Organization and Dynamics of NHEJ DNA repair condensates – XXL

In mammalian cells, DNA double-strand breaks are predominantly repaired by non-homologous end joining (NHEJ). Rapidity and untemplated adaptability to repair any kind of broken DNA end structures is the distinctive mechanistic features of NHEJ. Assembly of a flexible super structure — the synaptic scaffold, brings and holds broken DNA ends together during the repair process. Because the synaptic scaffold is dynamic, it is difficult to address how it evolves in space and time and at the same time supports protection, processing and ligation of the two broken DNA strands. Snapshots of its architecture, obtained by single particle cryo-electron microscopy analysis, recently shed light on its organizational dynamics. In complementing studies, we have used nuclear magnetic resonance to characterize the dynamics of the disordered regions of scaffolding proteins XLF and XRCC4 at atomic resolution. We discovered that XLF and XRCC4 form, together with DNA Ligase 4, a network of weak specific interactions that lead to liquid-liquid phase separation in vitro (XXL condensates). In this project, we hypothesize that NHEJ could be regulated in space and time by XXL condensates. To test this hypothesis, the XXL project will implement a multiscale and multidisciplinary approach that will integrate ensemble analysis of XXL condensates; analysis of single molecule behavior inside condensates and during interaction with external components; analysis of interactions of condensate components with external factors at atomic resolution; and we will further correlate in vitro findings to functional analysis. Results obtained from the analysis of XXL droplets will be correlated to results from analysis of XXL condensates at DNA damage sites in cell nuclei. New insights on how NHEJ is regulated and cooperates with other DSB repair pathways will be revealed, with implications for cancer biology, immunology, neurobiology and development of genome editing biotechnologies.