Dynamic Control of RNA-DNA Hybrid Formation Orchestrates DNA2 Activation at Stalled Forks by RNAPII and DDX39A

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 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 the protection of stalled forks in BRCA1/2-deficient cells, consequently conferring chemoresistance within this specific cellular context.