Dynamic control of RNA-DNA hybrid formation orchestrates DNA2 activation at stalled forks by RNAPII and DDX39A.

Stalled replication forks, susceptible to nucleolytic threats, necessitate protective mechanisms involving pivotal factors such as the tumor suppressors BRCA1 and BRCA2. Here, we demonstrate that, upon replication stress, RNA polymerase II (RNAPII) is recruited to stalled forks, actively promoting the transient formation of RNA-DNA hybrids. These hybrids act as safeguards, preventing premature engagement by the DNA2 nuclease and uncontrolled DNA2-mediated degradation of nascent DNA. Furthermore, we provide evidence that DExD box polypeptide 39A (DDX39A), serving as an RNA-DNA resolver, unwinds these structures and facilitates regulated DNA2 access to stalled forks. This orchestrated process enables controlled DNA2-dependent stalled fork processing and restart. Finally, we reveal that loss of DDX39A enhances stalled fork protection in BRCA1/2-deficient cells, consequently conferring chemoresistance. Our results suggest that the dynamic regulation of RNA-DNA hybrid formation at stalled forks by RNAPII and DDX39A precisely governs the timing of DNA2 activation, contributing to stalled fork protection, processing, and restart, ultimately promoting genome stability.