Etude du rôle de la réponse UV sur le contrôle de la réparation par excision de nucléotides (NER) des dommages à l’ADN : rôle des voies MAPK et de l’ADN polymérase eta
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 This observation, originally made in fibroblasts from xeroderma pigmentosum variant syndrome (XP-V) afflicted patients, was further confirmed in normal human cells, by abrogation of endogenous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.
UV eta MAPK NER CPD TLS Eta S-Phase Flow-cytometry Translesion