Abstract
Genome stability requires accurate repair of DNA double-strand breaks (DSBs), particularly those arising in actively transcribed chromatin, where transcription-coupled homologous recombination (TC-HR) is favored. R-loops, three-stranded RNA-DNA hybrid structures formed during transcription, have emerged as important intermediates in this process. FANCA is the most frequently mutated Fanconi anemia gene and a core component of the FA pathway, yet its role in R-loop metabolism during TC-HR remains poorly understood. This dissertation identifies FANCA as both a promoter of R-loop formation at the early stage of TC-HR and a factor required for subsequent R-loop resolution. Using purified proteins and topologically constrained DNA substrates, FANCA was shown to possess intrinsic RNA-DNA annealing and R-loop-forming activities on intact duplex DNA. FANCA also promotes D-loop formation and RAD51-dependent strand invasion, which are further enhanced by nascent RNA. In cells, FANCA localizes to ROS-induced DNA damage sites in a transcription-dependent manner and regulates dynamic R-loop formation and resolution in the DART system. Notably, the R-loop formation ability of FANCA is independent of FANCD2. Rescue experiments further show that wild-type FANCA restores TC-HR efficiency, whereas the L817P mutant, which retains cellular localization but lacks R-loop-forming activity in vitro, fails to rescue. FANCA depletion does not impair recruitment of RAD51, RAD52, or BRCA1, suggesting that FANCA promotes TC-HR by constructing the R-loop nucleic acid intermediate rather than by recruiting repair factors. Loss of FANCA sensitizes cancer cells to camptothecin and PARP inhibitors. Together, these findings establish FANCA as a regulator of the R-loop lifecycle during TC-HR and identify FANCA-dependent TC-HR as a potential therapeutic vulnerability in cancer.