Protein-mediated stabilization and nicking of the non-template DNA strand dramatically affect R-loop formation in vitro

R-loops are an important class of non-B DNA structures that form co-transcriptionally. Using invitro transcription and unbiased quantitative sequencing readouts, we show that the addition ofsingle-strand DNA binding proteins co-transcriptionally can drive a 3- to 5-fold increase of R-loop frequency without significant changes to R-loop distribution. We propose that this is causedby stabilizing and preventing the collapse of short nascent R-loops. This suggests that R-loopformation is highly dynamic and highlights single strand binding proteins as players in cellular R-loop regulation. We further show that non-template strand DNA nicks are powerful initiators ofR-loop formation, increasing R-loop frequencies by up to two orders of magnitude. Atomic forcemicroscopy (AFM) revealed that the non-template strand in nick-initiated structures is oftenflayed away from the RNA:DNA hybrid and engaged in self-pairing, creating unique forked R-loop features. DNA nicks, one of the most frequent DNA lesions in cells, are therefore potentialhotspots for opportunistic R-loop initiation and may cause the formation of a novel class of R-loops. Overall, this work highlights the importance of the displaced single-strand on R-loopinitiation and dynamics.