R‐loop formation during S phase is restricted by PrimPol‐mediated repriming

During DNA replication, conflicts with ongoing transcription are frequent and require careful management to avoid genetic instability. R‐loops, three‐stranded nucleic acid structures comprising a DNA:RNA hybrid and displaced single‐stranded DNA, are important drivers of damage arising from such conflicts. How R‐loops stall replication and the mechanisms that restrain their formation during S phase are incompletely understood. Here, we show in vivo how R‐loop formation drives a short purine‐rich repeat, (GAA)(10), to become a replication impediment that engages the repriming activity of the primase‐polymerase PrimPol. Further, the absence of PrimPol leads to significantly increased R‐loop formation around this repeat during S phase. We extend this observation by showing that PrimPol suppresses R‐loop formation in genes harbouring secondary structure‐forming sequences, exemplified by G quadruplex and H‐DNA motifs, across the genome in both avian and human cells. Thus, R‐loops promote the creation of replication blocks at susceptible structure‐forming sequences, while PrimPol‐dependent repriming limits the extent of unscheduled R‐loop formation at these sequences, mitigating their impact on replication.