Homologous recombination (HR) and nonhomologous end joining (NHEJ) are two distinct DNA double-stranded break (DSB) repair pathways. Here, we report that DNA-dependent protein kinase (DNA-PK), the core component of NHEJ, partnering with DNA-damage checkpoint kinases ataxia telangiectasia mutated (ATM) and ATM- and Rad3-related (ATR), regulates HR repair of DSBs. The regulation was accomplished through modulation of the p53 and replication protein A (RPA) interaction. We show that upon DNA damage, p53 and RPA were freed from a p53-RPA complex by simultaneous phosphorylations of RPA at the N-terminus of RPA32 subunit by DNA-PK and of p53 at Ser37 and Ser46 in a Chk1/Chk2-independent manner by ATR and ATM, respectively. Neither the phosphorylation of RPA nor of p53 alone could dissociate p53 and RPA. Furthermore, disruption of the release significantly compromised HR repair of DSBs. Our results reveal a mechanism for the crosstalk between HR repair and NHEJ through the co-regulation of p53-RPA interaction by DNA-PK, ATM and ATR.

No description available.

No description available.

La Poly(ADP-ribosyl)ation est une modification post-traductionnelle de protéines, impliquée dans un grand nombre de processus biologiques, dont la réparation de l’ADN. Alors que la fonction et le mode d’action de la Poly(ADP-ribose) (PAR) Polymérase 1 (PARP1), activée en réponse aux dommages de l’ADN sont bien compris, on en sait beaucoup moins sur la fonction et la régulation de l’enzyme de dégradation du PAR, la Poly(ADP-ribose) glycohydrolase (PARG). Dans le contexte de ce projet de thèse, nous décrivons de nouvelles lignées U2OS stables, déficientes pour toutes les isoformes de PARG, permettant la complémentation inductible avec chacun des isoformes de PARG. Ces modèles nous ont permis d’évaluer les contributions relatives des isoformes à la réparation de dommages à l’ADN. Nous avons identifié un nouveau partenaire cellulaire de PARG : la protéine-kinase dépendante des dommages à l’ADN (DNA-PK). Nous explorons l’interaction fonctionnelle de ces deux protéines dans le contexte de la réponse cellulaire à la camptothécine (CPT), un agent anticancéreux inhibant la topoisomérase I et provoquant l’activation simultanée de PARP1 et DNA-PK. / Poly (ADP-ribosyl) ation is a post-translational modification of proteins involved in a large number of biological processes, including DNA repair. While the function and mode of action of Poly (ADP-ribose) (PAR) Polymerase 1 (PARP1), activated in response to DNA damage, is well understood, much less is known about the function and regulation the PAR degrading enzyme, Poly (ADP-ribose) glycohydrolase (PARG). In the context of this thesis project, we describe new stable U2OS lines, deficient for all PARG isoforms, allowing the inducible complementation with each of the PARG isoforms. These models allowed us to evaluate the relative contributions of the isoforms to DNA damage repair. We have identified a new cellular partner of PARG: the DNA-dependent protein kinase-dependent kinase (DNA-PK). We explore the functional interaction between these two proteins in the context of the cellular response to camptothecin (CPT), an anticancer drug that inhibits topoisomerase I and induces the simultaneous activation of PARP1 and DNA-PK.

<p>Efficient and correct repair of DNA damage, especially DNA double-strand breaks (DSBs), is vital for the survival of individual cells and organisms. Defects in the DNA repair may lead to cell death or genomic instability and development of cancer. </p><p>The repair of DSBs in cell lines with different DSB rejoining capabilities was studied after exposure to ionising radiation. A new cell lysis protocol performed at 0ºC, which prevents the inclusion of non-true DSBs in the quantification of DSBs by pulsed-field gel electrophoresis (PFGE), was developed. Results showed that when the standard protocol at 50ºC was used, 30-40% of the initial yield of DSBs corresponds to artifactual DSBs. The lesions transformed to DSBs during incubation at 50ºC were repaired within 60-90 minutes <i>in vivo</i> and the repair was independent of DNA-PK, XRCC1 and PARP-1.</p><p>Non-homologous end-joining (NHEJ) is the major DSB repair pathway in mammalian cells. We show that DSBs are processed into long single-stranded DNA (ssDNA) ends after ≥1 h of repair in NHEJ deficient cells. The ssDNA was formed outside of the G₁ phase of the cell cycle and only in the absence of the NHEJ proteins DNA-PK and DNA Ligase IV/XRCC4. The generation of ssDNA had great influence on the quantification of DSBs by PFGE. The standard protocol caused hybridisation of the ssDNA ends, resulting in overestimation of the DSB repair capability in NHEJ deficient cells.</p><p>DSBs were also quantified by detection of phosphorylated H2AX (γ-H2AX) foci. A large number of γ-H2AX foci still remaining after 21 h of repair in an NHEJ deficient cell line confirmed the low repair capability determined by PFGE. Furthermore, in normal cells difficulty in repairing clustered breaks was observed as a large fraction of γ-H2AX foci remaining 24 h after irradiation with high-LET ions.</p>

Efficient and correct repair of DNA damage, especially DNA double-strand breaks (DSBs), is vital for the survival of individual cells and organisms. Defects in the DNA repair may lead to cell death or genomic instability and development of cancer. The repair of DSBs in cell lines with different DSB rejoining capabilities was studied after exposure to ionising radiation. A new cell lysis protocol performed at 0ºC, which prevents the inclusion of non-true DSBs in the quantification of DSBs by pulsed-field gel electrophoresis (PFGE), was developed. Results showed that when the standard protocol at 50ºC was used, 30-40% of the initial yield of DSBs corresponds to artifactual DSBs. The lesions transformed to DSBs during incubation at 50ºC were repaired within 60-90 minutes in vivo and the repair was independent of DNA-PK, XRCC1 and PARP-1. Non-homologous end-joining (NHEJ) is the major DSB repair pathway in mammalian cells. We show that DSBs are processed into long single-stranded DNA (ssDNA) ends after ≥1 h of repair in NHEJ deficient cells. The ssDNA was formed outside of the G1 phase of the cell cycle and only in the absence of the NHEJ proteins DNA-PK and DNA Ligase IV/XRCC4. The generation of ssDNA had great influence on the quantification of DSBs by PFGE. The standard protocol caused hybridisation of the ssDNA ends, resulting in overestimation of the DSB repair capability in NHEJ deficient cells. DSBs were also quantified by detection of phosphorylated H2AX (γ-H2AX) foci. A large number of γ-H2AX foci still remaining after 21 h of repair in an NHEJ deficient cell line confirmed the low repair capability determined by PFGE. Furthermore, in normal cells difficulty in repairing clustered breaks was observed as a large fraction of γ-H2AX foci remaining 24 h after irradiation with high-LET ions.

Vaccinia virus (VACV) is the most intensively studied orthopoxvirus and acts as an excellent model to investigate host-pathogen interactions. VACV encodes about 200 proteins, many of which modulate the immune response. This study focusses on two of these: C16 and C4, that share 43.7 % amino acid identity. Given the sequence similarity, we explored whether C16 and C4 have any shared functions, whilst also searching for novel functions. To gain mechanistic insight, we sought to identify binding partners and determine the residues responsible. C16 has two reported functions. Firstly, it inhibits DNA-PK-mediated DNA sensing, and this study found that C4 can perform this function as well. Like C16, C4 associates with the Ku heterodimer to block its binding to DNA leading to reduced production of cytokines and chemokines. For both proteins, the function localised to the C termini and was abrogated by mutating three residues. Secondly, C16 induces a hypoxic response by binding to PHD2. This function was mapped to the N-terminal 156 residues and a full length C16 mutant (D70K,D82K) lost the ability to induce a hypoxic response. In contrast, C4 did not bind PHD2. C4 inhibits NF-κB signalling by an unknown mechanism. Reporter gene assays showed that C16 also suppresses NF-κB activity and, intriguingly, this was carried out by both the N and C termini. C16 acts at or downstream of p65 and the N terminus of C16 associated with p65 independently of PHD2-binding. Conversely, C4 acted upstream of p65, did not display an interaction with p65, and the function was restricted to its C-terminal region. Novel binding partners were identified by a screen utilising tandem mass tagging and mass spectrometry, and selected hits were validated. The C terminus of C16 associated with VACV protein K1, a known NF-κB inhibitor. Additionally, C16 bound to the transcriptional regulator ARID4B. C4 did not interact with these proteins, but the N-terminal region of C4 associated with filamins A and B. The functional consequences of these interactions remain to be determined. In vivo, C4 and C16 share some redundancy in that a double deletion virus exhibits an attenuated virulence phenotype that is not observed by single deletion viruses in the intradermal model of infection. However, non-redundant functions also contribute to virulence in that both single deletion viruses display attenuated virulence compared to a wild-type Western Reserve virus in the intranasal model of infection. Data presented also reveal that C4 inhibits the recruitment of immune cells to the site of infection, as was previously described for C16. Overall, this investigation highlights the complexity of host-pathogen interactions showing that VACV encodes two multifunctional proteins with both shared and unique functions. Moreover, their inhibition of DNA-PK emphasises the importance of this PRR as a DNA sensor in vivo.

Adenoviruses have been extensively studied in the field of gene regulation, since their genes are subjected to a tightly controlled temporal expression during the virus lifetime. The early-to-late shift in adenoviral gene expression distinguishes two completely different programs in gene expression. The adenoviral L4-33K protein, which is the subject of this thesis, was previously implicated to be a key player in the transition from the early to the late phase of infection. Here we show that L4-33K activates late gene expression by functioning as a virus-encoded alternative RNA splicing factor activating splicing of transcripts containing weak 3’ splice sites; a feature common to the viral genes expressed at late times of infection. The splicing enhancer activity of L4-33K was mapped to a tiny arginine/serine (RS) repeat in the carboxyl-terminal domain of the protein. Also, the subcellular distribution to the nucleus with enrichment in the nuclear membrane and subnuclear redistribution to viral replication centers during a lytic infection was observed to depend on this motif. RS repeats are common features for the cellular splicing factors serine/arginine-rich (SR) proteins, which in turn are regulated by reversible phosphorylation. We further show that L4-33K is phosphorylated by two cellular protein kinases, the double-stranded DNA-dependent protein kinase (DNA-PK) and protein kinase A (PKA) in vitro. Interestingly, DNA-PK and PKA have opposite effects on the control of the temporally regulated L1 alternative RNA splicing. DNA-PK functions as an inhibitor of the late specific L1-IIIa pre-mRNA splicing whereas PKA functions as an activator of L1-IIIa pre-mRNA splicing. In summary, this thesis describes L4-33K as an SR protein related viral alternative splicing factor. A tiny RS repeat conveys splicing enhancer activity as well as redistribution of L4-33K to replication centers. Finally, DNA-PK and PKA that phosphorylates L4-33K are suggested to be novel regulatory factors controlling adenovirus alternative splicing.

La plupart des traitements anticancéreux, comme la chimiothérapie ou la radiothérapie, sont cytotoxiques et causent des dommages à l'ADN dans le but d’induire la mort des cellules tumorales. Cependant, l’efficacité d’activité de réparation de l'ADN des tumeurs entraine des résistances intrinsèques et acquises aux traitements. L'une des étapes précoces de la réparation de l’ADN est le recrutement de protéines au niveau du site de dommage. Ce recrutement est coordonné par une cascade de modifications et est contrôlé par des protéines senseurs telles que la protéine kinase ADN dépendante (DNA-PK) et / ou la poly (ADP- ribose) polymérase (PARP). Dans ce manuscrit, nous avons identifié et caractérisé le mécanisme d'action de petites molécules d'ADN (les siDNA), mimant des cassures double brin (appelé Dbait) ou simple brin (appelé Pbait), dans l’inhibition des voies de réparation des cassures simple brin (SSBR/BER). Nous démontrons que les molécules Dbait recrutent et activent à la fois PARP et DNA-PK, contrairement aux molécules Pbait qui ne recrutent que la PARP. L'étude comparative de ces deux molécules permet d'analyser les rôles respectifs des deux voies de signalisation: les deux molécules recrutent les protéines impliquées dans la voie de réparation des cassures simple brin (comme PARP, PCNA et XRCC1) et empêchent leurs recrutements aux niveaux des lésions chromosomiques. Les molécules Dbait inhibent par ailleurs le recrutement des protéines impliquées dans la voie de réparation des cassures double brin (NHEJ et HR). Pbait et Dbait désorganisent la réparation de l’ADN et sensibilisent les cellules tumorales aux traitements. L’inhibition de la réparation des cassures simple brin semble dépendre d’un piégeage des protéines directement sur les siDNA ou indirectement sur les polymères PAR. L’inhibition des voies de réparation des cassures double brin (DSB) semble par contre se faire de façon indirecte ; cette inhibition résulterait plutôt de la phosphorylation des protéines de réparation des DSB de part l’activation de DNA-PK. Les molécules Dbait et Pbait induisent un effet de létalité synthétique des cellules tumorales BRCA mutées. Cependant, la mutation BRCA semble être suffisante mais non nécessaire pour induire la sensibilité des cellules tumorales aux traitements Dbait. En effet, nous avons démontré que les molécules Dbait peuvent aussi sensibiliser les cellules ne présentant pas de mutation BRCA mais ayant toutefois une forte instabilité génétique. Nous avons trouvé une corrélation entre le niveau basal de protéines de réparation de l'ADN (ɣH2AX, PARP et PAR), le taux basal de cassures à l’ADN, la présence de micronoyaux (MN) et la sensibilité des cellules tumorales au traitement Dbait. Nous avons émis l’hypothèse que cette instabilité génétique, déterminé par la quantification de MN dans des biopsies tumorales, pourrait être un biomarqueur prédictif de l’effet du Dbait, non seulement dans les cancers du sein, mais aussi dans les glioblastomes, les mélanomes, les mélanomes uvéaux et les cancers du côlon. / Most conventional cancer treatments, such as chemotherapy or radiotherapy, are cytotoxic and cause DNA damages in the tumoral treated cells, which ultimately lead to their death. However, several intrinsic and acquired resistances of tumors to these treatments are due to the tumor efficient DNA repair activities. One of the major early steps of DNA repair is the recruitment of repair proteins at the damage site and this is coordinated by a cascade of modifications controlled by sensor proteins such as DNA-dependent protein kinase (DNA-PK) and/or poly (ADP-ribose) polymerase (PARP). In this manuscript, we identify and characterize the mechanism of action of short interfering DNA molecules (siDNA), mimicking double-strand breaks (called Dbait) or single-strand breaks (called Pbait) in Single Strand Break Repair pathway (SSBR/BER) inhibition. We demonstrate that Dbait bound and induced both PARP and DNA-PK activities, whereas Pbait acts only on PARP. The comparative study of the two molecules allows analysis of the respective roles of the two signaling pathways: both molecules recruit proteins involved in single-strand break repair (such as PARP, XRCC1 and PCNA) and prevent their recruitment at chromosomal damage. Dbait, but not Pbait, also inhibits recruitment of proteins involved in double-strand break (DSB) repair. By these ways, Pbait and Dbait disorganized DNA repair, thereby sensitizing cells to treatments. SSB repair inhibition depends upon a direct trapping of the main proteins on both molecules and an indirect trapping in PAR polymers. DSB repair inhibition may be indirect, resulting from the phosphorylation of DSB repair proteins by activated DNA-PK. The DNA repair inhibition by both molecules is confirmed by their synthetic lethality with BRCA mutations tumoral cell lines. However, BRCA mutation could be sufficient but not necessary to induce breast cancer cell lines and tumors sensitivity to Dbait treatment. In fact, we demonstrate that Dbait molecules could also have a stand-alone effect in BRCA wild type cells with a high genetic instability. We found a correlation between DNA repair proteins basal level (ɣH2AX, PARP and PAR), DNA break basal level, presence of micronucleus (MN) and tumoral cell lines sensitivity to Dbait treatment. We hypothesis that this genetic instability, determined by MN in tumor biopsies, could be a predictive biomarker of Dbait stand-alone effect, not only in breast cancer treatment, but also in glioblastoma, melanoma, uveal melanoma and colon cancer treatment.

Les cassures double-brin (DSB) sont des dommages dangereux de l’ADN et représentent un facteur de risque pour la stabilité du génome. Le maintien de l'intégrité du génome est essentiel pour les cellules souches adultes, qui sont responsables de la régénération des tissus endommagés et de l'homéostasie tissulaire tout au long de la vie. La régénération musculaire chez l'adulte repose sur les cellules souches musculaires (cellules satellites, SCs) qui possèdent une remarquable capacité de réparation des DSB, mais dont le mécanisme sous-jacent reste inconnu. Ce projet de thèse consistait à étudier comment la différenciation musculaire est affectée lorsque la réparation des DSB est altérée, et quels sont le(s) mécanisme(s) et les conséquences de ce défaut de réparation sur la régénération musculaire. Au cours de cette étude, il est apparu de façon originale que les facteurs de réparation des DSB peuvent affecter la myogenèse, indépendamment de leur fonction dans la réparation de l'ADN. La présente étude a porté sur le rôle de la protéine kinase dépendante de l'ADN (DNA-PK), un facteur crucial pour la réparation non-homologue des DSBs (NHEJ), au cours de la différenciation musculaire chez la souris. L’étude a ciblé l'activation des SCs et la régénération musculaire in vitro et in vivo et a également abordé la régulation de cette kinase. Le rôle "canonique" de la DNA-PK, et donc du NHEJ, dans les SCs a également été étudié en présence de lésions de l'ADN radio-induites. Le rôle d’ATM, une kinase qui orchestre les réponses cellulaires aux DSB, a également été abordé dans le contexte de la régénération musculaire. Ces résultats confirment la notion émergente du rôle multifonctionnel des protéines de réparation de l’ADN dans d’autres processus physiologiques que la réparation elle-même, ce qui m’a également permis de réaliser une étude bibliographique. Ce travail i) identifie de nouveaux régulateurs de la myogenèse et ii) contribue à la compréhension de la résistance des cellules souches musculaires au stress génotoxique. Ces résultats pourraient avoir des implications dans l'amélioration des thérapies cellulaires de la dysfonction musculaire en agissant sur les régulateurs nouvellement découverts. / DNA double-strand breaks (DSBs) are dangerous DNA damages and a risk factor for genome stability. The maintenance of genome integrity is crucial for adult stem cells that are responsible for regeneration of damaged tissues and tissue homeostasis throughout life. Muscle regeneration in the adult relies on muscle stem cells (satellite cells, SCs) that have a remarkable DSB repair activity, but the underlying mechanism is not known. The aims of the present PhD project were to investigate how muscle differentiation is affected when DSB repair is impaired, and which are the mechanism(s) and the consequences on muscle regeneration. During this study, a novel possibility has arisen, namely that DSB repair factors affects myogenesis independently of their DNA repair activity, suggesting a novel function, not previously anticipated, of these factors. The present study has addressed the role of DNA-dependent protein kinase (DNA-PK), a crucial factor in non-homologous end-joining (NHEJ) repair of DSBs, in muscle differentiation in the mouse. Studies have targeted SC activation and muscle regeneration in vitro and in vivo and also addressed the regulation of this kinase. In parallel the more “canonical” role of DNA-PK, and thereby of NHEJ, has been investigated in SCs via radiation-induced DNA damage. The role of ATM, a kinase that orchestrates cellular responses to DSBs in muscle regeneration has also been addressed. These results support the emerging notion of multifunctional repair proteins in a variety of physiological processes beyond the repair process itself, on which I have conducted a bibliographical study. This work i) identifies novel regulators of myogenesis, and ii) helps understanding the resistance of muscle stem cells to genotoxic stress. It has potential implications for improving cellular therapies for muscle dysfunction by acting on the newly discovered regulators.