Mechansim of RING-between-RING ubiquitin signaling in DNA repair

Genomic instability caused by defective repair of DNA double-strand breaks (DSBs) is a major contributor to cancer development. DSBs, among the most lethal forms of DNA damage, can arise from ionizing radiation, a common modality in cancer therapy. The repair of DSBs involves a tightly regulated network of proteins and post-translational modifications that coordinate recruitment and activation of DNA repair factors. Ubiquitin signaling plays a central role in assembling these repair complexes. Among E3 ubiquitin ligases, the RING- between-RING (RBR) family is the most recently identified and remains the least characterized, representing a critical gap in our understanding of DNA damage response (DDR) regulation. This project focuses on the RBR E3 ligase ARIH1, which we have identified as a novel DDR factor essential for DSB repair. Preliminary data show that ARIH1 is rapidly recruited to DNA lesions, facilitates homologous recombination, and is required for cell survival following genotoxic stress. We hypothesize that ARIH1 acts downstream of RNF168 to regulate DSB repair pathway choice by modulating DNA end resection at sites of damaged chromatin. Understanding how ARIH1 coordinates DDR is especially relevant to cancer therapy. Radiation-induced DNA damage is more toxic to cancer cells than normal cells due to underlying repair deficiencies. However, acquired resistance to radiation, often driven by restoration of repair capacity, remains a major clinical challenge. Targeting DDR components, particularly ubiquitin-dependent signaling hubs like ARIH1, offers a promising strategy to overcome such resistance and improve therapeutic response. To achieve our objectives, we will pursue the following specific aims: 1. Determine the molecular regulation of ARIH1 at damaged chromatin: We will define upstream signaling pathways and post-translational modifications (e.g., phosphorylation and ubiquitination) that govern ARIH1 recruitment and activation upon DNA damage. 2. Characterize the biological function of ARIH1 in genome stability maintenance: Using genetic and cell-based assays, we will assess the role of ARIH1 in pathway choice, DNA repair kinetics, and checkpoint activation following genotoxic stress. 3. Elucidate how ARIH1 regulates DNA end resection: We will use tunable dTAG-mediated ARIH1 depletion to dissect its role in promoting end resection and antagonizing resection-suppressive complexes. Additionally, we will explore the therapeutic implications of targeting ARIH1 in the context of radiation and PARP inhibitor resistance. Successful completion of this work will define a novel ubiquitin signaling axis in DSB repair, advancing our understanding of DDR regulation and resistance mechanisms. This may open new avenues for radiosensitization and combination therapies that exploit DNA repair vulnerabilities in cancer. Project Number: 1R01CA303071-01A1 | Fiscal Year: 2026 | NIH Institute/Center: National Cancer Institute (NCI) | Principal Investigator: Justin Leung | Institution: UNIVERSITY OF TEXAS HLTH SCIENCE CENTER, SAN ANTONIO, TX | Award Amount: $482,637 | Activity Code: R01 | Study Section: Molecular Genetics Study Section[MG] View on NIH RePORTER: https://reporter.nih.gov/project-details/11364749