Estudi dels mecanismes de reparació del dany oxidatiu en fase replicativa del DNA en humans: L'Anèmia de Fanconi i PCNA.

Student thesis: Doctoral thesis

Abstract

ATP is the molecular unit of currency of intracellular energy transfer. ATP is produced normally during the oxidative phosphorylation inside the mitocondria, the process induces by side effects reactive oxigen species (ROS) capable to induce damage to all cellular structures, including DNA. Cells have developed during evolution several mechanisms to control ROS damage. If these mechanisms fails, we consider the cell under oxidative stress conditions. The thesis sheds light on the DNA repair mechanisms in replicative phase of cell cycle under oxidative stress conditions. Is focused on the study of two DNA repair pathways: the Fanconi Anemia (FA) pathway and the role of proliferating cell nuclear antigen (PCNA) in response to oxidative insults. FA is a highly heterogenic rare genetic disease. Mutations in any of the 15 known FA gens cause FA. FA is characterized by bone marrow failure, chromosome fragility and increased cancer susceptibility. FA cells are sensitive to interstrand crosslinking (ICLs) agents. All 15 FA proteins cooperate in a common pathway to repair the ICLs lesions during replicative phases of cell cycle. We consider FA pathway activated when the central FA protein FANCD2 is monoubiquitinated in response to DNA damage. Until now, is not known any natural source of ICLs, either endogenous or exogenous, to explain FA phenotype. We tried to solve in the thesis if the endogenous oxidative damage could explain, at least partially, FA phenotype. Is known that FA cells present high level of the oxidated lesion 8-oxoG in their DNA. The results presented here discard a repair defect of FA cells in response to oxidative lesions such as 8-oxoG and other oxidative base lesions processed by BER. So and most probably, the high levels of 8-oxoG observed in FA cells are due to increase of 8-oxoG production rather than DNA repair defect of these kind of lesion. We showed here that DNA oxidative damage inducer H2O2 induces secondarily DNA double strand breaks and that this DNA lesion is processed by FA pathway. Ataxia telengectasia (A-T) is another rare genetic disease characterized by DNA repair defects caused by mutations in the cell cycle and DNA repair protein ATM. A-T shares some phenotypic common tracks with FA such as the increased levels of 8-oxoG in their DNA. Our work suggest a common and coordinated model between A-T and FA in response to oxidative damage inducer H2O2: ATM phosphorylates residue S222 of FANCD2 protein in response to DSBs induced by oxidative damage to correctly establish the cell cycle checkpoint in S-phase to repair the damage. This model could also explain why FANCD2 patients have more sever phenotype respect FA core complex patients. In summary, we showed here that FANCD2 has dual functions in response to oxidative damage inducer H2O2, is both monoubiquitinated through ATR and FA pathway and phosphorylated through ATM kinase for proper response to oxidative endogenous insults. The last part of the work describes a novel DNA repair pathway in response to oxidative damage mediated by PCNA and dependent on replicative phase of the cell cycle. PCNA is monoubiquitinated in response to oxidative DNA damage lesions by RAD18 ubiquitin ligase in a translesion synthesis (TLS) like process. Monoubiquitinated PCNA is then able to recruit TLS polymerases to bypass oxidative DNA lesions. We suggest here a putative and novel role for the unknown DNA polymerase iota to bypass such kind of oxidative lesions in an error free manner.
Date of Award26 Jan 2012
Original languageCatalan
Awarding Institution
  • Universitat Autònoma de Barcelona (UAB)
SupervisorJordi Surralles Calonge (Director)

Keywords

  • Fanconi Anemia
  • DNA

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