Genome Maintenance by HELZ, a Novel R-loop Resolvase - PROJECT SUMMARY R-loops, RNA-DNA hybrid containing structures, are critical for cellular processes, including DNA double-strand break (DSB) repair. Maintaining R-loop homeostasis—balancing their formation and resolution—is vital for genomic stability, and its dysregulation contributes to various diseases, including breast cancer. While the tumor suppressor BRCA2 and its essential partner DSS1 have recently been shown to regulate R-loop homeostasis, the precise mechanisms underlying this regulation, particularly during DSB repair, remain unclear. Importantly, R-loops are often upregulated in breast cancer, underscoring the need for a deeper understanding of their regulation to inform new therapeutic strategies. Using complementary genomic and proteomic approaches, we identified HELZ, a novel BRCA2-interacting RNA helicase, as a key regulator of R-loop resolution. Relatively little is known about HELZ, a class I superfamily RNA helicase, with only two published studies specifically focused on its function. For the first time, we have developed a robust system to express and purify full-length HELZ using insect cells. Our preliminary findings reveal that HELZ has high specificity for binding single-stranded RNA (ssRNA) and R-loops and demonstrates RNA-DNA hybrid unwinding activity with a preference for 5' ssRNA overhangs. Furthermore, HELZ directly interacts with the BRCA2-DSS1 complex, enhances R-loop resolution both globally and at DSBs, and promotes DSB repair by homologous recombination (HR) to maintain genomic stability in a manner dependent on R-loop resolution. Notably, HELZ is highly expressed in breast cancer, particularly triple-negative breast cancer (TNBC), where it contributes to resistance against DSB-inducing agents, including sacituzumab govitecan (SG), a novel antibody-drug conjugate (ADC). We propose that HELZ plays a critical role in preserving genome integrity by resolving R-loops in collaboration with BRCA2, thereby facilitating DSB repair and governing the resistance of breast cancer to SG and other DSB-inducing therapies. Insights into this mechanism may open new avenues for optimizing breast cancer treatment. This project seeks to define the molecular role of HELZ in R-loop resolution and its impact on treatment resistance in TNBC. In this project, we propose three specific aims: (1) determine the mechanisms by which HELZ resolves R-loops to facilitate DSB repair, (2) dissect how HELZ is regulated by BRCA2, DSS1, and other partners to promote R-loop resolution, and (3) evaluate the significance of HELZ and R-loops in governing resistance to SG and other DSB-inducing agents in TNBC. These studies integrate genetic, biochemical, cell biological, biophysical, and in vivo approaches to comprehensively investigate HELZ's function. This research is expected to advance our understanding of R-loop resolution in genome maintenance, elucidate how the BRCA2-HELZ interaction contributes to genomic stability, and identify HELZ-mediated R-loop resolution as a potential biomarker of treatment resistance as well as provide proof-of-concept for targeting HELZ as a therapeutic strategy to sensitize TNBC patients to DSB-inducing agents, addressing a critical unmet need in breast cancer treatment.
