The DExD/H-box protein Prp43 is essential for two biological processes: nucleoplasmic pre-mRNA splicing and nucleolar rRNA maturation. The biological basis for the temporal and spatial regulation of Prp43 remains elusive. The Spp382/Ntr1, Sqs1/Pfa1 and Pxr1/Gno1 G-patch proteins bind to and activate the Prp43 DExD/H box-helicase in pre-mRNA splicing (Spp382) and rRNA processing (Sqs1, Pxr1). These Prp43-interacting proteins each contain the G-patch domain, a conserved sequence of ~48 amino acids that includes 6 highly conserved glycine (G) residues. Five annotated G-patch proteins in baker’s yeast (i.e., Spp382, Pxr1, Spp2, Sqs1 and Ylr271) and with the possible exception of the uncharacterized Ylr271 protein, all are associated with ribonucleoprotein (RNP) complexes. Understanding the role of G-patch proteins in modulating the DExD/H box protein Prp43 biological function was the motivation of this thesis. The G-patch domain has been proposed as a protein-protein or a protein-RNA interaction module for RNP proteins. This study found that the three Prp43-associated G-patch domains interact with Prp43 in a yeast 2 hybrid (Y2H) assay but differ in apparent relative affinities. Using a systemic Y2H analysis, I identified the conserved Winged-helix (WH) domain in Prp43 as a major binding site for G-patch motif. Intriguingly, removal of the non-essential N-terminal domain (NTD) of Prp43 (amino acids 2-94), greatly improves G-patch binding, suggesting that the NTD may play a role in modulating enzyme activity by the G-patch effectors. I identify a second site within the Pxr1 that strongly binds Prp43 but, unlike the G-patch, is dispensable for Pxr1 function in vivo. By constructing chimeric proteins, I demonstrated that individual G-patch peptides differ in the ability to reconstitute Spp382 and Pxr1 function in support of pre-mRNA splicing and rRNA biogenesis, respectively. Through amino acid sequence comparisons and selective mutagenesis I identified several residues within the G-patch motif critical for Prp43-stimulated pre-mRNA splicing without greatly altering its ability to bind Prp43. These data lead me to propose that the G-patch motif is not a simple Prp43 binding interface but may contribute more directly to substrate selection or Prp43 enzyme activation in the biologically distinct pre-mRNA splicing and rRNA processing pathways.

Host organisms evolved antiviral responses, which can recognize the viral infection and deal with it. One of the frequent signs of viral infection in a cell is appearance of double-stranded RNA (dsRNA). One of the pathways responding to dsRNA is RNA interference (RNAi), which functions as the key antiviral defence system in invertebrates and plants. Mammals, however, utilize for antiviral defence a different dsRNA-sensing pathway called the interferon response. RNAi functions only in mammalian oocytes and early embryonal stages although its enzymatic machinery is present in all somatic cells, where it is employed in the microRNA pathway. A previous study indicated that the functionality of RNAi in mouse oocytes functions due to an oocyte-specific isoform of protein Dicer (DicerO ), which is truncated at the N-terminus. In my thesis, I aimed to assess whether DicerO processes RNAi substrates more efficiently in vitro than the full-length Dicer (DicerS ), which is found in somatic cells. Therefore, I developed Dicer purification protocol for obtaining both recombinant mouse Dicer isoforms of high purity. I examined their activity in a non-radioactive cleavage assay using RNA substrates with structural features characteristic of RNAi substrates. My results suggest that recombinant DicerO and DicerS do not...

Différents rôles de l'hélicase Pif1 ont été décrit dont le plus documenté est de décrocher la télomérase des télomères en déroulant les hybrides ARN/ADN formés entre l'ARN de la télomérase et l'ADN télomérique. Plus récemment, une nouvelle voie de signalisation des dommages à l'ADN a été mise en évidence, qui inhibe l'action de la télomérase au niveau d'une cassure de l'ADN via la phosphorylation de l'hélicase Pif1. Cette phosphorylation, dépendante de la kinase ATR (Mec1), inhibe la réparation aberrante de la cassure d'ADN par la télomérase. Nous étudions au sein de l’équipe la protéine RPA (Replication Protein A), affine de l'ADN simple-brin, qui recrute à la fois la protéine de recombinaison homologue Rad52 et la protéine Mec1 impliquée dans la cascade de signalisation des dommages de l'ADN. Lors de l'étude de différentes fonctions de l'hélicase Pif1, j'ai mis en évidence une interaction robuste entre Pif1 et RPA. J'ai identifié un allèle de RFA1, rfa1-D228Y, affectant l'interaction Pif1/RPA et montré, grâce à cet allèle, que cette interaction est impliquée dans le recrutement de Pif1 au niveau d'une cassure double-brins (CDB) induite de l'ADN. Enfin, il a été récemment mis en évidence un nouveau rôle de Pif1 dans la stabilité des G-Quadruplexes durant la réplication du brin avancé. En effet, les cellules pif1 présentent un taux d'instabilité du minisatellite CEB1 inséré sur le brin avancé d'environ 56%, correspondant à des réarrangements de l'ADN de type contractions ou expansions. Lors de l'étude de l'interaction Pif1/RPA, j'ai montré que la mutation rfa1-D228Y entraîne une instabilité du minisatellite CEB1 présent sur le brin avancé, similaire à celle observée avec la délétion pif1∆. Nous suggérons un modèle selon lequel RPA recruterait Pif1 au cours de différents processus cellulaires tels que la réponse des dommages à l'ADN ou la réplication des structures particulières de l'ADN telles que les G-Quadruplexes.En parallèle de cette étude, j’ai étudié le rôle de l'histone méthyltransférase Set1 spécifique de la lysine 4 de l'histone H3 dans la régulation de la taille des télomères. J’ai mis en évidence que le raccourcissement des télomères observé dans un mutant set1 est lié à l'absence de di- et tri-méthylation de H3K4 alors que la perte de monométhylation n'a aucun effet. Cependant, le défaut de la taille des télomères dans les cellules set1∆ n'est pas uniquement lié au défaut de méthylation de H3K4 mais semble impliquer une autre activité de Set1 qu’il reste à déterminer. Etonnamment, nous avons observé que la délétion de SET1 aggrave le raccourcissement des télomères des mutants dont les gènes sont impliqués dans la régulation positive de la taille des télomères et inversement, aggrave le rallongement des télomères de mutants dont les gènes sont impliqués dans la régulation négative des télomères. Nous postulons que l’inactivation de Set1 pourrait à la fois inhiber l’activation précoce des origines de réplication des régions subtélomériques et conduire à un sur-raccourcissement de la taille des télomères, à la fois affecter la synthèse du brin complémentaire dans un contexte où celle-ci est affectée (mutant rif1) et conduire à un sur-allongement des télomères. Une seconde hypothèse propose que Set1 régulerait la transcription deTERRA dans des cellules ayant les télomères déprotégés (mutant rif) entraînant le sur-allongement des télomères. / Different roles of Pif1 helicase have been described, the best documented being to remove telomerase from telomeres by unwinding the RNA/DNA hybrid between telomerase RNA and telomeric DNA. Recently, it was shown that the DNA damage signaling down-regulates telomerase action at a DNA break via Pif1 phosphorylation. Pif1 phosphorylation is dependent of the checkpoint kinase ATR (Mec1) and prevents the aberrant healing of broken DNA ends by telomerase. In our laboratory, we study RPA (Replication Protein A), a single-strand DNA binding protein which recruits the proteins involved in the DNA damage response and checkpoint regulation, such as the homologous recombination protein Rad52 and Mec1 involved in the DNA damage response. I have identified an allele of RFA1, rfa1-D228Y, that affects the Pif1/RPA interaction and showed using this allele that this interaction is implicated in the Pif1 recruitment at an induced double-strand break. Recently, a new role of Pif1 in the stability of G-quadruplex DNA during the leading strand replication has been described. pif1 cells show an instability about 56% of the human minisatellite CEB1 inserted on the leading strand. During my study of the Pif1/RPA interaction, I showed that the rfa1-D228Y mutant induced a similar instability of CEB1 minisatellite on the leading strand. We suggested that RPA would recruit Pif1 for many cellular processes such as DNA damage response or replication of secondary DNA structures such as G-Quadruplexes.In parallel, I have studied the role of the Set1 Histone methyltransferase which catalyse the methylation of the lysine 4 of histone H3, in the regulation of telomere length. I showed that the telomere shortening observed in set1 mutant is due to the loss of di- and tri-methylation of H3K4 while the loss of monomethylation has no effect. However, the short telomeres in set1∆ cells is not only due to the methylation defect shedding light on a new Set1 activity that remains to be fully characterized.. The SET1 deletion aggravates the telomere shortening of mutants which genes are involved in positive regulation of telomere length and conversely, aggravates the lengthening of mutants which genes are involved in negative regulation of telomere length. We postulated that inactivation of Set1 could affect at once activation of early-replication origins and leads to a telomere shortening, and affect synthesis of complementary strand in a context where this one is affected (mutant rif1) and leads to a telomere lengthening. A second hypothesis propose that Set1 would regulate TERRA transcription in cells with deprotected-telomere (rif mutant) leading to the lengthening of telomeres.

DNA is constantly subjected to chemical and mechanical damage. The ability to repair the lesions sustained is essential for all life. Double stranded DNA (dsDNA) breaks are especially toxic as both antiparallel strands of DNA are severed. The most high fidelity mechanism available to repair this damage is homologous recombination, a mechanism that uses homology from the sister chromatid to replace any lost information. Key proteins involved in maintaining genomic stability this way are conserved in all domains of life. One such component is the Mre11/Rad50 complex that is involved in the initial recognition of damage and recruitment of subsequent repair factors. Understanding the function of this DNA repair complex and any associated proteins has implications for human cancers and aging. The proteins of thermophilic archaea present an excellent opportunity to study these systems in a robust, tractable and eukaryote-like system. Archaea are in many ways biochemically unique, for example they are the only domain capable of methanogenesis. However archaea share a high level of homology with eukaryotes in many essential cellular processes such as DNA replication, homologous recombination and protein degradation. In thermophilic archaea the mre11/rad50 genes are clustered in an operon with the herA/nurA genes that form a helicase/nuclease complex. This has lead to speculation that the four proteins work together during homologous recombination to produce the 3' overhangs required by RadA to identify homology. As part of this investigation I have performed extensive bioinformatic searches of a variety of archaeal/bacterial systems. These analyses have revealed operonic linkages to other known recombinational helicase/nucleases, such as AddAB and RecBCD. These genomic linkages are especially prevalent in thermophilic organisms suggesting their functional relevance is particularly acute in organisms exposed to a high amount of genomic stress. Comparison of the evolutionary trees, constructed for each protein, makes a single genomic linkage event the most likely scenario, but cannot definitively exclude other possibilities. Exhaustive attempts were made to demonstrate an interaction between Mre11/Rad50 and HerA/NurA. Despite analysis by nickel/cobalt pulldown, immunoprecipitation, analytical gel filtration, ITC and OCTET an interaction could not be confirmed or definitively dismissed. However in the process an interesting Rad50 tetrameric assembly was identified and attempts were made to crystalize it. Hexameric helicases and translocases are key to the replication and DNA packaging of all cellular life and multiple viruses. The hexameric translocase HerA is a robust model for investigating the common features of multimeric ATPases as it is extremely stable and experimentally tractable. Here it is revealed that HerA exists in a dynamic equilibrium fluctuating between hexameric and heptameric forms with rapidly interchanging subunits. This equilibrium can be shifted to heptamer by buffering conditions or towards the hexamer by the physical interaction with the partnering nuclease NurA, raising the possibility that these alternate states may play a role in translocase assembly or function. A novel C-terminal brace, (revealed by a collaborative crystallographic structure) is investigated; as well as stabilizing the assembly, this brace reaches over the ATPase active site of its neighbouring subunit. It is seemingly involved in the conversion of energy generated by ATP hydrolysis into physical movement in the central channel of the hexamer. The regulation of homologous recombination is extremely important to prevent aberrant activity, resulting in mutations and genome reorganization. In eukaryotic organisms, it is well established that post-translational modifications and protein turnover at the proteosome play important roles in this control. In particular, there is significant interest currently in the ubiquination-proteasome destruction pathway as a mechanism for extracting DNA repair components from chromatin at the termination of the DNA repair process. To date no Ubiquitin proteins have been identified in the Archaea, however related proteins URMs/SAMPs (Ubiquitin Related Modifier/Small Archaeal Modifier Protein) have previously been identified. URMs are thought to have evolved from a common antecedent to eukaryotic ubiquitin and likely represent an evolutionary 'missing link' in the adaption of sulphur transfer proteins for covalent modifications. There has been speculation that Urm1 may play a similar role to ubiquitin in the proteasome degradation pathway and we have recently provided evidence to corroborate this. Here the potential for modification of Mre11/Rad50/HerA/NurA by Urm1 was investigated. Indeed Rad50 shows evidence of clear urmylation both in vivo and in vitro. Western blotting and mass spec analysis confirmed the covalent attachment of Urm1 to Rad50. Furthermore I present preliminary evidence that this urmylation can lead to the destruction of Rad50 via a direct physical interaction with the proteasome. This is the first evidence of such a regulatory system for Rad50. Investigating the urmylation and destruction of Rad50 was closely linked to investigating the archaeal proteasome, a close homologue of the eukaryotic proteasome. To date the majority of archaeal core proteasomes examined were believed to consist of only two subunits; alpha and beta. The subunits are arranged into heptameric rings, which then form an alpha/beta/beta/alpha stack with a single channel running through the centre of all four rings. Here we reveal that in Sulfolobus species the inner catalytic chambers are made up of mixed beta rings composed of two subunits. The first plays a crucial structural role but appears catalytically inert, while the second conveys catalytic activity. Here we investigate an inactive complex, containing only the structural beta subunit, and an active complex, containing both beta subunits. First, electron microscopy was performed on both complexes revealing the expected four-layered toroidal stack. Both complexes were subsequently investigated crystallographically. A 3.8 Å structure was determined for the inactive complex. As well as being one of the few archaeal core proteasome structures, this is also an important first step towards structurally investigating the novel three-subunit proteasome. The discovery of active and inactive beta subunits in the archaea brings them even closer to eukaryotic proteasomal systems, making the archaea even more valuable as model systems.

Pre-mRNA (precursor messenger RNA) splicing is a fundamental process in eukaryotic gene expression. In order to catalyse the excision of the intervening intronic sequence between two exons, the spliceosome is assembled stepwise on the pre-mRNA substrate. This ribonucleoprotein machine is extremely dynamic: both its activation and the progression through the catalytic stages require extensive compositional and structural remodelling. The first part of this thesis aims at understanding how the spliceosome is activated after assembly. When this work was started, the GTPase Snu114 was thought to activate the helicase Brr2 to unwind the U4/U6 snRNA duplex, which ultimately leads to the formation of the spliceosome active site. To explore the role of Snu114, a complex built from Snu114 and a part of Prp8 was expressed and analysed in its natural context, bound to U5 snRNA. However, before I was able to obtain highly diffracting crystals, the structure of Snu114 was determined in the context of a larger spliceosomal complex by electron cryo-microscopy by competitors. Regardless, the role of Snu114 in spliceosome activation remains elusive. In a short section of this thesis, genetic and biochemical analysis suggest Snu114 to be a pseudo-GTPase, precluding a role for Snu114-catalyzed GTP hydrolysis in activation. The second and larger part of the thesis describes the development of a novel, biochemical method to analyse spliceosome remodelling events that are caused by the eight spliceosomal helicases. Purified spliceosomes assembled on a defined RNA substrate are analysed by UV crosslinking and next-generation sequencing, which allows for the determination of the RNA helicase binding profile at nucleotide resolution. In vitro spliceosome iCLIP (individual-nucleotide resolution UV crosslinking and immunoprecipitation) was initially developed targeting the helicase Prp16 bound to spliceosomal complex C. The obtained binding profile shows that Prp16 contacts the intron, about 15 nucleotides downstream of the branch in the intron-lariat intermediate. Our finding supports the model of Prp16 acting at a distance to remodel the RNA and protein interactions in the catalytic core and thereby it promotes the transition towards a conformation of the spliceosome competent for second step catalysis. Control experiments, which locate SmB protein binding to known Sm sites in the spliceosomal snRNAs, validated the method. Preliminary results show that in vitro spliceosome iCLIP can be adapted to analyse additional spliceosomal helicases such as Prp22. Finally, I performed initial experiments that give promising directions towards time-resolved translocation profiles of helicases Brr2 and Prp16.

Activities of the DEAD box (Asp-Glu-Ala-Asp) family of proteins- including RNA-dependent ATPase and RNA helicase- function in all organisms to sculpt RNA-RNA duplex and RNA-protein complexes, ensuring that necessary rearrangements are rapidly and properly resolved during genetic information processing. Identified as a prototypic member of the DEAD box family and documented as an ATPase and RNA helicase, p68 plays essential and diverse functions in the control of gene expression ranging from pre-mRNA/rRNA processing and mRNA decay/stability to transcriptional activation and initiation. Despite the early implied roles in organ maturation and tumor progression, the functional contributions of p68 to growth/differentiation regulation and cancer development remain undefined. Here, we show c-Abl-dependent phosphorylation of p68 markedly associates with abnormal cell growth and cancer development. Importantly, we characterize an unanticipated signaling module through which p68 functionally contributes to Epithelial-Mesenchymal Transition (EMT) and cell proliferation. p68, which appears to be phosphorylated by c-Abl at tyrosine 593, consequently promotes an EMT through its ability to recruit â-catenin into cell nucleus via a canonic Wnt/â-catenin axis independent way; accordingly, phosphor-p68 (phosphorylated at tyrosine 593 residue) also stimulates tumor cell growth, which requires the ATPase activity of the protein. These findings define a potential mechanism whereby phosphor-p68 recruits â-catenin into cell nucleus in ATP hydrolysis driven fashion and cooperatively regulates transcriptional programs that control an EMT. The dissertation thus demonstrates a tight coordination between DEAD box RNA helicase and cancer development.

P68 RNA helicase is a RNA helicase and an ATPase belonging to the DEAD-box family. It is important for the growth of normal cells, and is implicated in diverse functions ranging from pre-mRNA splicing, transcriptional activation to cell proliferation, and early organ development. The protein is documented to be phosphorylated at several amino-acid residues. It was previously demonstrated in several cancer cell-lines that p68 gets phosphorylated at threonine residues during treatments with TNF-α and TRAIL. In this study, the role of threonine phosphorylation of p68 under the treatment of anti-cancer drug, oxaliplatin in the colon cancer cells is characterized. Oxaliplatin treatment activates p38 MAP-kinase, which subsequently phosphorylates p68 at T564 and/or T446. P68 phosphorylation, at least partially, influences the role of the drug on apoptosis induction. This study shows an important mechanism of action of the anti-cancer drug which could be used for improving cancer treatment. This study also shows that p68 is an important transcriptional regulator regulating transcription of the cytoskeletal gene TPPP/p25. Previous analyses revealed that p68 RNA helicase could regulate expression of genes responsible for controlling stability and dynamics of different cytoskeletons. P68 is found to regulate TPPP/p25 gene transcription by associating with the TPPP/p25 gene promoter. Expression of TPPP/p25 plays an important role in cellular differentiation while the involvement of p68 in the regulation of TPPP/p25 expression is an important event for neurite outgrowth. Loss of TPPP expression contributes to the development and progression of gliomas. Thus, our studies further enhance our understanding of the multiple cellular functions of p68 and its regulation of the cellular processes.

The Mre11/Rad50 complex has been implicated in the early steps of DNA double-strand break (DSB) repair through homologous recombination in several organisms. However, the enzymatic properties of this complex are incompatible with the generation of 3’ single-stranded DNA for recombinase loading and strand exchange. In thermophilic Archaea, the mre11 and rad50 genes cluster in an operon with genes encoding a bidirectional DNA helicase, HerA, and a 5’ to 3’ exonuclease, NurA, suggesting these four enzymes function in a common pathway. I show that purified Mre11 and Rad50 from Pyrococcus furiosus act cooperatively with HerA and NurA to resect the 5’ strand at a DNA end under physiological conditions in vitro where HerA and NurA alone do not show detectable activity. Furthermore, I demonstrate that HerA and NurA physically interact, and this interaction stimulates both helicase and nuclease activities. The products of HerA/NurA long-range resection are oligonucleotide products and HerA/NurA activity demonstrates both sequence specificity and a preference to cut at a specific distance from the DNA end. I demonstrate a novel activity of Mre11/Rad50 to make an endonucleolytic cut on the 5’ strand, which is consistent with a role for the Mre11 nuclease in the removal of 5’ protein conjugates. I also show that Mre11/Rad50 stimulates HerA/NurA-mediated resection through two different mechanisms. The first involves an initial Mre11 nucleolytic processing event of the DNA to generate a 3’ ssDNA overhang, which is then resected by HerA/NurA in the absence of Mre11/Rad50. The second mechanism likely involves local unwinding of the DNA end in a process dependent on Rad50 ATPase activity. I propose that this unwinding step facilitates binding of HerA/NurA to the DNA end and efficient resection of the break. Furthermore, the binding affinity of NurA for 3’ overhang and unwound DNA end substrates partially explains the efficiency of the two resection mechanisms. Lastly, 3’ single-stranded DNA generated by these enzymes can be used by the Archaeal RecA homolog RadA to catalyze strand exchange. This work elucidates how the conserved Mre11/Rad50 complex promotes DNA end resection in Archaea, and may serve as a model for DSB processing in eukaryotes. / text

Les cellules sont constamment exposées à des agents endommageant de l'ADN d'origine exogène, notamment les rayons ultraviolets, les irradiations γ, et l'exposition aux agents chimiques génotoxiques, mais également d'origine endogène générés par le métabolisme cellulaire. De plus en plus d'évidences montrent que la transcription est un processus biologique qui peut mettre en péril l'intégrité du génome. Un mécanisme actuellement très étudié qui lie la transcription à l'instabilité génomique est la formation des boucles R (R-loops), des structures hybrides ARN:ADN qui exposent un ADN simple brin déplacé. Ces structures aberrantes se présentent en tant que sous-produits de la transcription et/ou lors de l'interférence entre la réplication et la transcription, et plus récemment ils ont été montrées s'accumuler lorsque la biogénèse de l'ARNm est perturbée. La persistance des boucles R est une source importante d'instabilité génomique car elle peut générer des cassures double brin de l'ADN et favoriser la recombinaison. Pour faire face aux conséquences néfastes des endommagements de l'ADN, les cellules activent une cascade élaborée de voies de signalisation qui permet de coordonner la prolifération cellulaire avec la réparation de l'ADN. L'ensemble de ces acteurs moléculaires constitue un réseau de réponse aux dommages de l'ADN qui est indispensable pour la stabilité génomique. Récemment chez la levure, l'activation transitoire de ce réseau a été également proposée être important dans la coordination de la transcription et de la réplication, afin d'éviter d'une part des contraintes topologiques et d'autre part la formation de structures aberrantes générées lors de conflits entre ces deux processus cellulaires essentiels. Dans la perspective d'identifier des nouveaux gènes impliqués dans ce réseau de signalisation, un crible fonctionnel in vitro précédemment établi au laboratoire a conduit à l'identification de Ddx19, une hélicase à motif DEAD-box, en tant que nouvel élément répondant à l'endommagement de l'ADN. Ddx19 interagit avec le pore nucléaire via CAN/Nup214, et il est impliqué dans l'export des ARNm grâce à son activité hélicase et ATPase, stimulé par les facteurs IP6 et Gle1. Le présent travail de thèse dévoile une nouvelle fonction de Ddx19 distincte de son rôle connu dans l'export de l'ARNm. Je pu montrer que, lors de l'induction des dommages à l'ADN par les rayons UV, Ddx19 se relocalise transitoirement de la face cytoplasmique du nucléopore vers le noyau de façon dépendant d'ATR. L'inactivation de Ddx19 entraîne des endommagements spontanées dépendant de la prolifération, démontré par l'activation de la voie de signalisation d'ATM-Chk2 et la formation de foyers nucléaires de γH2AX et 53BP1. Ces phénotypes sont concomitants avec le ralentissement des fourches de réplication qui ne peuvent plus redémarrer après leur blocage par la camptothécine. En outre, les cellules déplétées de Ddx19 présentent une forte accumulation des boucles R nucléaires, enrichi dans le compartiment nucléolaire, et aussi autour de la périphérie nucléaire. Par ailleurs, ces cellules présentent une viabilité réduite et une létalité synthétique lorsque la déplétion de Ddx19 est combinée avec l'inhibition de l'expression de la topoisomérase I. Je propose Ddx19 comme deuxième hélicase nécessaire pour la résolution des boucles R, et qui fonctionne à côté mais de façon indépendante de la Senataxin, l'hélicase précédemment connue pour résoudre ces structures in vivo chez les cellules de mammifères. Je démontre que cette nouvelle fonction de Ddx19 ne dépend pas de son interaction avec le pore nucléaire, mais plutôt de son activité hélicase et d'un résidu de sérine phosphorylée par Chk1 qui stimule sa relocalisation vers le noyau. Ces données proposent Ddx19 en tant que nouvelle ARN hélicase qui facilite la coordination de la réplication et la transcription, médiée par ATR à travers de la résolution des boucles R, préservant ainsi l'intégrité du génome. / Cells are continuously challenged by DNA damage resulting from external cues as UV light, γ-irradiation and exposure to genotoxic chemicals, as well as from endogenous stress caused by cellular metabolism. Growing evidence points to transcription as a biological process that could adversely affect genome integrity. One currently highly investigated mechanism by which transcription can induce genome instability is through the formation of R-loops, RNA:DNA hybrid structures exposing a displaced single-stranded DNA tract. These aberrant structures occur as byproducts of transcription and/or upon interference between replication and transcription, and more recently were also shown to accumulate upon disruption of mRNA biogenesis and processing. Persistent unresolved R-loops are a potent source of genomic instability as they ultimately generate double strand breaks and promote recombination events. To deal with the deleterious consequences of DNA damage, cells activate elaborate DNA damage response (DDR) pathways to delay cell division and stimulate repair of lesions, thus preserving genome stability. Recently in yeast transient DDR activation has also been proposed to be important in the coordination of transcription and replication, in order to avoid topological constraints and the formation of aberrant structures generated upon collision of their machineries. By means of an in vitro screen aimed at identifying new DDR genes, we isolated Ddx19, a DEAD-Box helicase known to be involved in mRNA export, as a novel DNA damage responsive gene. Ddx19 interacts with the nucleopore complex via nucleoporin CAN/Nup214, and is involved in mRNA remodelling and export through its ATPase and helicase activities, stimulated by IP6 and the Gle1 factor. My present thesis work unravels a novel function of Ddx19 in preserving genome stability in mammalian cells, distinct from its known role in mRNA export. I show that upon UV-induced damage, Ddx19 transiently relocalizes from the cytoplasmic face of the nucleopore to the nucleus in an ATR-dependent manner. Downregulation of Ddx19 gives rise to spontaneous, proliferation-dependent DNA damage, as determined by the specific activation of the ATM-Chk2 pathway and formation of γH2AX and 53BP1 nuclear foci. This is concomitant with the slowing down of replication forks that are unable to restart after being stalled with camptothecin. In addition, cells depleted of Ddx19 display strong accumulation of nuclear R-loops, enriched in the nucleolar compartment, and around the nuclear periphery. Moreover, these cells show low viability and exhibited synthetic lethality when combined with inhibition of topoisomerase I expression. I propose Ddx19 as a second helicase required for R-loops resolution, functioning alongside but independently of Senataxin, the first known RNA helicase to resolve these structures in vivo in mammalian cells. I provide evidence that this new function of Ddx19 does not depend on its interaction with the nuclear pore, but rather on its helicase activity and on a serine residue phosphorylated by Chk1 which promotes its relocalization into the nucleus upon damage. These data put forward Ddx19 as a novel RNA helicase that facilitates ATR-dependent coordination of DNA replication and transcription through R-loops resolution, thus preserving genome integrity.

L'ADN mitochondrial (mtDNA) des plantes est caractérisé par les activités de recombinaison qui modulent sa structure. Ces activités sont nécessaires à son maintien et contribuent à son évolution rapide. Des facteurs contrôlant la recombinaison sont donc indispensables à la stabilité du mtDNA des plantes. Au cours de ma thèse j'ai identifié et caractérisé deux ADN hélicases présentes dans les organelles d'Arabidopsis thaliana. L'une d'entre elles est homologue à une hélicase bactérienne impliquée dans la réparation couplée à la transcription. Son rôle précis dans les organelles des plantes reste à déterminer. La deuxième hélicase, l'hélicase RECG1, a des rôles dans la réparation par recombinaison, la surveillance de la recombinaison ectopique impliquant des courtes séquences répétées, mais aussi dans la ségrégation du mtDNA. En effet, nous avons observé qu'en absence de RECG1 il y a une perte du contrôle de la recombinaison qui a pour conséquence la création de versions alternatives du mtDNA par recombinaison. L'analyse de leur ségrégation, induite par RECG1, nous a permis de modéliser comment de nouvelles configurations stables du mtDNA sont générées par le changement de stoechiométrie entre sous-génomes. Ce travail a permis de mieux comprendre les mécanismes de recombinaison et de ségrégation du mtDNA d'Arabidopsis. / The mitochondrial DNA (mtDNA) of flowering plants is characterized by the recombination activities that modulate its structure. These activities are required for the mtDNA maintenance, and drive its rapid structural evolution. The factors that control recombination are therefore essential for plant mtDNA stability. During my PhD, I identified and characterized two DNA helicases that are present in the organelles of Arabidopsisthaliana. One is the homologue of a bacterial helicase involved in transcription-coupled repair. Its role in the plant organelles is still not determined. The other one, the RECG1 helicase, has roles in recombination dependent repair, the surveillance of ectopic recombination involving short repeated sequences, and also the segregation of the mtDNA. We have found that in the absence of RECG1 there is loss of recombination control resulting in the occurrence of alternative versions of the mtDNA generated by recombination. The analysis oftheir segregation, induced by RECG1, allowed us to build a model to how new stable mtDNA configurations are generated by the stoichiometric shift of mtDNA sub-genomes. This work allowed us to better understand the recombination and segregation mechanisms that modulate the Arabidopsis mtDNA.