DNA Supercoiling is Required for Efficient Formation of R-loops

R-loops are branched nucleic acid structures formed during transcription. The nascent RNA reanneals with the template DNA and displaces the non-template DNA as single stranded. The displaced DNA is now more vulnerable to damage and thus R-loops contribute to genomic instability. From this perspective, R-loops control gene expression and are linked to several pathologies such as cancer and neurological disorders. Despite the importance of R-loops and their widespread on the human genome, it is unknown how chromatin structure and supercoiling impacts R-loop formation. DNA supercoiling refers to the twist in DNA helicity that impacts DNA conformation and torsion. DNA supercoiling is tightly balanced in the cell and regulates transcription, replication, protein recognition and can also contribute to DNA strain and instability yet its role in R-loop biogenesis is poorly understood. To address this gap, we used in vitro transcription (IVT) to reconstitute R-loops on plasmid DNA with distinct topologies containing the mouse antisense Igf2r RNA (Airn) gene, a hotspot for R-loop formation. We used restriction enzyme digestion before and after IVT to relax plasmid DNA (remove supercoiling) and assess the contribution of supercoiling on R-loop formation and stability. R-loops were visualized by agarose gel electrophoresis and revealed that linearization of plasmid DNA reduces their formation. Moreover, linearization of a plasmid containing an R-loop leads to a dramatic reduction in the R-loop species suggesting that supercoiling is required for optimal R-loop stability. Collectively, these observations indicate the requirement for supercoiling for efficient R-loop formation and that linear oligonucleotide fragments are a poor model for structural and mechanistic studies of R-loops.