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.