Fig. 4: CSB prevents R-loop formation induced by paused RNAPII at T-run.

a R-loop induced by T-run during transcription. Top: a schematic of the transcription reaction. Below: RNaseH1-mediated cleavage of high molecular weight transcripts from the T-run containing template, but not control template. Blue lines indicate RNase H-digested transcripts, while the black dot denotes RNA transcripts resulting from a template with incomplete ligation (ligation truncation). b Prevention of R-loop formation by Rad26. Top: a schematic of the transcription reaction. Below: RNaseH1-mediated cleavage of transcripts arrested at T-runs during RNAPII transcription in the absence of Rad26 (left), in the presence of the WT Rad26 (Middle), or an ATPase-deficient mutant rad26 (Right). c RNAPII backtracking during in vitro elongation. Top: a schematic of the transcription reaction. Below: the TFIIS assay conducted on the T-run containing template during various time points of RNAPII transcription. Experiments in a–c were repeated independently three times with similar results.