RNA/DNA Hybrid Interactome Identifies DXH9 as a Molecular Player in Transcriptional Termination and R-Loop-Associated DNA Damage

R-loops comprise an RNA/DNA hybrid and displaced single-stranded DNA. They play important biological roles and are implicated in pathology. Even so, proteins recognizing these structures are largely undefined. Using affinity purification with the S9.6 antibody coupled to mass spectrometry, we defined the RNA/DNA hybrid interactome in HeLa cells. This consists of known R-loop-associated factors SRSF1, FACT, and Top1, and yet uncharacterized interactors, including helicases, RNA processing, DNA repair, and chromatin factors. We validate specific examples of these interactors and characterize their involvement in R-loop biology. A top candidate DHX9 helicase promotes R-loop suppression and transcriptional termination. DHX9 interacts with PARP1, and both proteins prevent R-loop-associated DNA damage. DHX9 and other interactome helicases are overexpressed in cancer, linking R-loop-mediated DNA damage and disease. Our RNA/DNA hybrid interactome provides a powerful resource to study R-loop biology in health and disease. [Display omitted] •Mass spectrometry identifies the RNA/DNA hybrid interactome in human cells•Top RNA/DNA interactome candidate DHX9 promotes R-loop suppression•DHX9 regulates transcriptional termination•DHX9 interacts with PARP1 and prevents R-loop-associated DNA damage Cristini et al. use affinity purification and mass spectrometry to define the RNA/DNA interactome in human cells and validate it by revealing the role of DHX9 in transcriptional termination and R-loop-associated DNA damage.