UFMylation orchestrates chromatin engagement of core NHEJ components to promote DNA double-strand break repair

Wang, Z.; Foster, B. M.; da Costa, I. C.; Wu, Y.; Behera, D.; Conte, F.; Trotter, E. W.; Cabello-Lobato, M. J.; Choudhary, S.; Wiener, R.; Beli, P.; Smith, D. L.; Banks, W. H.; Bagley, S.; McKee, S.; Minnis, M.; Meyer, S.; Chaplin, A. K.; Doerner, W.; Mootz, H. D.; Hagan, I. M.; Galanty, Y.; Larrosa, I.; Cliff, M. J.; Schmidt, C. K.

doi:10.1101/2025.06.16.659844

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bioRxiv · 10.1101/2025.06.16.659844

UFMylation orchestrates chromatin engagement of core NHEJ components to promote DNA double-strand break repair

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Abstract

DNA double-strand breaks (DSBs) are highly cytotoxic lesions whose misrepair can lead to genomic instability, cancer and developmental disorders. Through systematic screening of understudied ubiquitin-like modifiers (UBLs), we identify UFM1 as a previously unrecognised regulator of non-homologous end-joining (NHEJ). Using a structure-guided chemical biology strategy, we develop a photo-crosslinkable UFM1 probe and, together with high-resolution NMR, uncover non-canonical UFM1-binding regions in core NHEJ components, including XRCC4. Mechanistically, proximity-dependent proteomics reveals Ku70 as a key UFMylation substrate, establishing a functional axis in which XRCC4 engages UFMylated Ku70 to promote the chromatin assembly of NHEJ factors. Perturbation of UFM1 signalling, via UFSP2 depletion or a hypomorphic UBA5 allele in patient-derived fibroblasts, impairs these processes, linking UFMylation defects to altered regulation of DSB repair. Our findings define a complete UFM1 signalling module in genome maintenance and uncover a molecular connection between hereditary UFMylation disorders and dysregulated DSB repair pathways.

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Wang, Z., Foster, B. M., da Costa, I. C., Wu, Y., Behera, D., Conte, F., Trotter, E. W., Cabello-Lobato, M. J., Choudhary, S., Wiener, R., Beli, P., Smith, D. L., Banks, W. H., Bagley, S., McKee, S., Minnis, M., Meyer, S., Chaplin, A. K., Doerner, W., Mootz, H. D., Hagan, I. M., Galanty, Y., Larrosa, I., Cliff, M. J., Schmidt, C. K.. 2025-06-17. UFMylation orchestrates chromatin engagement of core NHEJ components to promote DNA double-strand break repair. https://doi.org/10.1101/2025.06.16.659844

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