The E3 ubiquitin ligase RNF8 (RING finger protein 8) is a pivotal enzyme for DNA repair. However, RNF8 hyper‐accumulation is tumour‐promoting and positively correlates with genome instability, cancer cell invasion, metastasis and poor patient prognosis. Very little is known about the mechanisms regulating RNF8 homeostasis to preserve genome stability. Here, we identify the cellular machinery, composed of the p97/VCP ubiquitin‐dependent unfoldase/segregase and the Ataxin 3 (ATX3) deubiquitinase, which together form a physical and functional complex with RNF8 to regulate its proteasome‐dependent homeostasis under physiological conditions. Under genotoxic stress, when RNF8 is rapidly recruited to sites of DNA lesions, the p97–ATX3 machinery stimulates the extraction of RNF8 from chromatin to balance DNA repair pathway choice and promote cell survival after ionising radiation (IR). Inactivation of the p97–ATX3 complex affects the non‐homologous end joining DNA repair pathway and hypersensitises human cancer cells to IR. We propose that the p97–ATX3 complex is the essential machinery for regulation of RNF8 homeostasis under both physiological and genotoxic conditions and that targeting ATX3 may be a promising strategy to radio‐sensitise BRCA‐deficient cancers.

Highlights d SUMOylation and ubiquitylation enable DPC repair during DNA replication d Resolution of ubiquitylated and SUMOylated DPCs requires the protease SPRTN d Inactivation of SUMOylation after DPC formation activates homologous recombination d DPC SUMOylation and SPRTN prevent recombinationdependent genomic instability

Highlights d p97 interacts with MRE11 and regulates its retention on chromatin after IR d p97 inactivation causes excessive MRE11-mediated DNA end resection d p97-mediated MRE11 chromatin retention causes HR defects but efficient SSA d CB-5083 radiosensitizes cells and suppresses tumor growth in vivo after IR

In this review, we aim to discuss the molecular mechanisms behind adenosine triphosphate-mediated ischemic tissue injury and evaluate the role of extracellular adenosine triphosphate in ischemic injury in specific organs, in order to provide a greater understanding of the pathophysiology of this complex process. We also appraise potential future therapeutic strategies to limit damage in various organs, including the heart, brain, kidneys, and lungs.

The class I histone deacetylase inhibitor romidepsin sensitizes bladder cancer cells to ionizing radiation (IR) and delays tumor growth after IR. Treatment with romidepsin þ IR did not increase the normal tissue toxicity caused by radiation to the surrounding normal bowel incorporated in the radiation field acutely at 3.75 days after radiation or later at 29 weeks. Romidepsin treatment impaired both homologous recombination and nonhomologous end joining DNA repair pathways. Purpose: Muscle-invasive bladder cancer has a 40% to 60% 5-year survival rate with radical treatment by surgical removal of the bladder or radiation therapyebased bladder preservation techniques, including concurrent chemoradiation. Elderly patients cannot tolerate current chemoradiation therapy regimens and often receive only radiation therapy, which is less effective. We urgently need effective chemotherapy agents for use with radiation therapy combinations that are nontoxic to normal tissues and tolerated by elderly patients. Methods and Materials: We have identified histone deacetylase (HDAC) inhibitors as promising agents to study. Pan-HDAC inhibition, using panobinostat, is a good strategy for radiosensitization, but more selective agents may be more useful radiosensitizers in a clinical setting, resulting in fewer systemic side effects. Herein, we study the HDAC class I-selective agent romidepsin, which we predict to have fewer off-target effects than panobinostat while maintaining an effective level of tumor radiosensitization. Results: In vitro effects of romidepsin were assessed by clonogenic assay and showed that romidepsin was effective in the nanomolar range in different bladder cancer cells and radiosensitized these cells. The radiosensitizing effect of romidepsin was confirmed in vivo using superficial xenografts. The drug/irradiation combination treatment resulted in significant tumor growth delay but did not increase the severity of acute (3.75 days) intestinal normal tissue toxicity or late toxicity at 29 weeks.

No abstract

max 150 words) DNA-protein crosslinks (DPCs) are a specific type of DNA lesions where proteins are covalently attached to DNA. Unrepaired DPCs lead to genomic instability, cancer, neurodegeneration and accelerated ageing. DPC proteolysis was recently discovered as a specialised pathway for DPC repair. The DNA-dependent SPRTN protease and 26S proteasome emerged as as two independent proteolytic systems for DPC repair. DPCs are also repaired by homologous recombination (HR), a canonical DNA repair pathway. While studying the role of ubiquitin and SUMO in DPC repair, we identified mutually exclusive signalling mechanisms associated with DPC repair pathway choice. DPC modification by SUMO-1 favours SPRTN proteolysis as the preferred pathway for DPC repair. DPC SUMOylation counteracts DPC ubiquitination, which promotes DNA breaks and the switch toHR. We propose that modification of DPCs by SUMO-1 promotes SPRTN proteolysis, which is essential for DPC removal to prevent DNA replication defects, chromosomal recombination and genomic instability.

Summary DNA end resection converts broken ends of double-stranded DNA (dsDNA) to 3′-single-stranded DNA (3′-ssDNA). The extent of resection regulates DNA double-strand break (DSB) repair pathway choice and thereby genomic stability. Here, we characterize an optimized immunofluorescence (IF) microscopy-based protocol for measuring ssDNA in mammalian cells by labeling genomic DNA with 5-bromo-2′-deoxyuridine (BrdU). BrdU foci can be detected under non-denaturing conditions by anti-BrdU antibody, providing an accurate and reliable readout of DNA end resection in most mammalian cell lines. For complete details on the use and execution of this protocol, please refer to Kilgas et al. (2021).