Nuclear functions of CFAP20 in transcription and replication [DRIP-seq]

Fine-tuning DNA replication and transcription co-occurrence is vital to avoid collisions between their machineries. This is especially relevant near promoters, where RNAPII initiates transcription and often arrests, forming R-loops. Arrested RNAPII poses a roadblock for DNA replication, which evolutionarily initiates near promoters. The mechanisms that salvage arrested RNAPII during elongation to avoid conflicts with incoming replisomes remain unaddressed. In this study, employing genome-wide and proteomic approaches, we identify and characterize CFAP20 as part of a protective pathway that rescues arrested RNAPII in promoter-proximal regions, diverting it from the path of co-directional replisomes. CFAP20-deficient cells accumulate R-loops specifically near promoters, displaying defects in replication origin firing and fork progression. Co-depletion of the Mediator coactivator complex or removal of RNAPII engaged with R-loops rescues these replication phenotypes. Therefore, we propose that CFAP20 salvages arrested RNAPII under conditions of high Mediator-driven transcription to promote RNAPII elongation, thereby preventing collisions with co-directional replisomes. To explore the role of CFAP20 in R-loop resolution, we performed DRIP-seq experiments in RPE1 cells being either wild type (WT) or CFAP20-KO. NOTE: The records have been updated for data processing steps, contributor lists, replacement/addition of processed data files on May 30, 2025.