Cabello-Lobato, M. J. | Search bioRxiv

bioRxiv2025

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

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.

molecular biology↗

bioRxiv2021

Microarray screening reveals a non-conventional SUMO-binding mode linked to DNA repair by non-homologous end-joining

SUMOylation is critical for a plethora of cellular signalling pathways including the repair of DNA double-strand breaks (DSBs). If misrepaired, DSBs can lead to cancer, neurodegeneration, immunodeficiency and premature ageing. Based on systematic proteome microarray screening combined with widely applicable carbene footprinting and high-resolution structural profiling, we define two non-conventional SUMO2-binding modules on XRCC4, a DNA repair protein important for DSB repair by non-homologous end-joining (NHEJ). Mechanistically, interaction of SUMO2 with XRCC4 is incompatible with XRCC4 binding to at least two other NHEJ proteins - XLF and DNA ligase 4 (LIG4). These findings are consistent with SUMO2 interactions of XRCC4 acting as backup pathways at different stages of NHEJ, in the absence of these factors or their dysfunctioning. Such scenarios are not only relevant for carcinogenesis, but also for the design of precision anti-cancer medicines and the optimisation of CRISPR/Cas9-based gene editing. This work reveals insights into topology-specific SUMO recognition and its potential for modulating DSB repair by NHEJ. Moreover, it provides a rich resource on binary SUMO receptors that can be exploited for uncovering regulatory layers in a wide array of cellular processes.

biochemistry↗