The balance between R-loop levels and RNAPII occupancy dictates Transcription-Replication Conflict occurrence

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 to monitor a single R-loop prone locus in real time, we demonstrate that wild-type replisomes traverse R-loops with high efficiency.

Conversely, depleting R-loop processing factors, such as Rnase H enzymes or Mph1 and Sen1 helicases triggers replication fork stalling, particularly in head-on orientation. Paradoxically, 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.