Replisome progression regulates R-loop mediated transcriptional repression

ABSTRACT Maintaining cellular proliferation necessitates the synchronized activity of diverse molecular machineries operating in parallel on the genome. Wide-spread transcription and R-loop formation can interfere with genome duplication, causing transcription-replication conflicts (TRCs). Here, we use live-cell imaging for simultaneous monitoring of replication fork progression and transcription dynamics of an R-loop prone gene. While robust replisome progression through R-loops is observed in wild-type cells, it is impaired in RnaseH, Mph1 and Sen1 mutants. Intriguingly, we find that R-loop formation inhibits gene transcription, but this inhibition is reversed by replisome passage, demonstrating the dynamic crosstalk between replication, transcription and R-loops in a single cell cycle. Unexpectedly, we find that R-loops also have beneficial roles in reducing the density of RNAPII molecules on the gene, thereby preventing fork stalling upon high RNAPII occupancy. These findings illuminate that regulating R-loops and RNAPII density together is critical for preventing harmful TRCs.