GEOGenomicsSaccharomyces cerevisiaeGenome binding/occupancy profiling by high throughput sequencinghttps://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE328746GEOGSEfalseThe balance between R-loop levels and RNAPII occupancy dictates Transcription-Replication Conflict occurrenceMaintaining 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 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. Our findings reveal a dual nature of R-loops: while they can be obstacles, they 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.2026/04/29GSE328746GSM9688509GSM9688498GSM9688511GSM9688500GSM9688510GSM9688499GSM9688502GSM9688512GSM9688501GSM9688504GSM9688503GSM9688506GSM9688505GSM9688508GSM968850734739328746Saccharomyces cerevisiae