UC San Diego

R-loop, a three-stranded RNA/DNA structure, has been linked to induced genome in- stability and regulated gene expression. To enable precision analysis of R-loops in vivo, we develop an RNase-H-based approach; this reveals predominant R-loop formation near gene promoters with strong G/C skew and propensity to form G-quadruplex in non-template DNA, corroborating with all biochemically established properties of R-loops. Transcription perturbation experiments further indicate that R-loop induction correlates to transcriptional pausing. Interestingly, we note that most mapped R-loops are each linked to a nearby free RNA end; by using a ribozyme to co-transcriptionally cleave nascent RNA, we demonstrate that such a free RNA end coupled with a G/C-skewed sequence is necessary and sufficient to induce R-loop. These findings provide a topological solution for RNA invasion into duplex DNA and suggest an order for R-loop initiation and elongation in an opposite direction to that previously proposed.Increasing evidence suggests that R-loops participates in the transcriptional coupled re- pair (TCR) pathway, which all point to the interaction between the TCR signaling factor Cockayne syndrome group B (CSB) and R-loops. However, how CSB sense and bind R-loops remains unclear. We use R-ChIP, a high resolution and high accuracy R-loop profiling method, to reveal the underlying mechanism. Not simply in TCR, CSB is a universal regulator of R-loops during transcription, where its association is frequently detected at pol II pausing loci especially at gene body. Depletion of CSB induces a new type of R-loop associated with the poly T tract on non-template strand. CSB resolves the pol II pausing induced R-loops through pushing pol II forward is further validated by the in vitro transcription assay. Taken together, our results for the first time shows R-loops are induced at poly T tract, uncovering a unifying mechanism of R-loop formation at pol II pausing sites. Failure to resolve such transient R-loops during transcription by CSB, would impact long genes transcription and potentially cause more DNA damages.