Abstract R-loops, transcription-induced three-stranded nucleic acid structures, play essential roles in gene regulation but jeopardize genomic integrity when unresolved. BRCA2, a key tumor suppressor required for homologous recombination (HR) repair of DNA double-strand breaks (DSBs), also maintains R-loop homeostasis, though its mechanisms remain incompletely understood. To identify BRCA2-interacting factors involved in R-loop resolution during DNA repair, we employed biotin-based proximity labeling and mass spectrometry (BioID-MS), uncovering HELZ—a previously uncharacterized RNA-specific helicase—as a direct BRCA2 binding partner. BRCA2 enhances HELZ’s catalytic activity in vitro and promotes its recruitment to transcription-associated R-loops in vivo, particularly at DSB sites. Functionally, HELZ resolves co-transcriptional R-loops, facilitates DNA end resection, and promotes efficient HR. HELZ depletion results in R-loop accumulation, impaired HR capacity, and heightened sensitivity to genotoxic stress. Notably, HELZ activity is context-dependent and proves indispensable in hormone-driven malignancies such as estrogen receptor-positive (ER+) breast cancers, which exhibit transcriptional dysregulation and elevated R-loop burden. These findings establish HELZ as a critical mediator of BRCA2-dependent genome integrity in R-loop-rich chromatin environments. HELZ expression or function may serve as a biomarker for therapeutic response, and HELZ helicase activity offers a promising target to restore HR proficiency in transcriptionally dysregulated breast tumors. By integrating proximity proteomics with functional assays, our study identifies HELZ as a novel and therapeutically actionable regulator of R-loop resolution and BRCA2-mediated repair in breast cancer. Citation Format: W. Li, B. Wu, B. Gao, E. M. Irvin:, A. Arijit Ghosh, L. Eliaz, Y. Huang, Y. Kwon, C. M. Stiefel, T. T. Nguyen, D. Zhao, H. J. Suarez, T. Ni, S. Alejo, O. Fitzgerald, X. Song, E. V. Wasmuth, S. Zheng, J. Leung, X. Xue, H. Wang, J. Ji, L. Lan, W. Zhao. Helz-mediated r-loop resolution orchestrates brca2-dependent dna repair in hormone-driven breast cancers abstract. In: Proceedings of the San Antonio Breast Cancer Symposium 2025; 2025 Dec 9-12; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2026;32(4 Suppl):Abstract nr PS2-12-29.
Li et al. (Tue,) studied this question.