Ferrage, F. | bioRxiv Science

bioRxiv2026

Molecular characterization of MRI/CYREN reveals the Ku binding mode and the role of multimerization in stimulating the activity of NHEJ in DNA repair

Mammalian cells primarily repair DNA double-strand breaks through non-homologous end joining (NHEJ), a pathway that requires the Ku heterodimer, DNA-PKcs, XRCC4 complexed with DNA Ligase 4, and XLF as core components. In addition, several auxiliary proteins are involved in the regulation of NHEJ, whose importance has been underscored recently. Among them, MRI, also known as CYREN, is one of the most important auxiliary proteins. Despite its importance, the structural properties of MRI remain poorly characterized. In this study, we used solution NMR spectroscopy combined with cellular experiments to investigate two isoforms of human MRI (MRI1 and MRI2) at the residue level. Our findings reveal that both isoforms are predominantly disordered, and that the APLF-like Ku-binding motif (A-KBM) of MRI undergoes folding upon binding to the von Willebrand A domain of Ku80 (Ku80vWA). Moreover, an evolutionarily dominant leucine-to-methionine substitution in A-KBM significantly increases binding affinity for Ku80vWA by over 30 times without impacting cellular NHEJ efficiency. Importantly, we identified here a domain that drives MRI multimerization that is required for efficient NHEJ in cellular assays. This work further deciphers the increasingly recognized functional roles of disordered protein regions of the NHEJ machinery.

biophysics↗

bioRxiv2023

Multivalent interactions of the disordered regions of XLF and XRCC4 foster robust cellular NHEJ and drive the formation of ligation-boosting condensates in vitro

In mammalian cells, DNA double-strand breaks are predominantly repaired by non-homologous end joining (NHEJ). During repair, the Ku70/80 heterodimer (Ku), XRCC4 in complex with DNA Ligase 4 (X4L4), and XLF form a flexible scaffold that holds the broken DNA ends together. Insights into the architectural organization of the NHEJ scaffold and its regulation by the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) have recently been obtained by single-particle cryo-electron microscopy analysis. However, several regions, especially the C-terminal regions (CTRs) of the XRCC4 and XLF scaffolding proteins, have largely remained unresolved in experimental structures, which hampers the understanding of their functions. Here, we used magnetic resonance techniques and biochemical assays to comprehensively characterize the interactions and dynamics of the XRCC4 and XLF CTRs at atomic resolution. We show that the CTRs of XRCC4 and XLF are intrinsically disordered and form a network of multivalent heterotypic and homotypic interactions that promotes robust cellular NHEJ activity. Importantly, we demonstrate that the multivalent interactions of these CTRs led to the formation of XLF and X4L4 condensates in vitro which can recruit relevant effectors and critically stimulate DNA end ligation. Our work highlights the role of disordered regions in the mechanism and dynamics of NHEJ and lays the groundwork for the investigation of NHEJ protein disorder and its associated condensates inside cells with implications in cancer biology, immunology and the development of genome editing strategies.

biophysics↗