La réponse cellulaire aux ultra-violets (UV), ou réponse UV, est une réponse complexe et spécialisée dans l’adaptation et la tolérance des dommages aux UV. Celle-ci est initiée par un grand nombre d’évènements moléculaires et de signalisation nucléaire mais aussi au niveau de la membrane plasmique ou du cytoplasme. L’importance et l’influence exactes de ces évènements sur la réparation par excision de nucléotides (NER) des dommages UV à l’ADN sont encore mal comprises et doivent encore être méthodiquement démontrées. Dans cette thèse, grâce à l’utilisation d’une méthode sensible d’analyse de la réparation NER basée sur la cytométrie en flux, il est montré, dans un premier temps, que l’activité des voies MAPK (Mitogen-Activated Protein Kinases), qui sont des voies de signalisation de stress UV d’origine cytoplsamique, ne participent pas à l’efficacité de réparation NER des dommages UV dans les cellules humaines. En effet, l’abrogation de la signalisation MAPK, par inhibition pharmacologique, par utilisation de mutants dominant-négatifs ou par inhibition de leur expression endogène, ne révèlent aucun changement de la cinétique de réparation des dommages UV par excision de nucléotides. Cependant, l’utilisation de cette même méthode de réparation, mais cette fois, appliquée pour l’étude de réparation NER en fonction du cycle cellulaire, a permis de mettre en évidence la nécessité fonctionnelle de l’ADN polymérase translésionnelle eta (Pol η) dans la réparation NER des dommages UV, uniquement en phase S. Cette observation fut initialement caractérisée dans les cellules de patients affectés du syndrome variant de xérodermie pigmentaire (XP-V) puis, confirmée ensuite par l’inhibition de l’expression de Pol η endogène ou par la complémentation avec des mutants non-fonctionnels dans les cellules XP-V. Ces résultats indiquent que, contrairement à la réponse UV MAPK cytoplasmique, les évènements nucléaires comme la synthèse translésionnelle, peuvent influencer l’efficacité de réparation NER en phase S. Plus particulièrement, ces données établissent un lien possible entre la réparation NER en phase S et les niveaux de stress réplicatifs, révélé ici par la déficience fonctionnelle Pol η ou ATR. Les observations, présentées dans cette thèse, renforcent un rôle du point de contrôle S aux UV sur l’efficacité de la réparation NER et suggèrent que l’inhibition NER, observée en phase S dans les cellules XP-V, est modulée par le stress réplicatif. Un tel moyen de contrôle pourrait avoir une action plutôt protectrice pendant cette phase critique du cycle cellulaire. Mots clés: UV, translésionnelle, eta, MAPK, NER, CPD, cytométrie, phase-S, tolérance. / The UV-response is a complex cellular response to UV irradiation, which allows cellular adaptation and protection against deleterious effects of UV. This specialized response involves numerous molecular and signaling events from plasma membrane and from the nucleus represented, among others, by Mitogen-Activated Protein Kinase (MAPK) pathway activation and translesion synthesis respectively. More particularly, the exact role of these events on the removal UV-induced DNA damage by nucleotide excision repair (NER) in human cells is poorly understood and documented. By using a sensitive flow cytometry based-NER assay, presented and validated in this thesis, to quantify the removal of UV-DNA damage, it was unexpectedly found that Mitogen-Activated Protein Kinase (MAPK) signalling, originating from the the plasma membrane, does not regulate the efficiency of UV-induced DNA damage repair in human cells. Indeed, MAPK inhibition with pharmacological inhibitors, expression of short-hairpin RNA or dominant negative mutant, all together, substantiate fully the lack of effect of this signalling pathway on UV-damage removal by NER in human primaries and tumorous cells. Surprisingly, the same NER assay, applied to quantify the removal of UV-induced DNA damages as a function of the cell cycle, has shown a requirement of functional translesion synthesis polymerase eta (Pol η) for efficient UV-DNA damage repair in human cells uniquely during S-phase, where its function is required for the bypass of UV DNA damage. This observation, originally made in fibroblasts from xeroderma pigmentosum variant syndrome (XP-V) afflicted patients, was further confirmed in normal human cells, by abrogation of endogenous Pol η expression or by complementation with Pol η in XP-V cells. All together, the data presented here, indicate that MAPK signaling play no role in NER-mediated UV-damage removal, but highlight a role for UV-DNA damage tolerance response, as translesion synthesis, in regulation of NER efficiency. More particularly, these observations establish a potential link between the S-Phase Repair (SPR) of UV-DNA damages and replicative stress, revealed by a deficiency of Pol η or ATR. This SPR defects seen under acute replicative stress conditions could impact tumorogenesis or chemotherapy outcomes. Moreover, SPR defects, seen in XP-V cells could be controlled by replicative stress, and also reflect a protective coordination to reduce high risks of genetic or chromosomal aberrations that may occur during DNA replication upon UV exposure. Key Words: Translesion, UV, TLS, eta, MAPK, NER, S-phase, flow-cytometry, CPD.

Faithful replication of genomic DNA is crucial for the survival of a cell. In order to achieve high-level accuracy in copying its genome, all cells employ replicative DNA polymerases that have intrinsic high fidelity. When an error occurs on the template DNA strand, in the form of lesions caused by diverse chemicals, reactive oxygen species, or UV light, the high-fidelity replicative DNA polymerases are stalled. To bypass these replication blocks, cells harbor multiple specialized translesion DNA polymerases that are error-prone and therefore able to accommodate the lesions and continue DNA synthesis. As a result of their low fidelity, the translesion polymerases are associated with increased mutagenesis, drug resistance, and cancer. Therefore, the access of the translesion polymerases to DNA needs to be tightly controlled, but how this is achieved has been the subject of debate. This Thesis presents the development of a co-localization single-molecule spectroscopy (CoSMoS) method to directly visualize the loading of the Escherichia coli replicative polymerase on DNA, as well as the exchange between the replicative polymerase and the translesion polymerases Pol II and Pol IV. In contrast to the toolbelt model for the exchange between the polymerases, this work shows that the translesion polymerases Pol II and Pol IV do not form a stable complex with the replicative polymerase Pol IIIα on the β-clamp. Furthermore, we find that the sequential activities of the replication proteins: clamp loader, clamp, and Pol IIIα, are highly organized while the exchange with the translesion polymerases is disordered. This exchange is not determined by lesion-recognition but instead a concentration-dependent competition between the replicative and translesion polymerases for the hydrophobic groove on the surface of the β-clamp. Hence, our results provide a unique insight into the temporal organization of events in DNA replication and translesion synthesis.

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Bacterial DNA replication can stall at DNA lesions, leading to cell death if the damage fails to be repaired. To circumvent this, bacteria possess a mechanism called translesion DNA synthesis (TLS) to allow DNA damage bypass. The ImuABC TLS mutasome comprises the RecA domain-containing protein ImuA, the inactive polymerase ImuB, and the error-prone polymerase ImuC. ImuA and ImuB are necessary for the mutational function of ImuC that can lead to antimicrobial resistance (AMR) as seen in high-priority pathogens Pseudomonas aeruginosa and Mycobacterium tuberculosis. Understanding how ImuA and ImuB contribute to this function can lead to new targets for antimicrobial development. This research aims to discover the molecular functions of ImuA and ImuB homologs from Myxococcus xanthus through structural modelling and biochemical analyses. ImuA was discovered to be an ATPase whose activity is enhanced by DNA. Based on predicted structural models of the ATPase active site, I identified the critical residues needed for ATP hydrolysis, and found that the ImuA C-terminus regulates ATPase activity. Further, ImuA and ImuBNΔ34 (a soluble truncation of ImuB) display a preference for longer single-stranded DNA and overhang DNA substrates, and their affinity for DNA was quantified in vitro. To better understand how ImuA and ImuB assemble in the TLS mutasome, bacterial two-hybrid assays determined that ImuA and ImuB can self-interact and bind one another. Mass photometry revealed that ImuA is a monomer and ImuBNΔ34 is a trimer in vitro. ImuA and ImuBNΔ34 binding affinity was quantified in vitro at 1.69 μM ± 0.21 by microscale thermophoresis, and removal of the ImuA C-terminus weakens this interaction. Lastly, ImuA and ImuBNΔ34 secondary structures were quantified using circular dichroism spectroscopy, and ImuA was modified to enable crystallization for future structural studies. Together, this research provides a better understanding of ImuABC-mediated TLS, potentially leading to novel antibiotics to reduce the clinical burden of AMR. / Thesis / Master of Science (MSc) / The antimicrobial resistance (AMR) crisis is fueled by the emergence of multi-drug resistant microbes, posing a major threat to global health and disease treatment. Bacteria can develop resistance to antibiotics through mutations in the genome. When the genome becomes damaged, bacteria can acquire these mutations by an error-prone replication mechanism called translesion DNA synthesis (TLS). In some bacteria, TLS involves a specialized enzyme complex, consisting of proteins ImuA, ImuB and ImuC, allowing replication past bulky DNA damage and lesions. The goal of this thesis is to investigate how the ImuA and ImuB proteins contribute to the functioning of this mistake-making machinery. I used biochemical and biophysical methods to identify ImuA and ImuB interactions with each other and themselves. I discovered that ImuA is an enzyme that uses energy to enhance its binding to DNA, and determined the specific amino acids involved in this function.

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Genomic instability, in the form of gene mutations, insertions/deletions, and gene amplifications, is one of the hallmarks in many types of cancers and other inheritable genetic disorders. Trinucleotide repeat (TNR) disorders, such as Huntington’s disease (HD) and Myotonic dystrophy (DM) can be inherited and repeats may be extended through subsequent generations. However, it is not clear how the CAG repeats expand through generations in HD. Two possible repeat expansion mechanisms include: 1) polymerase mediated repeat extension; 2) persistent TNR hairpin structure formation persisting in the genome resulting in expansion after subsequent cell division. Recent in vitro studies suggested that a family A translesion polymerase, polymerase θ (Polθ), was able to synthesize DNA larger than the template DNA. Clinical and in vivo studies showed either overexpression or knock down of Polθ caused poor survival in breast cancer patients and genomic instability. However, the role of Polθ in TNR expansion remains unelucidated. Therefore, we hypothesize that Polθ can directly cause TNR expansion during DNA synthesis. The investigation of the functional properties of Polθ during DNA replication and TNR synthesis will provide insight for the mechanism of TNR expansion through generations.

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