The balance between R-loops and RNAPII occupancy determines the outcome of transcription-replication conflicts.

Maintaining genome integrity requires the precise coordination of replication and transcription. While R-loops are known to cause transcription-replication conflicts (TRCs), the precise nature of the barrier, whether it is the RNA:DNA hybrid itself, the stalled RNA Polymerase II (RNAPII), or a combination of both, remains elusive. Using live-cell imaging in yeast to monitor a single R-loop prone locus in real time, we demonstrate that wild-type replisomes traverse R-loop enriched transcription units with high efficiency. We find that while R-loops initially repress transcription, replisome passage prompts a robust recovery of transcription activity. Indeed, we discover that R-loops serve a critical regulatory function by suppressing excessive RNAPII occupancy. In an RNAPII mutant with high DNA affinity, the enzymatic removal of R-loops leads to a pile-up of RNAPII molecules, creating a barrier to replication, more severe than the R-loops themselves. Thus, our findings reveal a dual nature of R-loops: while they can be obstacles and repress transcription, they can also serve as a physiological buffer that prevents the formation of high-density RNAPII 'trains'. These findings establish that the precise regulation of both R-loop levels and RNAPII density is critical for preventing harmful TRCs and maintaining genome stability.