Firstly, we will produce cellular machineries that govern a major human double-strand break repair pathway by using cutting edge methodologies in protein expression and purification. Secondly, we will unveil the molecular mechanism of these machineries in action during repair with a large set of complementary structural and functional approaches
The first goal of the ANR project will be to tackle the challenge of the production of the seven core proteins that are central to recognize, to process and to ligate the two DNA extremities generated during a double-strand break. The second main goal will be to unveil the molecular mechanism of these machineries in action during repair with a large set of complementary approaches.
Task 1: Project management. A first “face to face” meeting with all partners is scheduled;Task 2 : The first productions of two complexes of respectively two and three proteins have been achieved; Task 3 & 4 Two complexes of the human double-strand repair pathway have been studied by microscopies and by high throughput crystallization screenings ;Task 5 a new DNA repair assay in cellulo has been designed.
In the first production assays, milligram amounts of two multiprotein complexes have been obtained. These two complexes have been used for crystallization assays and for microscopies analyses Several pharmaceutical companies have expressed their interest for the ANR NHEJ-complexes project.
Firstly, the expression and production works realized on the multiprotein complexes of the human repair pathway studied, confirmed the robustness of the methodology developed by Partner 4 ; Secondly, the promising results on production indicate that the structural analyses proposed in the project will benefit from large amount of the target multiprotein complexes.
A first manuscript is under preparation by Partner 3 on the microscopy analyses of the two first multiprotein complexes in presence of DNA. Partners 1 and 4 have published articles on other cellular machineries Gueneau, E et al, Apr 2013, Nat Struct Mol Biol: mismatch repair complexes; Bieniossek, C et al, Janv 2013, Nature: transcription factor TFIID complex.
DNA double strand breaks (DSBs) are a highly toxic form of cellular DNA damages, predominantly repaired in the mammalian cells by the Non-Homologous End-Joining (NHEJ) pathway. NHEJ is also involved in the rearrangement process of immunoglobulins and T-cell receptors genes, called V(D)J recombination. The NHEJ pathway includes at least seven proteins forming three distinct multiprotein complexes: the recognition complex (Ku70/Ku80), the ends processing complex (DNA-PKcs/Artemis) and the final ligation complex (Ligase IV/XRCC4/Cernunnos). In humans, dysfunction of the NHEJ pathway is associated with several severe combined immunodeficiencies (SCID). Partner 2 has previously discovered two core NHEJ factors, the processing DNA nuclease Artemis and the ligation factor Cernunnos (also called XLF), by complementing cells of SCID patients with human cDNAs (Moshous, 2001, Cell; Buck, 2005, Cell).
Though the structure-function relationships of the NHEJ factors have been well studied individually, in particular by Partners 1, 2 & 3 of this project, a major challenge is now to unveil the molecular mechanisms and interplay of the NHEJ multiprotein complexes at work all along the repair of DSB lesions. A major objective of the project will be to progressively produce the ligation complex, the recognition complex, the ends processing complex, and several combinations of these complexes. The proposal will benefit from the cutting-edge MultiBac methodology developed by Partner 4 for the expression of eukaryotic multiprotein complexes in insect cells. The MultiBac approach recently catalyzed breakthrough structural results on large multiprotein complexes (Imasaki, 2011, Nature; Yamada, 2011, Nature). Among the proteins forming the NHEJ complexes, DNA-PKcs is the most challenging to produce since it represents the largest human protein kinase with 4128 amino acids. Human proteins of such large size (dynein 520kDa, NF1 320kDa, Smg1 400 kDa) were recently produced successfully in high quality purity, for the first time, with the MultiBac approach. The second objective of the project is the structural and functional analysis of the purified NHEJ complexes obtained during this project, including complexes already successfully purified by Partner 1.
The NHEJ-complexes proposal combines in a highly integrated and complementary setup structural expertise, (i) crystallography & SAXS (Partner 1) and (ii) electron and atomic force microscopy (Partner 3), (iii) a large set of in vivo and in vitro DNA repair assays (Partner 2), and (iv) state-of-the-art protein production technologies (Partner 4). This project will benefit from the long-standing collaboration of Partners 1 and 2 on the human NHEJ ligation complex (Malivert, 2010, J Biol Chem). More recently, Partner 3 joined Partner 1 & 2 for the structural analysis of the XRCC4/Cernunnos complex by crystallography and electron microscopy. We recently discovered an unexpected filament arrangement of the XRCC4/Cernunnos complex (Ropars, 2011, PNAS). Our objective now is to achieve a significant advance on the complete ligation complex, LigIV/XRCC4/Cernunnos and to further extend our already successful strategy to decipher, for the first time, the entire core human NHEJ machineries. Our consortium possesses all essential tools and expertise to tackle this ambitious project. The paramount expected outcome of the proposal is a precise high-resolution description of the molecular mechanisms by which the NHEJ machineries recognize and repair the DSB lesions. This project addresses important societal issues since DNA repair mechanisms play a critical role in determining the sensitivity and resistance of tumor cells during drug treatment and irradiation (Friesen, 2008, Mol Biol Cell). Our project will set the stage for a targeted inhibition of NHEJ which in the future will directly and substantially enhance the efficacy of various anti-cancer treatments.
Monsieur Jean-Baptiste CHARBONNIER (UMR 8221) – JB.CHARBONNIER@cea.fr
The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.
CNRS UMR 8221
Inserm Inserm U768
CNRS UMR 8126
EMBL EMBL Grenoble
Help of the ANR 480,000 euros
Beginning and duration of the scientific project: September 2012 - 36 Months