DNA ‐protein crosslinks ( DPC s) are highly cytotoxic lesions that obstruct essential DNA transactions and whose resolution is critical for cell and organismal fitness. However, the mechanisms by which cells respond to and overcome DPC s remain incompletely understood. Recent studies unveiled a dedicated DPC repair pathway in higher eukaryotes involving the SprT‐type metalloprotease SPRTN / DVC 1, which proteolytically processes DPC s during DNA replication in a ubiquitin‐regulated manner. Here, we show that chemically induced and defined enzymatic DPC s trigger potent chromatin SUMO ylation responses targeting the crosslinked proteins and associated factors. Consequently, inhibiting SUMO ylation compromises DPC clearance and cellular fitness. We demonstrate that ACRC / GCNA family SprT proteases interact with SUMO and establish important physiological roles of Caenorhabditis elegans GCNA ‐1 and SUMO ylation in promoting germ cell and embryonic survival upon DPC formation. Our findings provide first global insights into signaling responses to DPC s and reveal an evolutionarily conserved function of SUMO ylation in facilitating responses to these lesions in metazoans that may complement replication‐coupled DPC resolution processes.
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 replicationdependent 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.
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 cycleregulatory 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.
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