Abstract 7118: FANCA promoteshomologous recombinationby facilitating DR-Loop formation

Abstract Homologous recombination (HR), a fundamental DNA double-strand break (DSB) repair pathway, is a universal mechanism employed by cells to accurately mend breaks in both strands of the DNA helix, ensuring genomic stability. Displacement loops, commonly referred to as D-loops, play a crucial role as intermediate structures in HR. Furthermore, recent studies indicate that the unique DNA: RNA three-stranded structure, known as R-loops, may serve as a starting point for homologous pairing and contribute significantly to enhancing HR efficiency by promoting D-loop formation. Besides, the formation of R-loops might assist in recruiting DNA repair proteins, especially those related to HR, thereby accelerating the repair of damaged sites.In this study, we find that highly purified human FANCA protein anneals synthetic single-stranded RNA (ssRNA) and ssDNA species to R-loops and binds R-loop substrates with high affinity, preferring guanine-rich sequences in vitro. In comparison, FANCA exhibits both a weaker capacity to form D-loops and a lower affinity for binding to D-loops in vitro. We then use the DART (damage at RNA transcription site) system to reveal the colocalization of FANCA with R-loops in a highly transcribed genomic locus upon DNA damage in cells. Next, we further demonstrate FANCA's participation in forming R-loops in the initial DSB repair stage and assisting in removing R-loops later. Additionally, we uncover FANCA's ability to aid in the generation of R-loops at specific DNA DSB sites within transcriptionally active regions in both Tet-DR-GFP U2OS cells and DIvA (DSB inducible via AsiSI) cells. Importantly, the R-loop formation ability of FANCA is independent of the Fanconi Anemia (FA) pathway. In conclusion, our findings indicate that FANCA, independent of the FA pathway, can enhance HR in non-transcriptionally active DNA DSB sites through D-loop formation. Furthermore, FANCA significantly elevates transcription-Coupled Homologous Recombination (TC-HR) efficiency by catalyzing the formation of DNA: RNA hybrids, generating temporary R-loops at DNA damage sites and thereby contributing to genomic stability maintenance. Citation Format: Fang Li, Haibo Yang, Boya Gao, Liang Luo, Alyan Zafar, Fenghua Yuan, Li Lan, Yanbin Zhang, Liang Luo, Alyan Zafar, Fenghua Yuan, Yanbin Zhang. FANCA promoteshomologous recombinationby facilitating DR-Loop formation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7118.