that both FUS and TDP43 co-localize with active RNA polymerase II at sites of DNA damage along with the DNA damage repair protein BRCA1, and that FUS and TDP43 participate in the prevention or repair of R-loop associated DNA damage.
Ideally, gaining a better understanding of the role(s) that FUS and TDP43 play in transcription-associated DNA damage will shed light on the mechanisms underlying ALS pathogenesis.
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Shotton, Priyasri. « A STUDY OF THE INTERPLAY BETWEEN SACCHAROMYCES CEREVISIAE TRANSCRIPTION AND DNA REPAIR ». OpenSIUC, 2012. https://opensiuc.lib.siu.edu/dissertations/552.
Texte intégralRésumé :
DNA is the storehouse of genetic information for the normal functioning of all cellular processes. This DNA is "read" by RNA Polymerases (RNAP) to carry out the transcription process, which is one of the most vital regulatory mechanisms within the cell for gene expression. There are three important RNA Polymerases available in the cell. RNAPI synthesizes ribosomal RNAs, while RNAPII makes mRNA precursors, snRNAs and most microRNAs, additionally RNAPIII takes care of tRNAs, small RNAs and 5S rRNAs that occur in the nucleus and the cytosol. The work in our lab focuses on the function of RNAPII transcription. It is not straightforward for RNAPII to transcribe the DNA, it needs to struggle with two major roadblocks that are in its path. First, is the nucleosomal barrier that is scattered across the coding regions of active genes. The mechanism to overcome this major obstacle is a very complex process that involves an extensive range of proteins which work collectively to help in the alteration of chromatin structure that ultimately facilitates transcriptional regulation. The second obstacle is that the DNA is also subject to constant onslaught by a variety of intrinsic and external damaging agents. Maintenance of normal physiological functions in addition to cell survival depends on the ability to preserve the genome integrity by protecting it from different mutations. Consequently, the cells have developed an extremely evolved repair mechanism known as the DNA damage response (DDR) system. This DDR pathway can restore various forms of damaged DNA that encompasses single base modifications, single strand damages and even the most toxic double strand breaks. Transcription-coupled DNA repair is involved in taking care of the single strand damages that can be induced by various chemicals including 4-nitroquinoline 1- oxide (4NQO) which forms bulky adducts on the DNA bases. On the other hand, double strand breaks are repaired via more complex and involved mechanisms that include non-homologous end joining and homologous recombination. Once the DNA is protected from injury it is prepared for transcription, replication and recombination. In my thesis, I have attempted to take a look at this intricate network and have studied the function of Rad26p, in yeast (associated with CSB-Cockayne syndrome B, in humans), a transcription coupled DNA repair factor, having an additional role in transcription. The results reveal that Rad26p plays a novel role in the regulation of elongation by modulating histone H2A-H2B dimer occupancy. Further, the study revealed how Rad26p is targeted to lesions for DNA repair. Next, I extended my study to the role of a nucleotide excision repair factor, Rad14p (associated with Xeroderma Pigmentosum, XPA- in humans), and for the first time demonstrated its function in transcription initiation in the absence of DNA damage. Finally, I analyzed whether repair of double strand breaks could be connected to transcription via an elegant method developed in my lab. This presented us with first time evidence that establishes that double strand break repair is faster on actively transcribing gene (ADH1) when compared to a nearly silent gene (MATα locus); thus, supporting our transcription-coupled DNA double strand break repair. The significance of all the proteins involved in my study is reflected by the various syndromes and diseases that arise due to mutations in their respective human homologues. Therefore, the results of my study will significantly advance our understanding of the regulatory mechanisms that are involved in the normal physiological functions of the cell which can be extended to humans.
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Gottlieb, Tanya M. « Biochemical characterisation and functional analysis of the DNA-dependent protein kinase ». Thesis, University of Cambridge, 1994. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.338306.
Texte intégral
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Feuerhahn, Sascha. « Mechanistic insights into the effect of oxidative lesions and anticancer drugs on RNA pol II transcription and transcription coupled repair ». Université Louis Pasteur (Strasbourg) (1971-2008), 2008. https://publication-theses.unistra.fr/restreint/theses_doctorat/2008/FEUERHAHN_Sascha_2008.pdf.
Texte intégralRésumé :
Le processus vital d’expression des gènes par l’ARN polymérase II (ARN pol II) peut être perturbé par les lésions portées par l’ADN. Une ARN pol II bloquée peut déclencher la réparation couplée à la transcription (TCR). Un acteur central de la TCR est la protéine du syndrome de Cockayne du groupe B (CSB), qui, quand elle est mutée, peut donner naissance au syndrome de Cockayne (CS). Dans cette thèse, l’effet de trois lésions oxydatives abondantes a été étudié en utilisant un système de transcription in vitro bien défini. Les lésions oxydatives peuvent bloquer la progression de l’ARN pol II qui est spécifique à chaque lésion. Par ailleurs, la présence d’un extrait nucléaire réduit ce blocage, et la purification de l’activité de franchissement mit en évidence le rôle du facteur de transcription TFIIF dans la synthèse translésionnelle d’ARN par l’ARN pol II. D’autres facteurs d’élongation, incluant CSB peuvent promouvoir le franchissement des lésions oxydatives de l’ADN avec une spécificité de lésion pour chaque facteur, qui peut donc avoir des implications dans le phénotype CS. Deuxièmement, le mécanisme méconnu du nouvel agent anticancéreux alkylant Trabectedin a été étudié. Dans cette thèse, il a été montré que la Trabectedin peut conduire à une persistance du complexe TCR due à l’inhibition de la coupure par l’endonucléase XPG et à la stimulation de la coupure par XPF-ERCC1. Un autre médicament anticancéreux apparenté est Zalypsis Dans cette thèse, ce compose a montré une efficacité pour inhiber les premières étapes de la transcription, qui pourraient avoir d’importantes implications dans le cancer du sein ou de la prostateThe vital process of gene expression by RNA polymerase II (RNA pol II) can be disturbed by DNA lesions. A stalled RNA pol II can trigger transcription coupled repair (TCR). A central player during TCR is the Cockayne syndrome group B protein (CSB), which, when mutated, can give rise to Cockayne syndrome (CS). In this thesis, the effect of three abundant oxidative lesions was investigated using a well defined in vitro transcription system. Oxidative lesions blocked RNA pol II progression to a lesion-specific extent. Importantly, the presence of nuclear extract alleviated this block, and the subsequent purification of this bypass activity from HeLa nuclear extract identified a role for the elongation factor TFIIF in RNA pol II translesion RNA synthesis. Other elongation factors, including CSB, were identified to promote bypass of oxidative DNA damage for a factor-specific subset of lesions, which could have implications for CS. Secondly, the poorly understood mechanism of the novel anticancer alkylating agent Trabectedin, which is more potent in cells proficient in TCR, was investigated in this thesis. It was found that Trabectedin can lead to a persisting TCR complex by the inhibition of the endonuclase cut of XPG and the stimulation of XPF-ERCC1 cutting during TCR. A related cancer therapeutic is Zalypsis. In this thesis, this compound was shown to efficiently inhibit early steps in transcription. Which could have important implications for breast or prostate cancer
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Taschner, M. J. « Transcription-coupled nucleotide excision repair and its regulation by the DNA damage checkpoint ». Thesis, University College London (University of London), 2009. http://discovery.ucl.ac.uk/18946/.
Texte intégralRésumé :
Elaborate DNA repair mechanisms have evolved, allowing cells to repair damages in their genomes. Nucleotide excision repair (NER) removes a variety of helix-distorting lesions, including those caused by ultraviolet (UV) irradiation. NER operates via two subpathways. Transcription-coupled repair (TC-NER) rapidly removes transcription-blocking lesions in the transcribed strand (TS) of active genes, and in the yeast Saccharomyces cerevisiae depends on the factors Rad26 and Rpb9. Lesions in untranscribed DNA, including the non-transcribed strand (NTS) of active genes are removed slower by global genome repair (GG-NER). Besides activating specific DNA repair systems, DNA damage also leads to a global cellular response, known as the DNA damage checkpoint (DDC). Cell-cycle progression is temporarily stopped after DNA damage to allow sufficient time for repair and prevent replication or segregation of damaged chromosomes. The DDC is a complex signal transduction cascade involving a number of protein kinases, the central players in budding yeast being Mec1 and Tel1, the homologues of human ATR and ATM, respectively. Besides inhibiting cell-cycle progression, accumulating evidence suggests that DNA repair systems are also influenced by the checkpoint. I have investigated the rates of repair of UV lesions in checkpoint deficient strains of Saccharomyces cerevisiae and found that NER is significantly inhibited on both strands of an active gene in the absence of Mec1. The effect on NTS repair seems to be caused by deficient de novo synthesis of repair factors, whereas TC-NER is influenced mainly by post-translational modification of one or more pre-existing proteins. I have characterised a checkpoint-dependent phosphorylation of Rad26, and have shown using point mutants that this phosphorylation increases the TC-NER capacity of cells, establishing a new link between NER and the checkpoint. In addition to these results about the interplay between the DDC and NER pathways, preliminary data from two unrelated projects will be presented. One was an attempt to establish a system for analysis of NER factor recruitment to an artificial, highly UV-damage-prone DNA sequence. The other focussed on the regulation of UV-induced degradation of Rpb1, the largest RNA Polymerase II (RNAPII) subunit, by the DDC.
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Gopaul, Diyavarshini. « Study of the molecular mechanisms linking transcription and DNA repair in Saccharomyces cerevisiae ». Thesis, Université Paris-Saclay (ComUE), 2018. http://www.theses.fr/2018SACLS347.
Texte intégralRésumé :
La voie de réparation par excision de nucléotides (NER) répare les lésions qui distordent la double hélice d’ADN notamment ceux induits par l’irradiation UV. Le NER est subdivisé en deux sous-voies : GG-NER (Global Genome Repair) et TC-NER (Transcription-Coupled Repair). La sous-voie GG-NER enlève les dommages à l’ADN dans l’ensemble du génome. La sous-voie TC-NER répare les dommages sur le brin transcrit qui interfèrent avec la progression de l’ARN Pol II. Les défauts de la voie NER peuvent conduire à l’apparition de pathologies graves. Par exemple, des mutations dans le gène XPG, codant une 3’ endonucléase impliquée dans la voie NER, peuvent mener au xeroderma pigmentosum (XP) associé ou non au syndrome de Cockayne (CS).Récemment, le laboratoire a découvert un lien fonctionnel entre Rad2, homologue chez la levure Saccharomyces cerevisiae de la protéine XPG humaine, et le Médiateur (Eyboulet et al., 2013). Le Médiateur est un complexe multiprotéique nécessaire à la régulation de la transcription dépendante de l’ARN Pol II. Cette étude a suggéré que le Médiateur est impliqué dans la sous-voie TC-NER en facilitant le recrutement de Rad2 au niveau des régions transcrites.Mon projet de thèse visait à étudier les mécanismes moléculaires qui lient la transcription et la réparation de l’ADN. Plus précisément, d’investiguer le lien fonctionnel entre le Médiateur et la machinerie du NER chez S. cerevisiae.Lors du TC-NER, l’ARN Pol II est le premier facteur signalant le dommage à l’ADN. De plus, le Médiateur et Rad2 interagissent avec l’ARN Pol II. Pour déterminer le lien fonctionnel entre ces composants, nous avons utilisé des approches de génétique et génomique dans les mutants de TFIIH (kin28), de l’ARN Pol II (rpb9) and du Médiateur (med17). Nos résultats nous ont permis de proposer un modèle dans lequel Rad2 est recruté au niveau des régions régulatrices enrichies par le Médiateur, et Rad2 est ensuite transféré au niveau des régions transcrites de manière dépendante à l’ARN Pol II. De plus, ces résultats suggèrent que le rôle du Médiateur dans la transcription est fortement lié à son rôle dans la réparation de l’ADN.Ensuite, nous avons montré que le lien entre le Médiateur et la machinerie du NER peut être étendu à d’autres protéines du NER notamment en démontrant une interaction physique entre le Médiateur et Rad1/XPF, Rad10/ERCC1 ou Rad26/CSB, en l’absence des UV. Tout comme Rad2, nous avons démontré que Rad1 et Rad10 n’ont pas de rôle majeur dans la transcription. Pour approfondir le lien entre ces protéines du NER et le Médiateur, des expériences de ChIP-sequencing ont été réalisées. Nous avons observé que le Médiateur est présent au niveau de certaines régions qui sont aussi enrichies par ces protéines du NER. Après l’induction des dommages par UV, les interactions entre le Médiateur et la machinerie du NER reste inchangées par rapport aux conditions en l’absence des UV. De plus grâce à nos expériences de ChIP, nous avons observé un changement de la liaison à la chromatine des protéines du NER et du Médiateur après l’irradiation aux UV. Des expériences de ChIP-sequencing seront réalisées pour avoir une vue globale de ces changements.En conclusion, nous avons solidifié le lien fonctionnel entre Rad2, le Médiateur et l’ARN Pol II par rapport à la réparation couplée à la transcription. Nous avons aussi démontré que le Médiateur interagit avec d’autres protéines du NER (Rad1/XPF, Rad10/ERCC1 et Rad26/CSB) et colocalise avec eux sur certaines régions de la chromatine. En somme, notre projet place le Médiateur à l’interface de la transcription et de la réparation de l’ADN, deux processus essentiels dont les défauts peuvent mener à des pathologies gravesNucleotide excision repair (NER) is a well conserved pathway that removes helix-distorting DNA lesions such as those arising upon UV irradiation. Global genome repair subpathway (GG-NER) removes the DNA lesions in the genome overall, and transcription-coupled repair (TC-NER) removes the DNA lesions interfering with Pol II progression through actively-transcribed regions. Defects in the NER pathway may lead to severe human pathologies. For instance, mutations in human XPG gene, encoding a 3’ endonuclease essential for NER, give rise to xeroderma pigmentosum (XP) sometimes associated with Cockayne syndrome (CS). Recently, the laboratory discovered a functional link between Rad2/XPG and Mediator in Saccharomyces cerevisiae (Eyboulet et al., 2013). Mediator is a large multisubunit complex essential for transcription regulation. We suggest that Mediator is involved in TC-NER by facilitating Rad2 recruitment to transcribed genes.My PhD work aimed at addressing the molecular mechanisms of this link between transcription and DNA repair, especially by investigating the functional interplay between Mediator and the NER machinery in yeast Saccharomyces cerevisiae.RNA Pol II is the first complex of TC-NER that encounters the DNA damage. Moreover, both Mediator and Rad2/XPG interact with Pol II. However, a functional interplay between all these components related to TC-NER remained to be determined. Using genetic and genomic approaches, in particular ChIP-sequencing in TFIIH (kin28), RNA Pol II (rpb9) and Mediator (med17) mutants, our work led us to propose a model where Rad2 shuttles between Mediator on upstream activating sequence (UAS) and RNA Pol II on transcribed regions (Georges, Gopaul et al., under review). Our results also suggest that Mediator functions in transcription and DNA repair are closely related.Moreover, we showed that Mediator’s link to NER can be extended to other NER proteins. Indeed, we identified a physical interaction between Mediator and other NER proteins, including Rad1/XPF, Rad10/ERCC1 and Rad26/CSB in the absence of UV irradiation. Similarly to Rad2, we demonstrated that Rad1 and Rad10 do not have a major role in yeast transcription. To further study the functional link between Mediator and the NER machinery, we obtained the genomic distribution of different NER proteins by ChIP-sequencing. We found that some promoter regions are co-occupied by Mediator and these NER proteins, and that relationships between Mediator and these NER proteins are more complex than between Mediator and Rad2. We also investigated if physical interactions between Mediator and NER proteins are modified after UV, we did not observe any significant change. Furthermore, we observed that the chromatin binding profiles of NER proteins and Mediator are modified after UV-irradiation. ChIP-sequencing will be carried out to get a genome-wide view of their chromatin binding profiles.In conclusion, we have strengthened the link between Rad2/XPG, Mediator and RNA Pol II, providing mechanistic insights into functional interplay between these components related to transcription-coupled repair, and showed that the link between Mediator and the NER machinery can be extended to other proteins. Taken together, our results suggest a close relation between Mediator functions in transcription and in NER, two fundamental processes dysfunction of which leads to human diseases
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Tamburini, Beth Ann. « Characterization of chromatin dynamics during DNA repair and transcriptional regulation / ». Connect to full text via ProQuest. IP filtered, 2006.
Trouver le texte intégralRésumé :
Thesis (Ph.D. in Molecular Biology) -- University of Colorado at Denver and Health Sciences Center, 2006.Typescript. Includes bibliographical references (leaves 137-151). Free to UCDHSC affiliates. Online version available via ProQuest Digital Dissertations;
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Mello, Jill Ann 1966. « Transcription and mismatch repair in the mechanism of action of the anticancer drug cisplatin ». Thesis, Massachusetts Institute of Technology, 1997. http://hdl.handle.net/1721.1/42993.
Texte intégralRésumé :
Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, 1997.Vita.
Includes bibliographical references (leaves 188-212).
cis-Diamminedichloroplatinum(II) (cis-DDP or cisplatin) is a powerful cytotoxin and anticancer therapeutic, used most effectively in the treatment of testicular and ovarian cancers. By contrast, the geometric isomer of cisplatin, trans- DDP, is comparatively non-toxic and fails to show significant antitumor activity. Cisplatin is believed to derive its cytotoxic effects from processes triggered by its reaction with DNA. The formation of cisplatin adducts can elicit many cellular responses, including inhibition of both DNA replication and transcription. Cisplatin DNA adducts are also specifically recognized by various proteins within the cell, and such cisplatin-damage recognition proteins have been previously suggested to play a role in the clinical efficacy of the drug. To date, however, the precise mechanism by which cisplatin lesions mediate the cytotoxic and antitumor activities of cisplatin remains elusive. The work in this dissertation evaluated two possible mechanisms by which cisplatin might exert its cytotoxic effects that had been heretofore largely unexplored. The first aspect of this work evaluated a model wherein the differential cytotoxic and antitumor activities of cisplatin and trans-DDP may result from a greater ability of cisplatin DNA damage to inhibit RNA transcription. A nonreplicating plasmid harboring the [beta]-galactosidase ([beta]-gal) reporter gene was modified in vitro with either of the two platinum compounds and transfected into human or hamster cell lines. The use of cell lines both proficient and deficient in nucleotide excision repair allowed the examination of transcriptional bypass independent of excision repair for each platinum compound. A two to three fold higher level of transcription was observed in both cell lines from plasmids containing trans-DDP adducts as compared to plasmids modified by cis-DDP. This difference in transcriptional activity was not decreased in human and rodent nucleotide excision repair deficient cell lines, indicating that more efficient excision repair of the trans- DDP adducts was not the cause of its lower ability to block transcription. The possibility that trans-DDP lesions are preferentially bypassed by RNA polymerase was examined by monitoring the elongation of [beta]-gal mRNA on damaged templates in vivo. Nascent [beta]-gal mRNA transcripts were recovered from nucleotide excision repair deficient xeroderma pigmentosum A cells transfected with platinated plasmids, and the extent of RNA synthesis was measured by using ribonuclease protection. The results showed that four-fold more trans-DDP than cis-DDP adducts were required to inhibit transcription elongation by 63 %. RNA polymerase II translocated past a single, representative DNA adduct of cisplatin and trans-DDP in vivo with an efficiency of 0- 16% and 60-76%, respectively. These data support the view that inhibition of transcription may contribute to the greater cytotoxicity of cis-DDP compared with its trans isomer. The second aspect of this work evaluated a possible novel role of the human mismatch repair protein, hMSH2, as a cisplatin-damage recognition protein. The interaction of purified recombinant hMSH2 with DNA containing adducts of cisplatin and various cisplatin analogs was examined in vitro by using an electrophoretic gel mobility shift assay. The results showed that hMSH2 recognizes and binds specifically to DNA adducts of cisplatin. This protein displayed affinity for DNA modified by therapeutically effective platinum complexes, but not for that modified by clinically inactive platinum compounds such as trans-DDP. Recognition by hMSH2 was dictated, in part, by the major intrastrand DNA adduct formed by cisplatin. The results also show that hMSH2 is overexpressed in testicular and ovarian tissue, tissues in which tumors are best treated by cisplatin. These results complement a growing body of literature correlating mismatch repair activity with cisplatin toxicity in Escherichia coli and mammalian cells. Viewed together, these observations are consistent with a model whereby mismatch repair plays an active role in potentiating cisplatin DNA lesion toxicity. Further, these results may provide insight into a previously undiscovered mechanism by which tumor cells may acquire resistance to CISplatin.
by Jill Ann Mello.
Ph.D.
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Liu, Hairong. « DNA repair and transcription of the yeast MFA2 gene : roles of Tup1p, Gcn5p and Rad16p ». Thesis, Cardiff University, 2005. http://orca.cf.ac.uk/54096/.
Texte intégralRésumé :
To clarify the role of one of the general chromatin remodelling factors-the histone acetylase Gcn5p in NER, I undertook experiments with
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Oksenych, Valentyn. « Molecular mechanisms of recruitment of transcription/repair factor TFIIH to the sites of damaged DNA ». Strasbourg, 2009. https://publication-theses.unistra.fr/public/theses_doctorat/2009/OKSENYCH_Valentyn_2009.pdf.
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Yang, Margaret Hwae-Ling. « Mutations flanking the DNA channel through RNA polymerase II affect transcription-coupled repair in Saccharomyces cerevisiae / ». view abstract or download file of text, 2007. http://proquest.umi.com/pqdweb?did=1417800941&sid=1&Fmt=2&clientId=11238&RQT=309&VName=PQD.
Texte intégralRésumé :
Thesis (Ph. D.)--University of Oregon, 2007.Typescript. Includes vita and abstract. Includes bibliographical references (leaves 80-87). Also available for download via the World Wide Web; free to University of Oregon users.
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O'Connell, Charlotte. « Roles of histone deacetylases and histone phosphorylation in transcription and nucleotide excision repair in Saccharomyces cerevisiae ». Thesis, Cardiff University, 2007. http://orca.cf.ac.uk/54298/.
Texte intégralRésumé :
Repair of UV-induced damage in the cell is crucial to maintain genome integrity and stability. Acetylation of the histone ammo-terminal tails plays key roles in transcription and DNA repair pathways. Acetylation states are determined by a balance between the activities of histone acetyltransferases (HATs) and histone deacerylases (HDACs). This study examined the roles of histone deacetylation in transcription and nucleotide excision repair (NER) in
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Ruff, C. A. D. B. « The role of growth associated transcription factors c-Jun and ATMIN in neuronal regeneration and repair ». Thesis, University College London (University of London), 2010. http://discovery.ucl.ac.uk/19813/.
Texte intégralRésumé :
Subsequent to neuronal injury, increased levels of protein synthesis accompany associated rises in transcription factor expression. Two transcription factors involved in the cellular stress response are c-Jun and ATMIN. Neuron-specific deletion of c-Jun resulted in defects in perineuronal sprouting, lymphocyte recruitment and microglial activation. Motoneurons also exhibited an atrophic phenotype, reduced target reinnervation and resistance to cell death. Additionally, mutants lacking Jun expression in peripheral Schwann Cells exhibited decreased regeneration and target muscle reinnervation, with accompanying deficits in cell survival, which highlights the dual role of c-Jun in response to stress. Homozygous deletion of JNK1, JNK2 or JNK3, or substitution of the c-Jun N-terminal serine phosphoacceptor sites (ser63&73), with alanines (JunAA), did not produce a difference in response to injury. This evidence indicates that N-terminal phosphorylation of ser63&73 does not play an essential function for axonal regeneration in vivo, while the whole c-Jun is clearly needed to successfully mount a regenerative response. ATMINΔN mutants, which have CNS-specific Nestin::Cre-mediated ATMIN deletion, exhibited higher transcription factor expression (c-Jun, Activating Transcription Factor-3/ATF3) in facial motoneurons – both baseline and following peripheral facial axotomy - and increased central post-traumatic sprouting of CGRP-and galanin-immunoreactive motoneurites. Although there was no effect on gross functional recovery or neuronal cell death, retrograde transport of florescent markers (Fluoro-Gold, MiniRuby) revealed augmented branching under normal conditions and during the reinnervation of peripheral motor targets. In the spinal cord injury model, ATMINΔn animals showed increased numbers of corticospinal tract (CST) axons projecting to both dorsal horns and contralateral CST, as well as bilateral impairment in precise co-ordinated motor behaviour following dorsolateral hemisection. This highlights the role of ATMIN as an important regulator of axonal guidance and pruning in neuronal development and regeneration. Altogether, these findings demonstrate the essential roles of c-Jun and ATMIN in the neuronal stress response following nerve transection.
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Assenmacher, Nora. « Structural and biochemical analysis of the UvrA binding module of the bacterial transcription repair coupling factor Mfd ». [S.l.] : [s.n.], 2006. http://edoc.ub.uni-muenchen.de/archive/00006371.
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Aßenmacher, Nora. « Structural and biochemical analysis of the UvrA binding module of the bacterial transcription repair coupling factor Mfd ». Diss., lmu, 2006. http://nbn-resolving.de/urn:nbn:de:bvb:19-63719.
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Bollins, Jack Michael. « The non-canonical roles of the transcription-repair coupling factor, Mfd, in Escherichia coli and Campylobacter jejuni ». Thesis, University of Bristol, 2017. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.738545.
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Lee, Sungkeun. « Molecular genetic analysis of nucleotide excision repair genes in Dictyostelium discoideum / ». free to MU campus, to others for purchase, 1997. http://wwwlib.umi.com/cr/mo/fullcit?p9841209.
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Traboulsi, Hussein. « Relationship between the transcription/DNA repair factor TFIIH and the Peroxisome Proliferator-Activated Receptor coactivator 1α PGC-1α ». Strasbourg, 2011. http://www.theses.fr/2011STRA6063.
Texte intégralRésumé :
Des mutations dans la sous unité XPD du facteur de transcription IIH (TFIIH) conduit à des maladies génétiques rares incluant trichothiodystrophy TTD. Connaissant que la sous unité MAT1 de TFIIH est nécessaire pour une activité complète de l’activateur proliferator activated receptor g coactivator 1a (PGC-1a), on a décidé de regarder l’influence de la mutation R722W dans XPD sur l’activité de PGC-1a dans les hépatocytes TTD. Nos résultats montrent que le facteur transcriptionnel TFIIH est nécessaire pour l’activité hépatique du cofacteur PGC-1α dans la voie de la néoglucogenèse. En plus on montre que la mutation R722W dans la sous unité XPD du TFIIH perturbe l’interaction du PGC-1α avec SIRT1, dérégulant par ca l’activité du PGC-1α. Etant PGC-1α un régulateur important du métabolisme du glucose et des lipides, nos résultats permet donc de suggérer un lien entre les symptômes liés à un défaut du métabolisme observés chez les patients TTD et le dérèglement du comportement du PGC-1α. Des études ultérieures seront nécessaires pour mieux comprendre le mécanisme d’activation du PGC-1α par TFIIH, surtout voir si ca implique un lien directe entre SIRT1 et TFIIH. Il est ainsi important d’analyser si d’autres voies du PGC-1α sont aussi affectéesMutations in the subunit XPD of the transcription factor IIH (TFIIH) lead to genetic disorders including trichothiodystrophy TTD. Knowing that subunit MAT1 of TFIIH is required for the full function of the proliferator activated receptor γ coactivator 1 α (PGC-1α), we decided to study the influence of the mutation R722W in XPD on PGC-1α activity. Using immortalized hepatocytes isolated from TTD mutant mice, we investigated the expression of PGC-1α target genes involved in gluconeogenesis. We observed that these genes are downregulated in TTD hepatocytes. Moreover, we found that XPD mutation disrupted the interaction between PGC-1α and the deacetylase Sirtuin1 (SIRT1) leading to reduced activation of PGC-1α. We also showed that PGC-1α and SIRT1 both interact with TFIIH. We thus established that TFIIH is required for full PGC-1α activity in the gluconeogenesis pathway, and more likely through modulation of SIRT1 activity. Therefore, our results suggest that the deregulation in TTD likely results from the inability of the mutated TFIIH to fully participate in recruitment of PGC-1α and/or SIRT1 to the DNA
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Phillips, Jennifer Elizabeth. « Runx2-Genetically Engineered Dermal Fibroblasts for Orthopaedic Tissue Repair ». Diss., Georgia Institute of Technology, 2007. http://hdl.handle.net/1853/19818.
Texte intégralRésumé :
Tissue engineering has emerged as a promising alternative to conventional orthopaedic grafting therapies. The general paradigm for this approach, in which phenotype-specific cells and/or bioactive growth factors are integrated into polymeric matrices, has been successfully applied in recent years toward the development of bone, ligament, and cartilage tissues in vitro and in vivo. Despite these advances, an optimal cell source for skeletal tissue repair and regeneration has not been identified. Furthermore, the lack of robust, functional orthopaedic tissue interfaces, such as the bone-ligament enthesis, severely limits the integration and biological performance of engineered tissue substitutes. This works aims to address these limitations by spatially controlling the genetic modification and differentiation of fibroblasts into a mineralizing osteoblastic phenotype within three-dimensional polymeric matrices. The overall objective of this project was to investigate transcription factor-based gene therapy strategies for the differentiation of fibroblasts into a mineralizing cell source for orthopaedic tissue engineering applications. Our central hypothesis was that fibroblasts genetically engineered to express Runx2 via conventional and biomaterial-mediated ex vivo gene transfer approaches will differentiate into a mineralizing osteoblastic phenotype. We have demonstrated that a combination of retroviral Runx2 overexpression and glucocorticoid hormone treatment synergistically induces osteoblastic differentiation and biological mineral deposition in primary dermal fibroblasts cultured in monolayer. We report for the first time that glucocorticoids induce osteoblastic differentiation in this model system by modulating the phosphorylation state of a negative regulatory serine residue (Ser125) on Runx2 through an MKP-1-dependent mechanism. Furthermore, we utilized these Runx2-genetically engineered fibroblasts to create mineralized templates for bone repair in vitro and in vivo. Finally, we engineered a heterogeneous bone-soft tissue interface with a novel biomaterial-mediated gene transfer approach. Overall, these results are significant toward the ultimate goal of regenerating complex, higher-order orthopaedic grafting templates which mimic the cellular and microstructural characteristics of native tissue. Cellular therapies based on primary dermal fibroblasts would be particularly beneficial for patients with a compromised ability to recruit progenitors to the sight of injury as result of traumatic injury, radiation treatment, or osteodegenerative disease.
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Iltis, Izarn. « Rôles transcriptionnels des facteurs NER ». Phd thesis, Université de Strasbourg, 2012. http://tel.archives-ouvertes.fr/tel-00849966.
Texte intégralRésumé :
Lors de la vie, des mécanismes de réparation de l'ADN sont mis en oeuvre lors d'agressions, pour protéger le génome. La réparation par excision de nucléotides (NER) est l'un de ces mécanismes. Des mutations des facteurs NER sont à l'origine de 3 maladies génétiques humaines: Xeroderma pigmentosum (XP), la trichothiodystrophie (TTD) et le syndrome de Cockayne (CS). Certains de leurs signes cliniques ne sont pas expliqués par un défaut de réparation de l'ADN. Des études suggèrent que ces facteurs interviennent dans d'autres processus, notamment lors de l'expression des gènes. Durant ma thèse, je me suis intéressé aux rôles des facteurs NER dans la transcription. En effet, j'ai montré que ces facteurs, dit de réparation, étaient recrutés avec la machinerie transcriptionnelle au niveau du promoteur et du terminateur de gènes activés. Ils influencent l'environnement chromatinien des gènes activés (boucles de chromatine et modifications post-‐ traductionnelles des histones). Ma thèse apporte une meilleure compréhension du processus de transcription des gènes activés, permettant de mieux comprendre certaines anomalies associées aux yndromes XP, CS et TTD.
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Yu, Sung-Lim. « Analysis of the response of nucleotide excision repair genes in Dictyostelium discoideum / ». free to MU campus, to others for purchase, 1997. http://wwwlib.umi.com/cr/mo/fullcit?p9841196.
Texte intégral
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Hardy, Robert George. « Alterations in cadherin and catenin expression in colonic neoplasia, injury and repair : regulation of P-cadherin transcription in the colon ». Thesis, University of Birmingham, 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.396450.
Texte intégral
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Bourseguin, Julie. « Etude des relations biochimiques, moléculaires et fonctionnelles entre le facteur de transcription MiTF et la voie de réparation FANC ». Thesis, Université Paris-Saclay (ComUE), 2016. http://www.theses.fr/2016SACLS271.
Texte intégralRésumé :
Les protéines et les voies impliquées dans la réponse aux dommages de l'ADN (DDR), permettant de maintenir la stabilité génétique et de préserver la fidélité de la réplication, agissent non seulement comme l'initiation de la cancérogenèse mais peuvent également jouer un rôle majeur dans la progression tumorale et dans la résistance aux thérapies. La voie FANC joue un rôle central dans la stabilité génétique lors d'un stress réplicatif. La perte de fonction de cette voie est la cause d'un syndrome de fragilité chromosomique et de prédisposition au cancer appelé Anémie de Fanconi (FA).Nous avons démontré que les protéines FANC était surexprimées et suractivées dans les mélanome métastatiques exprimant le facteur de transcription MITF, un oncogène exprimé dans 80% des cas de mélanome. Nous avons identifié MITF comme un régulateur majeur de l'expression des transcrits codant les protéines de la voie FANC dans les cellules de mélanomes et montré que les cellules déplétées pour MITF présentaient les caractéristiques des cellules FA, i.e., une hypersensibilité aux agents pontants l'ADN. De plus, la voie FANC module également la migration des cellules de mélanome. Nos observations montrent le rôle central de cette voie de réparation dans la résistance des cellules de mélanomes aux dommages de l'ADN. Cette voie serait donc une nouvelle cible thérapeutique dans le traitement du mélanome.Nous avons également observé que la perte de fonction de la voie FANC augmente l'expression de MiTF dans des cellules FA et des cellules déplétées pour les protéines FANC. Nous avons montré que la voie FANC régule négativement l'expression de MITF au niveau transcriptionnel. Des résultats préliminaires montre que FANCD2 pourrait être associés au promoteur de MiTF au niveau d'un site de fixation de NF-kB, où il pourrait empêcher son action. La déplétion de MiTF conduit à une sensibilité aux agents pontants l'ADN dans des cellules contrôles mais pas dans des cellules FA, suggérant que MITF jouerait un rôle dans la DDR en régulant l'expression des protéines FANC. Enfin, nous avons montré que l'expression de MiTF est induite en réponse aux stimuli pro-inflammatoire. Enfin, l'expression altérée de MITF pourrait expliquer des défauts de pigmentation et la microphthalmie rapportés chez les patients FA. L'ensemble de ces données, à la fois validée et préliminaire, supporte l'existence d'une relation épistatique entre MiTF et la voie FANC. Cet voie aurait un rôle important à la fois dans la résistance au mélanome métastatique et dans certaines caractéristiques pathologiques de l'anémie de FanconiProteins and pathways involved in DNA damage response (DDR), maintaining genetic stability and safeguarding DNA replication, act not only as caretakers against cancer initiation but also play a major role in sustaining cancer progression and resistance to pharmacological-based therapies. The FANC pathway is central in maintaining genetic stability under conditions of replication stress and its loss-of-function is causative of the cancer predisposition and chromosome fragility syndrome Fanconi Anemia (FA).We demonstrate here that FANC proteins are over-expressed and over-activated in metastatic melanoma cells expressing the oncogenic microphthalmia-associated transcription factor (MiTF), which high expression is maintained in 80% of melanoma cases. We identified MiTF as a critical regulator of the expression of the mRNAs coding key proteins of the FANC pathway in melanoma cells and demonstrated that MiTF-silenced cells display the primary characteristics of FA cells, i.e., the cellular and chromosomal hypersensitivity to DNA interstrand crosslink- inducing agents. Moreover, FANC pathway also modulates melanoma cell migration. Our observations point to a central role of the FANC pathway in cellular and chromosomal resistance to DNA damage in melanoma cells. Thus, the FANC pathway appears as a promising new therapeutic target for melanoma treatment.Inversely, we observed that FANC pathway loss-of-function is associated to increased expression of MiTF in both FA patient-derived and siRNA-downregulated cells. We demonstrated that the FANC pathway negatively regulates MiTF expression at the mRNA level and have obtained preliminary data suggesting that FANCD2 associates to the MiTF promoter, impeding the action of the NF-kB transcription factor. MiTF depletion increases MMC sensitivity in FANC pathway proficient cells, but does not modify the sensitivity of FA cells, supporting the hypothesis that MiTF acts on the DDR by regulating the expression of FANC proteins. Finally, we demonstrated that MiTF expression is induced in response to inflammatory stimuli, like TNF-a. Thus, altered MiTF expression in FA could be involved in the pigmentation defects and microphthalmia reported in patients.In conclusion, we will present a corpus of both validated and yet preliminary data that strongly supports the existence of an epistatic relationship between MiTF and the FANC pathway. This circuitry appears to have an important role in melanoma resistance to chemotherapies and in some FA pathological traits
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Richards, Jodi D. « Helicases and DNA dependent ATPases of Sulfolobus solfataricus ». Thesis, University of St Andrews, 2008. http://hdl.handle.net/10023/474.
Texte intégralRésumé :
DNA is susceptible to various types of damage as a result of normal cellular metabolism or from environmental sources. In order to maintain genome stability a number of different, partially overlapping DNA repair pathways have evolved to tackle specific lesions or distortions in the DNA. Nucleotide excision repair (NER) is highly conserved throughout eukarya, bacteria and archaea and predominantly targets lesions that result from exposure to UV light, for example cyclobutane pyrimidine dimers and 6-4 photoproducts. The majority of archaea possess homologous of the eukaryotic repair genes and this thesis describes the isolation and the characterization of two XPB homologues identified in the crenarchaeon Sulfolobus solfataricus, SsoXPB1 and SsoXPB2. Human XPB is one of 10 proteins that make up the TFIIH transcription complex. The activity of XPB is tightly controlled by protein interactions, in particular with p52, which stimulates the ATPase activity of XPB. Rather than a conventional helicase, human XPB is thought to act as an ATP dependent conformational switch. Consistent with human XPB, however, the S. solfataricus proteins were unable to catalyse strand separation and the identification of an archaeal protein partner, Bax1, for SsoXPB2 was one of the focuses of this project. In order to maintain genome stability, the DNA must be replicated accurately with each cell cycle. When the advancing replication fork stalls at a lesion or a DNA break, it is crucial that the fork is reset and that replication continues to completion. The helicase Hel308 is thought to clear the lagging strand template of a stalled replication fork in order for replication restart to proceed via homologous recombination (HR). Although the specific function of Hel308 is not well understood, the possibilities are described in this thesis. Strand exchange proceeds to form a D-loop, followed by branch migration to increase regions of heterology during the synapsis stage of HR. No motors for branch migration have previously been recognised in archaea, although the identification of a possible candidate was investigated during this project.
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Vesin, Rose-Marie. « Etude du rôle du facteur de transcription Helios dans les cellules souches hématopoïétiques ». Thesis, Strasbourg, 2014. http://www.theses.fr/2014STRAJ117/document.
Texte intégralRésumé :
Les cellules souches hématopoïétiques (CSH) sont à l’origine de l’ensemble des cellules hématopoïétiques, mais les mécanismes responsables de leurs réponses au stress ne sont que partiellement compris. J’ai étudié le rôle du facteur de transcription Helios dans les CSH, dans lesquelles il est fortement exprimé. J’ai trouvé que les CSH de souris nulles pour Helios (He-/-) possèdent un plus fort potentiel de reconstitution que les CSH WT dans des expériences de transplantations en série. De manière frappante, les souris âgées de 2 ans possèdent 8 fois plus de CSH fonctionnelles par rapport aux souris vieilles WT. De plus, le pool de CSH de long terme de souris He-/- vieilles ressemblent aux CSH WT jeunes en termes de phénotype et de fréquences. Les CSH He-/- vieilles présentent une dérégulation de gènes impliqués dans le vieillissement. De plus,les CSH He-/- jeunes sous-expriment des gènes codant des protéines impliquées dans la réparationde l’ADN ainsi que dans la voie p53. Quand les CSH He-/- et WT sont traitées avec différents agents radiomimétiques qui induisent des cassures simple et double brins à l’ADN, tels que la néocarzinostatine, la camptothécine ou l’étoposide, l’entrée en apoptose, en sénescence et l’arrêt de la prolifération des CSH He-/- sont altérées. Ce phénotype est accompagné d’une faible induction des gènes cibles de p53 et d’une altération du dégagement des foyers gammaH2AX. De plus, j’ai montré que Helios agit en synergie avec p53 pour réguler les réponses aux dommages à l’ADN des CSH. Mes résultats suggèrent qu’en synergie avec p53, Helios contrôle le vieillissement des CSH en prévenant l’accumulation des dommages de l’ADN des CSHHematopoietic stem cells (HSCs) give rise to all blood cell lineages but the mechanisms responsible of HSCs responses to stress remain partially understood. I studied the role of the transcription factor Helios in HSCs, where Helios is highly and specifically expressed. I found that HSCs from young Helios null mice (He-/-) reconstitute the hematopoietic system of irradiated recipient mice similarly to HSCs from WT mice in primary transplantations, but out-perform WT cells in secondary and tertiary transplantations. Strikingly, HSCs from 2-year-old He-/- mice had 8-fold higher reconstitution potential than old WT HSCs in primary transplantations. Moreover, the pool of long-term HSCs in old He-/- mice resembles that of young WT animals in both phenotype and frequency. HSCs from old He-/- mice present a down regulation of genes involved in aging. Further, young He-/- HSCs express reduced mRNA levels of genes encoding DNA repair proteins as well as those associated with thep53 pathway. When He-/- and WT HSCs were subjected to DNA damage by different agents like neocarzinostatin, camptothecin, or etoposide, DNA damage-induced apoptosis, senescence and cell cycle arrest were significantly impaired in He-/- HSCs. This phenotype was accompanied by a poor induction of p53 target genes and impaired clearance of gammaH2AX foci. Furthermore, I found that Helios synergies with p53 to regulate the DNA damage responses of HSCs. My results suggest that,in synergy with p53, Helios controls HSC aging by preventing the accumulation of DNA damage in these cells
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Eyboulet, Fanny. « Mécanismes de l'activation de la transcription in vivo par le Médiateur ». Thesis, Paris 11, 2014. http://www.theses.fr/2014PA112177/document.
Texte intégralRésumé :
Chez les eucaryotes, la synthèse des ARN messagers (ARNm) est un processus hautement régulé en réponse à la fixation d’activateurs spécifiques sur des régions régulatrices. Cette étape permet le recrutement de co-activateurs, des facteurs généraux de la transcription (GTFs) et de l’ARN polymérase II (Pol II) pour former le complexe de préinitiation (PIC). Le Médiateur est un complexe co-activateur essentiel à ce processus et bien qu’il ait fait l’objet de nombreuses études ces dernières années, sa complexité a empêché de parvenir à une compréhension détaillée de son mécanisme de fonctionnement in vivo. Au cours de ma thèse, je me suis intéressée à la sous-unité Med17 qui joue un rôle central au sein du module de tête du Médiateur et interagit directement avec la Pol II. Nous avons construit une collection de mutants thermosensibles de cette sous-unité chez la levure Saccharomyces cerevisiae, que nous avons ensuite caractérisés par différentes approches de biologie moléculaire et génomique fonctionnelle. Nos analyses par ChIP-seq montrent que le Médiateur influence indépendamment le recrutement et/ou la stabilisation de la TBP ainsi que des modules cœur et kinase de TFIIH sur le génome. Ces résultats indiquent que, contrairement à la séquence d’assemblage linéaire observée in vitro, l’assemblage du PIC in vivo est un processus à plusieurs étapes non-séquentielles et que le Médiateur est important pour orchestrer l’arrivée des différents composants du PIC. Par ailleurs, nous avons mis en évidence un contact direct entre le Médiateur et Rad2/XPG, une endonucléase qui intervient dans la réparation de l’ADN. Une analyse à l’échelle du génome a révélé que cette protéine est présente sur les gènes de classe II, en absence de stress génotoxique et que sa localisation génomique corrèle avec celle du Médiateur. Nous avons ainsi démontré que le Médiateur est important pour le recrutement de Rad2, suggérant un nouveau rôle pour ce complexe dans la réparation de l’ADN, en plus de son rôle de co-activateur dans la transcription par la Pol IIIn eukaryotes, the synthesis of messenger RNA (mRNA) is highly regulated in response to the binding of specific activators to regulatory regions. This step allows the recruitment of coactivators, general transcription factors (GTFs) and RNA polymerase II (Pol II) to form the preinitiation complex (PIC). Mediator is a co-activator complex essential to this process and although it has been studied intensively during the last few years, its complexity has precluded a detailed understanding of the molecular mechanisms of its function in vivo. During my PhD, I focused on the Med17 subunit which plays a central role within the Mediator head module and interacts directly with Pol II. We obtained a large collection of temperature-sensitive mutants of this subunit in the yeast Saccharomyces cerevisiae, and then characterized these mutants by different molecular biology and functional genomics approaches. Our ChIP-seq analyses show that Mediator influences independently the recruitment and/or the stabilization of TBP as well as TFIIH core and kinase modules on the genome. These results indicate that, unlike a linear sequence observed in vitro, in vivo the PIC assembly is a non-sequential multistep process and that Mediator is important to orchestrate the recruitment of different PIC components. Furthermore, we identified a direct contact between Mediator and Rad2/XPG, an endonuclease involved in DNA repair. A genome-wide analysis reveals that this protein is present on class II genes in the absence of genotoxic stress, and that its genomic localization correlates with that of Mediator. We thus demonstrated that Mediator is important for Rad2 recruitment, suggesting a new role for this complex in DNA repair, in addition to its co-activator role in Pol II transcription
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Soret, Christine. « Mise en évidence d'une fonction non-transcriptionnelle du facteur de transcription homéotique Cdx2 ». Thesis, Strasbourg, 2014. http://www.theses.fr/2014STRAJ109/document.
Texte intégralRésumé :
Le cancer colorectal (CCR) représente la 2ème cause de mortalité par cancer dans les pays industrialisés. De nouveaux traitements permettant de bloquer l’évolution de la maladie sont nécessaires. Il est donc important de mieux connaitre les acteurs impliqués dans la cancérogenèse. Lors du développement du cancer, des gènes suppresseurs de tumeur sont inhibés et des oncogènes sont activés, perturbant ainsi l’équilibre des cellules et les transformant. Au cours de ma thèse, je me suis intéressée à deux gènes homéotiques qui possèdent des rôles opposés dans les CCR. Cdx2 exerce un rôle suppresseur de tumeur, alors que HoxB7 agit comme un oncogène. Mon travail de thèse a permis (i) de mettre en évidence une nouvelle fonction non-transcriptionnelle de Cdx2 : inhibiteur de la réparation des cassures de l'ADN spécifiquement dans le côlon, (ii) et de révéler que le niveau d'expression des gènes Cdx2 et Hoxb7 au sein de la tumeur peut avoir une importance pour le choix du traitement des CCRColorectal cancer is the 2nd cause of mortality by cancer in industrialized countries. New treatments allowing to prevent the evolution of the disease are needed. It is important to better understand the actors implicated in carcinogenesis. During cancer development, tumor suppressor genes are inhibited and oncogenes are activated, thus disrupting the homeostasis of the tissue and transforming the cells. During my thesis, I have been interested in two genes having two opposite functions in CCR : Cdx2 is a tumor suppressor while Hoxb7 acts as an oncogene. My work allows to highlight (i) a new non-transcriptional function of Cdx2 : inhibitor of the reparation of DNA breaks specifically in the colon, (ii) and that the expression level of Cdx2 and Hoxb7 genes inside the tumor can have an importance in the choice of the CCR treatment
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Semer, Maryssa. « Le facteur de réparation XPC est un cofacteur de l'ARN polymérase II régulant les modifications post-traductionnelles des histones lors de la transcription ». Thesis, Strasbourg, 2018. http://www.theses.fr/2018STRAJ040/document.
Texte intégralRésumé :
La voie de réparation NER implique une cascade de complexes protéiques dont le senseur des dommages de l’ADN (XPC/HR23B). Des mutations dans les gènes de la NER (TTD-A, XPA-G, XPV, CSA et CSB), sont associées à des maladies génétiques humaines dont le Xeroderma Pigmentosum (XP), la Trichothiodystrophie (TTD) et le syndrome de Cockayne (CS). L’ensemble des symptômes des patients ne peut être expliqué seulement par un défaut de la réparation de l’ADN. Or depuis quelques années, il a été prouvé que les facteurs de la NER sont aussi impliqués lors de la transcription. Dans le cadre de ma thèse, je me suis particulièrement intéressé à la protéines XPC en déterminant son rôle transcriptionnel à l’échelle génomique afin de mieux comprendre les conséquences de sa dérégulation dans un contexte pathologique. En ce sens, mon second objectif a été de caractériser au niveau moléculaire l’étiologie de nouveaux patients XP en analysant de manière combinée les évènements moléculaires de la NER et la transcription associés à XPC. Nos différentes approches expérimentales ont permis d’identifier au niveau génomique un ensemble de gènes sont les promoteurs sont régulés aussi bien positivement que négativement par XPC dans un contexte RAR dépendant. De plus, nous montrons que XPC interagit avec KAT2A contenu dans le complexe ATAC, ainsi que qu’avec le facteur de transcription E2F1, le facteur de remodelage de la chromatine BRD2 et le variant d’histone H2A.Z. Via KAT2A, ce complexe va acétyler non seulement H2A.Z mais également H3K9 au niveau des promoteurs ciblés par E2F1NER involves a cascade of protein complexes including the DNA damage sensor (XPC/HR23B). Mutations in NER genes (TTD-A, XPA-G, XPV, CSA and CSB) are associated with human genetic diseases including Xeroderma pigmentosum (XP), Trichothiodystrophy (TTD) and Cockayne Syndrome (CS). All the symptoms can only be explained by a defect of the DNA repair. However all the symptoms can only be explained by a defect of the DNA repair. However, it has been proven that NER factors are also involved in transcription. As the genomic scale to better understand the consequences of its deregulation in a pathological context. In this sense, my second goal has been to characterize at the molecular level the etiology of new XP patients by analyzing in a combined way the molecular events of the NER and the transcription associated with XPC. Our different experimental approaches have made it possible to identify at genomic level a set of gene whose promoters are regulated both positively and negatively by XPC in a dependent RAR context. In addition, we show that XPC interacts with KAT2A contained in the ATAC complex, as well as with the transcription factor E2F1, the chromatin remodeling factor BRD2, and the histone variant H2A.Z. Via KAT2A, this complex will acetylate not only H2A.Z but also H3K9 at promoters targeted by E2F1
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Palassin, Pascale. « Etude du rôle du corégulateur transcriptionnel RIP140 dans le contrôle de l'instabilité microsatellitaire des cancers colorectaux héréditaires ». Thesis, Montpellier, 2017. http://www.theses.fr/2017MONTT054/document.
Texte intégralRésumé :
Le corégulateur transcriptionnel RIP140 est un facteur ubiquitaire majeur impliqué dans la régulation de nombreux processus physiopathologiques, qui possède la capacité d’être un coactivateur ou un corépresseur des voies de signalisation selon son recrutement sur les gènes cible. Des résultats du laboratoire ont montré que RIP140 est un facteur de bon pronostic de la tumorigenèse intestinale sporadique. Ce travail s’intéresse à l’implication de ce facteur de transcription dans les cancers colorectaux familiaux et, plus particulièrement, en lien avec le syndrome de Lynch (LS). Le syndrome de Lynch est une prédisposition héréditaire aux cancers, majoritairement colorectaux, caractérisés par un défaut du système de réparation des mésappariements de l’ADN (Mismatch Repair, MMR), dû à une première mutation germinale d’un des gènes de ce système. La perte de fonctionnalité MMR est responsable du phénotype d’instabilité microsatellitaire (MSI). Cependant, il existe des formes familiales de cancers colorectaux, avec MSI, où il n’est pas retrouvé d’atteinte germinale ou somatique de l’un des gènes du système MMR. Ce sont les syndromes apparentés au syndrome de Lynch (Lynch Like Syndrome, LLS) dont la prise en charge est identique à celle du LS. L’utilisation de modèles murins et de lignées cellulaires colorectales, présentant des modulations d’expression de RIP140 ont permis de mettre en évidence l’effet positif de ce corégulateur sur la régulation transcriptionnelle de l’expression des gènes du système MMR, MSH2 et MSH6. La validité fonctionnelle de cette régulation a été explorée par des analyses d’instabilité microsatellitaire et de sensibilité à différentes molécules cytotoxiques. Des cohortes de tumeurs ont permis de confirmer la corrélation d’expression entre RIP140 et les gènes MSH2 et MSH6 chez les patients. En outre, la régulation de l’expression par RIP140 d’une polymérase translésionnelle particulière, la polymérase Polκ, a été étudiée. Cette polymérase assure la réplication des séquences microsatellitaires du génome. Nous avons démontré que RIP140 stimule l’expression du gène POLK dans nos modèles cellulaires et que son expression est corrélée à celle de RIP140 au sein des tumeurs colorectales humaines. Enfin, par séquençage de différentes lignées cellulaires, nous avons mis en évidence une mutation de RIP140 qui entraîne un décalage du cadre de lecture et génère une protéine tronquée avec perte de deux domaines répresseurs de la protéine. Un séquençage à très haut débit nous a permis de rechercher cette mutation parmi des échantillons de tumeurs colorectales avec MSI. Cette mutation est retrouvée dans 19% des tumeurs, notamment LLS (16,2%), où elle est associée à une moins bonne survie globale. Elle affecte les propriétés antiprolifératives et transrépressives de RIP140 ainsi que les régulations positives des gènes MSH2, MSH6 et POLK. Le développement d’un outil anticorps spécifique de cette mutation serait extrêmement utile pour suivre l’expression de la forme mutée au sein des tumeurs et des premiers essais ont été réalisés en ce sens. En conclusion de ce travail, RIP140 contrôle l’expression de gènes majeurs impliqués dans le maintien de l’intégrité du génome et une mutation de ce corégulateur transcriptionnel pourrait être responsable de l’instabilité microsatellitaire de certaines tumeurs où des altérations des gènes MMR ne sont pas retrouvées. Des études cliniques sur des cohortes plus conséquentes seront nécessaires pour valider son intérêt en tant que marqueur utilisable dans la prise en charge des patientsThe transcriptional coregulator RIP140 is an ubiquitous cofactor playing a major role in the regulation of many physiopathological processes. It can either act as a coactivator or as a corepressor of signaling pathways depending on its recruitment on target genes. It has been shown that RIP140 is a good prognostic marker in sporadic intestinal tumorigenesis. This work focuses on its role in familial colorectal cancers and particularly in relation to the Lynch syndrome (LS). Lynch syndrome is a hereditary cancer predisposition, mostly colorectal, characterized by a defect in the Mismatch Repair (MMR) system, due to a first germline mutation of one gene of this system. Loss of MMR function induces a microsatellite instability (MSI) phenotype. However, there are some MSI familial colorectal cancers, where neither germinal nor somatic alteration of one MMR gene is found. They are referred to as Lynch like Syndrome (LLS) and their overall management is identical to that of LS. Murine models and colorectal cell lines, harboring modulations of RIP140 expression, allowed us to demonstrate the positive transcriptional regulation of the MMR genes, MSH2 and MSH6 by RIP140. Functional validation of this regulation was explored by microsatellite instability and sensitivity to various cytotoxic drugs analyses. A positive correlation has been confirmed between RIP140 and MSH2 and MSH6 gene expression in a cohort of 396 patients. Moreover, the transcriptional regulation by RIP140 of a specialized translesional DNA polymerase, the Polκ polymerase, has been investigated. Polκ ensures microsatellite sequences replication. We have demonstrated that RIP140 positively stimulates the expression of the POLK gene in our cell models and which appears correlated with that of RIP140 in human colorectal tumors. Finally, by sequencing different cell lines, we found a frameshift mutation of RIP140, generating a truncated protein with loss of the last two repression domains. High-throughput sequencing allowed us to look for this mutation in patient MSI colorectal tumor samples. This mutation was found in 19% of these tumors, especially LLS (16,2%), where it has been associated with lower overall survival. This mutation affects the antiproliferative and transrepressive properties of RIP140, as well as the positive regulation of the MSH2, MSH6 and POLK gene. Development of a specific antibody for this mutation would be extremely useful in following the expression of this mutated form within tumors and first tests have been already carried out. In conclusion, RIP140 controls expression of major genes involved in genome integrity maintenance and a mutation of this transcriptional coregulator could be responsible for microsatellite instability of some tumors where alterations of MMR genes are not found. Clinical studies on larger cohorts will be necessary to validate its interest as a marker usable in patient management
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Xie, Jenny X. « Regulation of BACH1/FANCJ Function in DNA Damage Repair : A Dissertation ». eScholarship@UMMS, 2009. https://escholarship.umassmed.edu/gsbs_diss/435.
Texte intégralRésumé :
The DNA damage response (DDR) pathway is a complicated network of interacting proteins that function to sense and remove DNA damage. Upon exposure to DNA damage, a signaling cascade is generated. The damage is either removed, restoring the original genetic sequence, or apoptosis is activated. In the absence of DDR, cells are unable to effectively process DNA damage. Unprocessed DNA damage can lead to chromosomal changes, gene mutations, and malignant transformation. Thus, the proteins involved in DDR are critical for maintaining genomic stability.
One essential DDR protein is the BRCA1 Associated C-terminal Helicase, BACH1. BACH1 was initially identified through its direct association with the BRCT domain of the Breast Cancer Associated Gene, BRCA1. Similar to BRCA1, germline mutations in BACH1were identified in patients with early onset breast cancer. Interestingly, the disease-associated mutations in BACH1 were shown to have altered helicase activity in vitro, providing a direct link between BACH1 helicase activity and disease development. The correlation between BACH1 and cancer predisposition was further confirmed by the identification of BACH1 as the cancer syndrome Fanconi anemia (FA) gene product, FANCJ. Similar to other FA proteins, suppression of FANCJ leads to decreased homologous recombination, enhanced sensitivity to DNA interstrand crosslinking (ICL) agents, and chromosomal instability.
In an effort to further understand the function of FANCJ in DDR, FANCJ was shown to directly associate with the mismatch repair (MMR) protein MLH1. This interaction is facilitated by lysines 141 and 142 within the helicase domain of FANCJ. Importantly, the FANCJ/MLH1 interaction is critical for ICL repair. Furthermore, in an attempt to dissect the binding site of FANCJ on MLH1, we discovered an HNPCC associated MLH1 mutation (L607H) that has intact mismatch repair, but lacks FANCJ interaction. In contrast to the MLH1 interaction, the FANCJ/BRCA1 interaction was not required for correcting the cellular defects in FANCJ null cells. Thus, in an effort to understand the functional significance of the FANCJ/BRCA1 interaction, we discovered that FANCJ promotes Pol η dependent translesion synthesis (TLS) bypass when uncoupled from BRCA1. In this thesis, we provide evidence suggesting that FANCJ and MLH1 are functionally linked and that the interaction of these proteins is critical for repair choice.
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Ali, Nsrein. « Rôle du facteur de transcription HIF-1α dans la physiologie cutanée et dans la réponse à l'exposition UV ». Thesis, Vandoeuvre-les-Nancy, INPL, 2010. http://www.theses.fr/2010INPL041N/document.
Texte intégralRésumé :
Le facteur de transcription HIF-1 est un hétérodimère composé d’une sous-unité α et d’une sous-unité ß. HIF-1 est capable de reconnaître une séquence consensus appelée HRE (HIF Response Element) et de réguler l’expression de plus de 200 gènes cibles impliqués dans divers mécanismes cellulaires. Nous nous intéressons à étudier le rôle de HIF-1α dans la peau, d’une part dans la régulation des enzymes de la réparation de l’ADN suite à l’irradiation UVB, d’autre part dans la physiologie cutanée.Nos résultats montrent bien que HIF-1α régule l’expression des gènes participant à la réparation de l’ADN (XPC et XPD). Ces gènes contiennent dans leurs régions promotrices des HRE de HIF-1α. La quantification de l’immunoprécipitation de chromatine révèle des HRE putatifs dans les gènes codant pour d'autres protéines de la réparation de l'ADN (XPB, XPG, CSA et CSB), ce qui suggère que HIF-1α est un régulateur clé de la machinerie de réparation de l'ADN. Nous avons prouvé que HIF-1α est indispensable à l’adhésion des kératinocytes par sa régulation exercée sur la laminine-332 et les intégrines (α6 et ß1). L’absence de l’expression de HIF-1α empêche aussi la reconstruction des épidermes à partir des kératinocytes humains. Nos résultats ont montré que les souris invalidées pour HIF-1α développent avec l’âge un phénotype d’inflammation dans plusieurs régions. Ces souris sont très sensibles au moindre stress consécutif à une blessure et une irradiation UVB. L’induction de l’inhibition de HIF-1α dans des souris inductibles avec le tamoxifène indique un détachement de l’épiderme au niveau des couches supra-basales. Ces souris meurent deux semaines après injection du tamoxifèneThe transcription factor HIF-1 is a heterodimer composed of an α and ß subunit. HIF-1 is capable of recognizing a consensus sequence called HRE (hypoxia Response Element) and regulate the expression of more than 200 target genes involved in various cellular mechanisms. We are interested in studying the role of HIF-1α in the skin physiology.Our results show that HIF-1α regulates the expression of two main factors (XPC and XPD) involved in nucleotide excision repair through binding on HRE in their promoter regions. Quantitative chromatin immunoprecipitation assays further revealed putative HREs in the genes encoding other DNA repair proteins (XPB, XPG, CSA and CSB), suggesting that HIF-1α is a key regulator of the DNA repair machinery. We proved that HIF-1α is essential for keratinocyte adhesion through its regulation exerted on laminin-332 and integrins (α6, ß1). The lack of HIF-1α expression also prevents the reconstruction of epidermis by human keratinocytes. Our results showed that mice constitutively depleted for HIF-1α in their epidermis develop with age a phenotype of inflammation in several regions. These mice are very sensitive to the stress resulting from wound injury and UVB irradiation. HIF-1α depletion in the epidermis of inducible mice using tamoxifen results in a detachment of the epidermis in suprabasal layers. These mice die within two weeks after injection of tamoxifen
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Charles, Richard John Lalith. « FACT, réparation par excision de bases et fixation du facteur de transcription NF-kB sur la chromatine ». Thesis, Grenoble, 2012. http://www.theses.fr/2012GRENV034/document.
Texte intégralRésumé :
FACT est une protéine clé, qui joue de multiples rôles, y compris dans la transcription et la réparation de l'ADN endommagé. Néanmoins, comment FACT participe à la réparation et à la transcription de la chromatine n'est pas élucidé. Dans ce travail nous avons tout d'abord étudié le rôle de FACT dans le processus de réparation par excision de base (BER). Nous avons utilisé des nucléosomes reconstitués avec de l'ADN à uracile incorporé au hasard. Nous avons trouvé que l'enzyme UDG est capable d'enlever les uraciles localisés du côté de la solution et pas les uraciles se trouvant en face de l'octamère d'histone. La présence simultanée de FACT et de RSC (facteur de remodelage de la chromatine, impliqué dans la réparation) permet un enlèvement efficace des uraciles localisés du côté de l'octamère d'histone par l'UDG. De plus, l'action concertée de FACT et RSC contribue à l'enlèvement de la lésion oxidative 8-oxoG, autrement inaccessible, de la matrice nucléosomale par l'enzyme OGG1. Ce résultat est obtenu grâce à une activité « co-remodelatrice » de la protéine FACT. Dans ce travail nous décrivons pour la première fois cette nouvelle propriété de FACT et nous montrons par une série d'expériences biochimiques que FACT est capable de stimuler l'activité de remodelage du RSC. Nos expériences montrent que la présence de FACT augmente l'efficacité de RSC à transformer l'énergie libérée par l'hydrolyse de l'ATP en travail « mécanique ». Les données obtenues suggèrent une nature stochastique du BER in vivo, FACT étant un facteur clé dans le processus de réparation. Nous avons également investigué l'implication de l'activité co-remodelatrice de FACT dans la fixation de NF-kB aux matrices nucléosomales. La production de nucléosomes remodelés, mais non - mobilisés (remosomes) n'est pas suffisante pour promouvoir la fixation de NF-kB. Pourtant, la mobilisation des nucléosomes par l'intermédiaire de RSC permet une interaction efficace entre NF-kB et l'ADN nucléosomal. Toutes ces données sont essentielles pour le décryptage du mécanisme moléculaire par lequel FACT agit dans le BER et dans la transcription médiée par NF-kBFACT is a vital protein which has multiple roles including one in transcription and repair of damaged DNA. However, how FACT assists repair and transcription remains elusive. In this work, we have first studied the role of FACT in Base Excision Repair (BER). We used nucleosomes containing DNA with randomly incorporated uracil. We found that the enzyme UDG is able to remove uracils facing the solution and not the uracils facing the histone octamer. The simultaneous presence of FACT and RSC (a chromatin remodeler involved in repair) allows, however, a very efficient removal of uracil facing the histone octamer by UDG. In addition, the concerted action of FACT and RSC permits the removal of the otherwise un-accessible oxidative lesion 8-oxoG from nucleosomal templates by OGG1. This was achieved thanks to the co-remodeling activity of FACT. Here we described for the first time this novel property of FACT and we show in a series of biochemical experiments that FACT is able to boost the remodeling activity of RSC. The experiments reveal that the presence of FACT increases the efficiency of RSC to transform the energy freed by ATP hydrolysis into “mechanical” work. The presented data suggest a stochastic nature of BER functioning in vivo, FACT being a key factor in the repair process. The implication of the co-remodeling activity of FACT in NF-kB factor binding to nucleosomal templates was also investigated. The generation of remodeled, but not mobilized nucleosomes (remosomes), was not sufficient to promote NF-kB binding. However, the RSC-induced nucleosome mobilization allows efficient NF-kB interaction with nucleosomal DNA. Our data are instrumental in deciphering the molecular mechanism of FACT implication in BER and NF-kB mediated transcription
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Hillukkala, T. (Tomi). « Roles of DNA polymerase epsilon and TopBP1 in DNA replication and damage response ». Doctoral thesis, University of Oulu, 2006. http://urn.fi/urn:isbn:9514282922.
Texte intégralRésumé :
Abstract
During DNA replication cells accurately copy their DNA to transfer the genetic information to daughter cells. DNA polymerases synthesise the new DNA strand using the old strand as a template. Other functions of DNA polymerases are recombination linked and DNA iamage repair linked DNA synthesis, regulation of replication complex formation and regulation of transcription – a process in which the genetic information is transformed into an RNA sequence needed to guide protein synthesis.
In this study, the TopBP1 protein was shown to associate with DNA polymerase epsilon. TopBP1 contains eight BRCT domains mediating interactions between phosphorylated proteins and is a human homolog of bakers yeast Dpb11 and fission yeast Cut5. These yeast proteins act on DNA replication and cell cycle arrest after DNA damage. TopBP1 was found to be phosphorylated and expressed in elevated amounts during S phase suggesting an involvement in DNA replication. This was directly demonstrated by DNA synthesis inhibition by a competing TopBP1 fragment and by an antibody targeted to block TopBP1.
Ultraviolet irradiation damages DNA and decreases the amount of TopBP1 in the nucleus. The transcription factor Miz-1 was found to associate with TopBP1 and was released from this interaction after UV damage. Free Miz-1 activated the expression of the cell cycle arresting proteins p15 and p21 cooperatively with other transcription factors and allowed extra time for DNA damage repair.
TopBP1 was also found to interact with the breast cancer susceptibility protein 1 and both proteins localised together to arrested DNA synthesis apparatuses. The interaction of TopBP1 with the damage recognition and processing protein Rad9 is still further evidence of a link between TopBP1 and DNA damage.
DNA polymerase epsilon forms a complex with Cdc45, a protein involved in DNA replication initiation and elongation. This complex does not interact with Cdc45 complexed with DNA polymerase delta, suggesting that these complexes synthesise DNA independently of each other. Our results are in agreement with the view that polymerase epsilon synthesises the first strand of DNA and polymerase delta the other.
Finally,DNA polymerase epsilon binds to the RNA synthesising form of RNA polymerase II and nascent transcripts. The physiological meaning of this interaction needs to be determined.
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Guintini, Laetitia. « Étude de la réparation des lésions induites par les UVs dans les extrémités chromosomiques de la levure Saccharomyces cerevisiae ». Thèse, Université de Sherbrooke, 2016. http://hdl.handle.net/11143/9524.
Texte intégralRésumé :
Résumé : Les télomères sont des structures nucléoprotéiques spécialisées qui assurent la stabilité du génome en protégeant les extrémités chromosomiques. Afin d’empêcher des activités indésirables, la réparation des dommages à l’ADN doit être convenablement régulée au niveau des télomères. Pourtant, il existe peu d’études de la réparation des dommages induits par les ultraviolets (UVs) dans un contexte télomérique. Le mécanisme de réparation par excision de nucléotides (NER pour « Nucleotide Excision Repair ») permet d’éliminer les photoproduits. La NER est un mécanisme très bien conservé de la levure à l’humain. Elle est divisée en deux sous voies : une réparation globale du génome (GG-NER) et une réparation couplée à la transcription (TC-NER) plus rapide et plus efficace. Dans notre modèle d’étude, la levure Saccharomyces cerevisiae, une forme compactée de la chromatine nommée plus fréquemment « hétérochromatine » a été décrite. Cette structure particulière est présente entre autres, au niveau des régions sous-télomériques des extrémités chromosomiques. La formation de cette chromatine particulière implique quatre protéines nommées Sir (« Silent Information Regulator »). Elle présente différentes marques épigénétiques dont l’effet est de réprimer la transcription. L’accès aux dommages par la machinerie de réparation est-il limité par cette chromatine compacte ? Nous avons donc étudié la réparation des lésions induites par les UVs dans différentes régions associées aux télomères, en absence ou en présence de protéines Sir. Nos données ont démontré une modulation de la NER par la chromatine, dépendante des nucléosomes stabilisés par les Sir, dans les régions sous-télomériques. La NER était moins efficace dans les extrémités chromosomiques que dans les régions plus proches du centromère. Cet effet était dépendant du complexe YKu de la coiffe télomérique, mais pas dépendant des protéines Sir. La transcription télomériques pourrait aider la réparation des photoproduits, par l’intermédiaire de la sous-voie de TC-NER, prévenant ainsi la formation de mutations dans les extrémités chromosomiques. Des ARN non codants nommés TERRA sont produits mais leur rôle n’est pas encore clair. Par nos analyses, nous avons confirmé que la transcription des TERRA faciliterait la NER dans les différentes régions sous-télomériques.Abstract : Telomeric DNA is made of short tandem repeats located at the ends of chromosomes and their maintenance is critical to prevent genome instability. DNA lesions constitute a serious risk to genome integrity. Thus, DNA repair mechanisms are required for continuous and unabridged cell divisions. The nucleotide excision repair (NER) pathway removes bulky DNA lesions such as UV-induced photoproducts, like the cyclobutane pyrimidine dimers (CPD). NER is divided in two sub-pathways: global genome repair (GGR) and the faster transcription-coupled repair (TCR), which only differ in how they recognize UV-induced lesions. In eukaryotes, NER must find and repair DNA lesions that are buried in nucleosomes. In the yeast S. cerevisiae, genes positioned close to telomeres are silenced by a heterochromatin-like structure that is formed by silent information regulator proteins (Sir). To determine if nucleosomes and chromatin in subtelomeric regions affect the efficiency of NER, we studied the repair of photoproducts in different telomere-associated regions in both, WT and SIR genes deleted cells (sirΔ). We found that NER efficiency was modulated by the presence of nucleosomes on the subtelomeric type X element. In addition, in absence of Sir proteins, NER efficiency increased and was not modulated by nucleosomes, indicating that nucleosome positioning was less defined in sirΔ cells. Remarkably, in telomeric restriction fragment, NER was less efficient at telomeres than in the subtelomere type Y’ element. We suggest that low NER efficiency at the very end of chromosomes results from attachment sites to the nuclear periphery. Our data indicate that NER in sub-telomeric chromatin is modulated by Sir proteins stabilized-nucleosomes, and that NER is inhibited in telomeric chromatin by the presence of YKu, independently from the presence of Sir proteins. It was recently shown that the chromosome ends are transcribed and a non-coding RNA, called TERRA, is produced. Currently the precise functions of TERRA are not understood. Our second goal is to help understand the function of TERRA. We think that transcription at the chromosome ends could facilitate the removal of DNA lesions from heterochromatin by TCR, which would prevent the formation of mutations and, ultimately, chromosome shortening. Our data showed that TC-NER is effective in Y’ element and the telomere. Without Sir proteins, TERRA transcription is found in a particular region at the end of the X element. The transcription of TERRA could improve the repair of UV-induced lesions.
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Khobta, Andriy [Verfasser]. « Consequences of DNA damage for gene transcription : direct effects of modified nucleobases and the role of base excision repair = Folgen von DNA-Schäden für die Gentranskription / Andriy Khobta ». Mainz : Universitätsbibliothek Mainz, 2014. http://d-nb.info/1070334405/34.
Texte intégral
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Hochhauser, Daniel. « Transcriptional regulation of topoisomerase II ». Thesis, University of Oxford, 1993. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.333178.
Texte intégral
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Senarisoy, Muge. « Etudes des fonctions du facteur de transcription YB-1, de l'ADN glycosylase hNTH1 et de la topoisomerase humaine I dans le contexte de la résistance aux drogues et en relation avec les voies de réparation de l'ADN ». Thesis, Université Grenoble Alpes (ComUE), 2018. http://www.theses.fr/2018GREAV064/document.
Texte intégralRésumé :
La résistance acquise aux traitements anticancéreux représente un problème clinique majeur. Les voies de réparation de l'ADN fournissent un mécanisme de résistance, mais celle-ci peut aussi résulter de mutations ou d'une expression réduite de la protéine ciblée. La surexpression ou la localisation nucléaire de la Y-Box binding (YB-1) protéine est considérée comme un marqueur pronostique de chimiorésistance de la tumeur. YB-1 interagit avec plusieurs partenaires ; dans cette étude, nous nous sommes concentrés sur son interaction avec l'enzyme de réparation de l'ADN NTH1 (hNTH1) et l'ADN topoisomérase I (hTopoI), deux enzymes stimulées par YB-1. L'abondance du complexe hNTH1/YB-1 est accrue dans les cellules tumorales résistantes au cisplatine. La TopoI humaine est une enzyme essentielle impliquée dans la régulation cellulaire du surenroulement de l'ADN et est la cible de plusieurs agents anticancéreux. YB-1 augmente la sensibilité à l'inhibiteur de TopoI, la camptothécine, dans les tumeurs. Nous avons caractérisé les complexes YB-1/hNTH1 et YB-1/hTopoI in vitro et in vivo en utilisant des mesures de transfert d'énergie par résonance en fluorescence (ou FRET) pour identifier et développer de nouvelles stratégies pour le traitement de tumeurs chimio-résistantes. Nous avons développé et optimisé un biosenseur original basé sur le FRET pour cribler deux chimiothèques de taille moyenne afin d’identifier des inhibiteurs potentiels du complexe hNTH1/YB-1. Plusieurs «hits» ont été identifiés qui réduisent de façon significative le niveau de FRET de notre biosenseur. Pour certains de ces composés, nous avons reproduit ces résultats à partir de poudres, effectué des courbes dose-réponse et validé leurs actions en tant qu'inhibiteurs de l'interface hNTH1/YB-1 en utilisant d'autres tests d’interactions. Ensemble nos résultats démontrent que YB-1 interagit directement et stimule des enzymes de la réparation de l'ADN et du relaxation de l’ADN, et que cibler l’interface YB-1/hNTH1 représente une nouvelle stratégie intéressante pour le développement de traitements anticancéreuxAcquired resistance to anti-cancer therapy is common and is a major clinical issue. Functional DNA repair pathways provide a common mechanism for drug resistance, but it can also result from mutations or reduced expression of the targeted protein. The overexpression or nuclear localisation of the multifunctional Y-box binding protein (YB-1) is considered as a prognostic marker for drug resistance in tumours. YB-1 has several interaction partners in cells; in this study, we have focused on its interaction with the human DNA repair enzyme NTH1 (hNTH1) and human DNA topoisomerase I (hTopoI), two enzymes that have been shown to be stimulated by YB-1. The abundance of the hNTH1/YB-1 complex was shown to increase in cisplatin-resistant tumour cells. Human TopoI is an essential enzyme involved in cellular regulation of DNA supercoiling and is the target of several anti-cancer agents. YB-1 enhances the activity of hTopoI and its sensitivity to hTopoI inhibitor, camptothecin in tumour cells. We have characterised the YB-1/hNTH1 and YB-1/hTopoI complexes in vitro and in vivo using Fluorescence Resonance Energy Transfer (FRET) measurements to identify and develop new strategies for the treatment of drug-resistant tumours. We also designed and optimised an original FRET-based biosensor to screen two medium-sized chemical libraries in order to find potential inhibitors of the hNTH1/YB-1 complex. Several “hits” were identified that significantly reduced the FRET level of our biosensor. For some of these compounds, we have reproduced these results starting from powders, have performed dose-response curves and have validated their actions as inhibitors of the hNTH1/YB-1 interface using alternative binding assays. Taken together, our results demonstrate that YB-1 directly interacts and stimulates a DNA repair and a DNA relaxing enzyme and targeting the YB-1/hNTH1 interface represents an interesting new strategy for the development of anti-cancer drugs