Kub5‐Hera Deficiency Promotes R‐Loop‐Induced Genomic Instability and Carcinogenesis Following Whole‐Body Exposure to Ionizing Radiation

Abstract

Formation of persistent R‐loops (a structure containing an RNA:DNA hybrid and a displaced single‐stranded DNA) during transcription are now emerging as a direct threat to genomic stability with links to cancer and various neurodegenerative, autoimmune, and developmental disorders. However, the molecular mechanisms by which R‐loops are resolved or aberrantly formed to induce DNA damage and genomic instability remains unclear. Aberrant accumulation of these nucleic acid structures through compromised transcription and exposure to genotoxic agents (e.g., ionizing radiation, IR) imposes a roadblock to ongoing replication and transcription causing genomic instability by generating double‐stranded DNA breaks (DSBs). Kub5‐Hera (Ku70‐binding protein 5‐Hera, K‐H; also known as RPRD1B, Regulation of nuclear pre‐mRNA‐domain‐containing protein 1B) is a novel factor discovered in our lab that is involved in transcription termination and DNA repair. We found that loss of K‐H increases R‐loop‐induced DSBs that activates the error‐prone alternative non‐homologous end‐joining (alt‐EJ or back‐up NHEJ) due to defects in classical NHEJ (c‐NHEJ) and homologous recombination (HR). Using our transgenic mouse model, we discovered that a complete knockout of K‐H is embryonic lethal suggesting a critical role for this protein in cellular development and proliferation. Interestingly, deletion of one K‐H allele in mice caused rapid development of cancer‐related deaths over time after whole‐body exposure to IR in a dose‐dependent manner compared to their wild‐type littermates. Using targeted gene silencing, western blot, proximity ligation and immunofluorescent assays in our cell line models and mouse tissues, we showed that depletion of K‐H increases the formation of R‐loop‐induced double‐stranded breaks (DSBs) and PARP1 activity due to a compromised homologous recombination (HR). Loss of K‐H in cells increases the error‐prone activity of PARP1 in alt‐EJ to possibly repair harmful R‐loop‐induced single‐stranded breaks (SSBs) and DSBs due to HR and c‐NHEJ deficiency, which could provide the mechanism of carcinogenesis in mice after exposure to IR. Overall, our studies offer new mechanistic insights into carcinogenesis caused by K‐H genetic alterations and persistent R‐loops, as well as identify novel strategies to leverage Kub5‐Hera expression or the formation of lethal R‐loop‐induced‐DSBs for innovative therapeutic approaches.