Fusarium oxysporum is a filamentous fungus that is known to invade over a hundred different hosts and poses a major threat to the economy and food supply world-wide. Poly (Adenosine diphosphate-Ribose) Polymerase (PARP) is a family of regulatory proteins that affect change in the cell through transfer of ADP-Ribose moieties onto target molecules. The most well-studied PARP protein is the human PARP1, a PARylating nuclear protein that serves as our model PARP protein. F. oxysporum was found to contain a large expansion of PARP catalytic-domain-containing proteins compared to other filamentous fungi. We utilized in silico multiple sequence alignments and domain predictions to identify a human PARP1 homolog termed foPARP1 that was conserved within the core chromosomes in all three strains within our comparative system. Our in silico predictions also stated that only one strain, an Arabidopsis pathogen, Fo5176, contained several other predicted catalytically active PARP homologs within the accessory chromosome. To test the effect that foPARP1 knockout would have on DNA damage tolerance, we created a foParp1 knockout and found that only strains Fol4287 and Fo5176 had a significant reduction in tolerance upon being plated with methyl methanesulfonate (MMS), a DNA alkylating agent. To test how global PARylation trends would be affected by foParp1 knockout, we utilized immunodot-blotting with PAR antibodies to assess PARylation in total protein extracts. We found that all strains of the comparative system had the capacity to catalyze the synthesis of long PAR chains, while only Fo47 and Fo5176 had a significant PARylation increase when exposed to MMS, and no samples had a significant increase in PARylation within the foParp1 knockouts. Finally, we utilized RNA-Sequencing to determine the transcriptional impacts that foParp1 knockout would have and found aberrant DNA repair pathways and disruptions in stress responses. Taken together, we conclude that foPARP1 is in fact a functional PARP1 homolog and exhibits similar post-transcriptional modification and transcriptional impacts as its human counterpart. However, we were not able to correlate PARP copy number with DNA stress tolerance, and further research would be needed to assess the full function of the PARP expansion.

Pendant longtemps, la chromatine a été uniquement décrite comme un moyen de compacter près de deux mètres d’ADN dans un noyau de quelques micromètres de diamètre. On sait aujourd’hui que la chromatine représente en fait un élément majeur de régulation de toutes les fonctions nucléaires impliquant l’ADN. Dans le contexte de dommages de l’ADN induits par irradiations UV, la chromatine endommagée subit une décondensation rapide et transitoire qui l’amène à occuper un volume 1,5 fois plus grand que son volume initial. Cette relaxation chromatinienne est associée à une plus grande accessibilité de l’ADN. Néanmoins, le lien entre ces deux effets découlant de la présence de dommages, n’a pas été établi, ni caractérisé. En couplant l’imagerie de cellules vivantes à l’induction de dommages ciblés au sein de noyaux cellulaires par micro-irradiation laser, ces travaux ont permis de mettre en évidence le rôle majeur de PARP1 dans la réponse chromatinienne aux dommages de l’ADN. En effet, certaines conclusions contradictoires présentes dans la littérature scientifique concernant l’action de PARP1 sur la chromatine ont été réconciliées en démontrant que PARP1 seul peut se lier à la chromatine et entraîner une plus forte compaction de celle-ci, tandis que son activité catalytique de PARylation va, quant à elle, conduire à une décompaction de la structure chromatinienne. Cette étude s’est aussi intéressée à la dynamique particulière de l’histone H1 suite aux dommages de l’ADN. En effet, celui-ci est rapidement exclu des zones de dommages par un mécanisme encore inconnu, et les éléments apportés ici suggèrent que H1 pourrait jouer un rôle dans la décondensation de la chromatine suite aux dommages de l’ADN. Pour finir, des techniques de photo-perturbation et de spectroscopie de corrélation de fluorescence ont été employées pour comprendre et caractériser l’environnement moléculaire que constitue la chromatine endommagée et décondensée. Bien qu’une augmentation significative des interactions entre la chromatine et certains de ses partenaires d’interactions soit observée au sein des zones endommagées, aucun changement en termes d’encombrement moléculaire n’a pu être mis en évidence à ce niveau qui pourrait expliquer une plus grande accessibilité de l’ADN. / For a long time, chromatin was only described as a mean to fit the two-meters long DNA molecule into a nucleus of only a few microns. It is admitted today that chromatin actually represents a key element in the regulation of all nuclear functions dependent on DNA. In the context of UV-induced DNA damage, chromatin undergoes a rapid and transient relaxation which leads to an expansion of the damaged area to 1.5 times its original size. While this chromatin response to damage is associated with a higher DNA accessibility, the link between those two phenomena, as well as the mechanisms driving them, are still poorly understood. Using live-cell imaging and laser micro-irradiation to induce DNA damage on specific nuclear areas, this work allowed to gain hindsight on the predominant role played by PARP1 in the DNA damage-induced chromatin relaxation. Indeed, showing that PARP1 at DNA damage sites can both induce chromatin compaction through its recruitment at DNA breaks or chromatin decondensation through its PARylation activity helped reconcile its apparent opposite effects described in the literature. A focus was also made on the linker histone H1, as it displays a peculiar behavior upon DNA damage, being rapidly released from the site of DNA lesions. Even if the driving force behind H1 release from damaged chromatin areas has not been identified yet, its behavior suggests that H1 might play a part in chromatin relaxation or in increasing DNA accessibility upon DNA damage. Lastly, combining photo-activation techniques and fluorescence correlation spectroscopy, experiments were performed in order to understand the physical environment that damaged, relaxed chromatin constitutes. We report here that, while enhanced binding of random DNA binding factors is observed in the damaged chromatin area, no significant change is observed in the macromolecular crowding levels that could potentially explain this enhanced binding, as well as a higher DNA accessibility.

Adenosine diphosphate (ADP) ribosylation is a post-translational modification dependent on the transfer of ADPr units from nicotinamide adenine dinucleotide (NAD+) on to a plethora of biomolecules (i.e., proteins, DNA, RNA, etc.) in response to physiological stressors (i.e., nutrient deprivation, oxidative stress, DNA strand breaks). Poly-ADP-ribosylation (PARylation) is primarily mediated by the family of poly(ADP-ribose) polymerases (PARPs) and enzymatically degraded (dePARylation) by hydrolases such as poly(ADP-ribose) glycohydrolase (PARG). This thesis characterizes the role of poly(ADP-ribose) polymerase 1 (PARP1) and PARG in the skeletal muscle of healthy mature mice under normal physiological conditions. Specifically, we validate the deletion of Parp1 and Parg in inducible skeletal muscle-specific KO mouse models followed by performing general phenotyping of both male and female mice. The thesis concludes that under normal physiological conditions the activity of Parp1 or Parg in (de)PARylation is dispensable for maintaining skeletal muscle mass, function, and homeostasis in healthy mature mice.

L’ADN cellulaire qu’il soit nucléaire ou mitochondrial est constamment soumis à l’action de stress d’origine exogène ou même endogène à la base d’altérations plus ou moins profondes de sa structure. Ces modifications chimiques sont très variées et peuvent aller de l’oxydation d’une base aux cassures double-brins de la molécule d’ADN. Ces dernières sont considérées comme les dommages les plus agressifs pour la cellule car peuvent conduire à la perte d’information et donc à la mort cellulaire. Parmi les systèmes de surveillance de la stabilité du génome figure la Poly(ADP-ribosyl)ation (PARylation). Cette modification post-traductionnelle est assurée essentiellement par les protéines PARP et PARG et est caractérisée par l’incorporation des polymères d’ADP ribose (pADPr) sur des protéines cibles. La PARylation constitue un élément clé dans plusieurs voies de maintien de l’intégrité génomique (BER, NHEJ, HR). La PARylation est aussi décrite au niveau de la mitochondrie mais son rôle dans la gestion des DSBs de l’ADNmt n’est pas connu. Le travail, objet de cette thèse, consiste à étudier le rôle de la PARylation dans le cas des DSB au niveau général chez la drosophile et ensuite de comprendre les mécanismes de gestion des DSB mitochondriales et évaluer l’implication de la PARylation dans ce processus. Nos résultats montrent que : (1) le comportement de la PARylation ne varie pas au cours du processus de cassures et de réparation de l’ADN nucléaire, alors que l’expression des ARNm de PARP-I et PARP-II augmente durant la phase de réparation ; (2) les cassures de l’ADN mitochondrial, induites par la bléomycine, entraînent une augmentation du nombre de copies de l’ADNmt. Cette augmentation transitoire de la quantité de l’ADNmt est observée durant la phase des dommages et retourne à la valeur initiale durant la phase de la réparation. Ce comportement semble être régulé par PARP. L’ensemble de ces résultats suggère que la réparation des DSBs est indépendante de la PARylation au niveau nucléaire mais que la présence de PARP est importante. De plus, PARP semble avoir un rôle dans la régulation de la réplication de l’ADNmt en réponse à un stress génotoxique. / Both nuclear and mitochondrial DNA alterationsarise following exposure to environmental and endogenous stresses. These genomic alterations are various, ranging from base oxidation to DNA strand breaks, single- and double-strand breaks. These damages are highly detrimental to the cell because they can lead to loss of genetic information and thus to cell death. However, cells have developed various mechanisms to counteract this biological issue and to lead up to a complex DNA damage response (DDR). The Poly (ADP- ribosyl) ation (PARylation) is among these DDR systems. This post-translational modification is mainly carried out by PARP and PARG proteins and is characterized by the incorporation of polymers of ADP-ribose on target proteins. The majority of the PARylationfunctions are related to cellular stress response, particulary in response to genomic damages where it is implicated in many DNA integrity pathways such as Base Excision Repair, Non Homologous End Joining and Homologous Recombination. In contrast to the nucleus, PARylation is also described in the mitochondria but its role in mtDNA integrityis still a heavily debate issue, particularly in case of mtDNA DSBs.To understand it, we used Drosophila model wherePARP-B isoform (human PARP-1 ortholog) is the only enzymatically active form in Drosophila PARP family. The aim of this thesis is to study the role of PARylation in response to DSBs induction in nucleus and mitochondrial DNAand then to understand the mechanisms involved in mtDNA integrity and to evaluate the role of PARylation in this process. Our results show that PARylation level remains stable during DSBs induction and also during repair process,contrary to what is shown in Human cells.However, PARP-I and PARP-II mRNA expression increase during repair period. In mitochondria compartment,our data show an increase of mtDNA copy number in presence of mtDNA DSBs. This increased level returns to normal during repair period and seems to be dependent on PARP. All these results suggest that DSBs repair is PARylation independent at the nuclear level but that the presence of PARP is important. In addition, PARP appears to have a role in the regulation of mtDNA replication in response to genotoxic stress.