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Analyse des interactions ADN lésé / protéines : Optimisations méthodologiques et applications aux dommages de l'ADN engendrés par les dérivés du platine

Christophe Bounaix Morand Du Puch 1 
1 LAN - Laboratoire Lésions des Acides Nucléiques
SCIB - UMR E3 - Service de Chimie Inorganique et Biologique : DSM/INAC/SCIB
Abstract : DNA lesions contribute to the alteration of DNA structure, thereby inhibiting essential cellular processes. Such alterations may be beneficial for chemotherapies, for example in the case of platinum anticancer agents. They generate bulky adducts that, if not repaired, ultimately cause apoptosis. A better understanding of the biological response to such molecules can be obtained through the study of proteins that directly interact with the damages. These proteins constitute the DNA lesions interactome. This thesis presents the development of tools aiming at increasing the list of platinum adduct-associated proteins. Firstly, we designed a ligand fishing system made of damaged plasmids immobilized onto magnetic beads. Three platinum drugs were selected for our study: cisplatin, oxaliplatin and satraplatin. Following exposure of the trap to nuclear extracts from HeLa cancer cells and identification of retained proteins by proteomics, we obtained already known candidates (HMGB1, hUBF, FACT complex) but also 29 new members of the platinated-DNA interactome. Among them, we noted the presence of PNUTS, TOX4 and WDR82, which associate to form the recently-discovered PTW/PP complex. Their capture was then confirmed with a second model, namely breast cancer cell line MDA MB 231, and the biological consequences of such an interaction now need to be elucidated. Secondly, we adapted a SPRi biochip to the study of platinum-damaged DNA/proteins interactions. Affinity of HMGB1 and newly characterized TOX4 for adducts generated by our three platinum drugs could be validated thanks to the biochip. Finally, we used our tools, as well as analytical chemistry and biochemistry methods, to evaluate the role of DDB2 (a factor involved in the recognition of UV-induced lesions) in the repair of cisplatin adducts. Our experiments using MDA MB 231 cells differentially expressing DDB2 showed that this protein is not responsible for the repair of platinum damages. Instead, it appears to act as a positive mediator of their cytotoxicity. In the near future, the abovementioned microsystems will be adapted to the study of the interactome of other DNA lesions.
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Submitted on : Thursday, December 23, 2010 - 10:12:18 AM
Last modification on : Thursday, November 3, 2022 - 8:35:35 PM
Long-term archiving on: : Thursday, March 24, 2011 - 2:42:27 AM


  • HAL Id : tel-00549987, version 1



Christophe Bounaix Morand Du Puch. Analyse des interactions ADN lésé / protéines : Optimisations méthodologiques et applications aux dommages de l'ADN engendrés par les dérivés du platine. Sciences du Vivant [q-bio]. Université Joseph-Fourier - Grenoble I, 2010. Français. ⟨NNT : ⟩. ⟨tel-00549987⟩



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