Systematic discovery of UFM1 receptors reveals a regulatory module in DNA repair directing non-homologous end-joining
- Zijuan Wang
- Benjamin M. Foster
- Isabelle C. da Costa
- Yue Wu
- Deepak Behera
- Francesca Conte
- Eleanor W. Trotter
- Felicia Wednesday Lopezcolorado
- Maria Jose Cabello-Lobato
- Shweta Choudhary
- Reuven Wiener
- Petra Beli
- Duncan L. Smith
- William H. Banks
- Steven Bagley
- Shane McKee
- Meenakshi Minnis
- Stefan Meyer
- Amanda K. Chaplin
- Wolfgang Dörner
- Henning D. Mootz
- Iain M. Hagan
- Yaron Galanty
- Jeremy M. Stark
- Igor Larrosa
- Matthew J. Cliff
- Christine K. Schmidt
2026-06-15
Posttranslational modifications with ubiquitin-like modifiers (UBLs) are critical for genome maintenance, yet many remain mechanistically uncharacterised. Here, we identify UFM1 as a key regulator of non-homologous end-joining (NHEJ), a major DNA double-strand break repair pathway. Using a structure-guided chemical biology approach, we develop a photo-crosslinkable UFM1 probe and, in combination with NMR, map non-canonical UFM1-binding interfaces in core NHEJ factors, including the disordered XRCC4 tail. Mechanistically, proximity-dependent proteomics and functional assays identify Ku70 as a crucial UFMylation substrate and reveal a UFM1-dependent axis in which XRCC4 engages UFMylated Ku70 to stabilise NHEJ complex assembly on chromatin. Disruption of this molecular mechanism via UFSP2 depletion or a hypomorphic UBA5 variant in patient-derived cells impairs NHEJ function, linking UFMylation defects to compromised genome integrity processes. Our findings define a complete UFM1 signalling module in DNA repair and establish a generalisable framework for dissecting low-affinity UBL networks with broad functional and disease relevance.