Binding of FANCI-FANCD2 Complex to RNA and R-Loops Stimulates Robust FANCD2 Monoubiquitination

Summary Fanconi anemia (FA) is characterized by developmental abnormalities, bone marrow failure, and cancer predisposition. FA cells are hypersensitive to DNA replicative stress and accumulate co-transcriptional R-loops. Here, we use the Damage At RNA Transcription assay to reveal colocalization of FANCD2 with R-loops in a highly transcribed genomic locus upon DNA damage. We further demonstrate that highly purified human FANCI-FANCD2 (ID2) complex binds synthetic single-stranded RNA (ssRNA) and R-loop substrates with high affinity, preferring guanine-rich sequences. Importantly, we elucidate that human ID2 binds an R-loop structure via recognition of the displaced ssDNA and ssRNA but not the RNA:DNA hybrids. Finally, a series of RNA and R-loop substrates are found to strongly stimulate ID2 monoubiquitination, with activity corresponding to their binding affinity. In summary, our results support a mechanism whereby the ID2 complex suppresses the formation of pathogenic R-loops by binding ssRNA and ssDNA species, thereby activating the FA pathway.