The E3 ubiquitin ligase DTX3L and the deubiquitinase USP28 fine-tune DNA double strand repair through mutual regulation of their protein levels

Summary The DNA damage response (DDR) relies on a complex protein network to maintain genomic integrity, yet the interplay between post-translational modifiers remains poorly understood. Here, we uncover a novel regulatory axis between the E3 ubiquitin ligase DTX3L and the deubiquitinase USP28 at DNA double-strand breaks (DSBs). Our results reveal a sophisticated feedback mechanism in which DTX3L ubiquitinates USP28, leading to its proteasomal degradation, while USP28 counteracts by deubiquitinating both itself and DTX3L. This cross-regulation fine-tunes DSB repair in multiple pathways, including non-homologous end joining (NHEJ), homologous recombination (HR), single-strand annealing (SSA), and microhomology-mediated end joining (MMEJ). Strikingly, the detrimental effects of USP28 depletion on these repair pathways were rescued by concurrent DTX3L knockdown. Collectively, our work uncovers a novel layer of DDR regulation in which DTX3L and USP28’s antagonistic activities calibrate cellular responses to genotoxic stress, thus identifying promising therapeutic targets to combat diseases associated with genomic instability. Highlights DTX3L and USP28 physically interact and colocalize in cellular sub-compartments, with the N-terminal D1-D3 domains of DTX3L primarily mediating the interaction DTX3L ubiquitinates USP28 for degradation, while USP28 deubiquitinates itself and DTX3L, creating a sophisticated feedback mechanism. The DTX3L-USP28 circuit influences levels of key proteins like HIF-1α, p53, and c-MYC, suggesting broader impacts on cellular stress responses. DTX3L and USP28 cooperatively regulate multiple DSB repair pathways, including NHEJ, HR, SSA, and MMEJ, with USP28 depletion effects rescued by DTX3L silencing.