Abstract
SummaryR-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.
Highlights
R-loops consist of an RNA/DNA hybrid and a displaced nontemplate DNA strand
We demonstrate that a top candidate DHX9 helicase prevents R-loop accumulation in vivo and is required for transcriptional termination
We describe a role of DHX9 in maintaining genome stability
Summary
R-loops consist of an RNA/DNA hybrid and a displaced nontemplate DNA strand These structures are thermodynamically stable and can arise during transcription, where they contribute to gene regulation at multiple levels (Ginno et al, 2012; SkourtiStathaki et al, 2011; Yang et al, 2014). They are involved in immunoglobulin class switch recombination, DNA replication, and regulation of DNA and histone modifications (Aguilera and Garcıa-Muse, 2012; Skourti-Stathaki and Proudfoot, 2014). A growing body of evidence connects R-loops to processes that are deregulated in cancer, including DNA repair, replication, and gene expression of tumor-promoting genes (Bhatia et al, 2014; Boque-Sastre et al, 2015; Hatchi et al, 2015; Kotsantis et al, 2016; Schwab et al, 2015; Stork et al, 2016; Yang et al, 2014)
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