CFAP20 salvages arrested RNAPII from the path of co-directional replisomes

Fine-tuning DNA replication and transcription is crucial to prevent collisions between their machineries, especially at promoters where RNA polymerase II (RNAPII) initiates transcription and forms R-loops. Arrested RNAPII can obstruct DNA replication, which often starts near promoters. The mechanisms that salvage arrested RNAPII during elongation to avoid conflicts with incoming replisomes are unknown. Here, we identify CFAP20 as a key player in rescuing arrested RNAPII in promoter-proximal regions, preventing conflicts with co-directional replisomes. CFAP20-deficient cells accumulate R-loops near promoters and exhibit defects in replication origin firing and fork progression. Co-depleting the Mediator coactivator complex or removing RNAPII engaged with R-loops rescues these defects. Thus, CFAP20 salvages arrested RNAPII under conditions of high Mediator-driven transcription to promote RNAPII elongation, thereby preventing collisions with co-directional replisomes. Overall design: Wild-type and CFAP20-KO RPE-1 cells were subjected to scEdU-seq as previously described by van den Berg 2024