Concerted SUMO-targeted ubiquitin ligase activities of TOPORS and RNF4 are essential for stress management and cell proliferation.

Protein SUMOylation provides a principal driving force for cellular stress responses, including DNA-protein crosslink (DPC) repair and arsenic-induced PML body degradation. In this study, using genome-scale screens, we identified the human E3 ligase TOPORS as a key effector of SUMO-dependent DPC resolution. We demonstrate that TOPORS promotes DPC repair by functioning as a SUMO-targeted ubiquitin ligase (STUbL), combining ubiquitin ligase activity through its RING domain with poly-SUMO binding via SUMO-interacting motifs, analogous to the STUbL RNF4. Mechanistically, TOPORS is a SUMO1-selective STUbL that complements RNF4 in generating complex ubiquitin landscapes on SUMOylated targets, including DPCs and PML, stimulating efficient p97/VCP unfoldase recruitment and proteasomal degradation. Combined loss of TOPORS and RNF4 is synthetic lethal even in unstressed cells, involving defective clearance of SUMOylated proteins from chromatin accompanied by cell cycle arrest and apoptosis. Our findings establish TOPORS as a STUbL whose parallel action with RNF4 defines a general mechanistic principle in crucial cellular processes governed by direct SUMO-ubiquitin crosstalk.

Mechanism and function of DNA replication-independent DNA-protein crosslink repair via the SUMO-RNF4 pathway.

DNA-protein crosslinks (DPCs) obstruct essential DNA transactions, posing a serious threat to genome stability and functionality. DPCs are proteolytically processed in a ubiquitin- and DNA replication-dependent manner by SPRTN and the proteasome but can also be resolved via targeted SUMOylation. However, the mechanistic basis of SUMO-mediated DPC resolution and its interplay with replication-coupled DPC repair remain unclear. Here, we show that the SUMO-targeted ubiquitin ligase RNF4 defines a major pathway for ubiquitylation and proteasomal clearance of SUMOylated DPCs in the absence of DNA replication. Importantly, SUMO modifications of DPCs neither stimulate nor inhibit their rapid DNA replication-coupled proteolysis. Instead, DPC SUMOylation provides a critical salvage mechanism to remove DPCs formed after DNA replication, as DPCs on duplex DNA do not activate interphase DNA damage checkpoints. Consequently, in the absence of the SUMO-RNF4 pathway cells are able to enter mitosis with a high load of unresolved DPCs, leading to defective chromosome segregation and cell death. Collectively, these findings provide mechanistic insights into SUMO-driven pathways underlying replication-independent DPC resolution and highlight their critical importance in maintaining chromosome stability and cellular fitness.

Conditional gene inactivation by combining tetracycline-mediated transcriptional repression and auxin-inducible degron-mediated degradation.

Characterizing the functions of essential cell cycle control genes requires tight and rapid inducible gene inactivation. Drawbacks of current conditional depletion approaches include slow responses and incomplete depletion. We demonstrated that by integrating the tetracycline-controlled promoter system and the auxin-inducible degron (AID) system together, AID-tagged proteins can be downregulated more efficiently than the individual technology alone. When used in conjunction with CRISPR-Cas9-mediated disruption of the endogenous locus, this system facilitates the analysis of essential genes by allowing rapid and tight conditional depletion, as we have demonstrated using several cell cycle-regulatory genes including cyclin A, CDK2, and TRIP13. The vectors constructed in this study allow expression of AID-fusion proteins under the control of tetracycline-controlled promoters and should be useful in studies requiring rapid and tight suppression of gene expression in mammalian cells.