The team’s biological questions focus on understanding, at a mechanistic level, the key pathways involved in repairing DNA double-strand breaks and replication-stalling lesions, particularly the Homologous Recombination and Non-Homologous End Joining repair pathways. We combine strong molecular biology and genetics approaches to investigate the regulation of these repair pathways in cells. In parallel, we bring unique expertise and employ original methodological strategies, notably the use of Electron Microscopy (EM) and Atomic Force Microscopy (AFM) to study DNA repair factors during reconstituted biochemical reactions, as well as to visualize DNA intermediates enriched from cells. Additionally, we have developed Cryo-EM techniques to determine, at very high resolution, the structures of nucleoprotein complexes central to DNA repair, providing deep insights into the molecular mechanisms that maintain genome stability.
Cryo-EM Analysis of Replication Forks with RAD52 and RAD51 :
In this project, the team has purified DNA replication-fork substrates that are sufficiently long for visualization by positive-stain electron microscopy. These substrates are used to study the interaction between RAD51 and RAD52 at replication forks. The goal is to characterize the molecular structures of their binding modes, the formation of mixed filaments, and their dynamics. These experiments provide key insights into the crucial roles of RAD51 and RAD52 in protecting replication forks during replication stress.
NHEJ Machinery in Action, XRCC4 Filament as a Scaffold Beyond NHEJ :
XRCC4 is a central protein in the Non-Homologous End Joining (NHEJ) pathway for repairing double-strand breaks (DSBs). After purifying the full-length XRCC4 protein, the team determined the structure of XRCC4 filaments, which form a helical assembly. This structure has broad implications for coordinating other proteins within the NHEJ ligation complex. Additionally, XRCC4 filaments serve as molecular anchors for cytoskeleton-associated proteins such as IFFO1. Cryo-EM is now being used to map the molecular details of these interactions, identifying critical residues and domains to guide small peptide design.
Structural Studies of BRCA2, RAD51, and Partners FIGNL1 and RAD54 :
Through collaboration with researchers at CEA, a new family of binding motifs was identified. The team is solving atomic-resolution Cryo-EM structures of peptides containing these motifs from FIGNL1 or RAD54 bound to RAD51 and its meiotic homolog DMC1. This work aims to reveal the critical molecular interactions involved and understand how mutations in these domains affect function. Furthermore, these motifs may offer new avenues for therapeutic targeting.
Regulation of HR Factors by Degrons and Phosphodegrons :
Using ovarian cancer (OC) as a model (in collaboration with the OG team), the group aims to identify phosphodegrons in HR proteins specifically regulated during replication stress—a hallmark of OC. The project aims to fully characterize these regulatory mechanisms and determine if it correlates with changes HR protein levels, which often change in aggressive tumors. Targeting the regulatory circuit controlling protein levels represents a promising therapeutic strategy, inducing selective protein degradation that leads to loss of function and lethality in specific genetic backgrounds.
We specialize in using Cryo-EM to unravel complex and dynamic nucleoprotein assemblies. Our deep expertise in EM and the biochemistry of DNA-related complexes allows us to expertly optimize conditions for even the most challenging targets.
We’re experts in reconstituting DNA repair reactions of the Homologous Recombination and NHEJ pathways. With a solid mix of biochemical know-how and hands-on experience, we fine-tune conditions and we can observe them by Electron Microscopy to get insights on the heteregeneity of molecular intermediates.
We use genetic and molecular biology tools to study the regulators and modifications of key players in Homologous Recombination and NHEJ. By working with model organisms and cancer cells, we gain deeper insight into how these repair pathways function in real biological contexts.
