Safeguarding replication fork stability in transcriptionally active regions is crucial for precise DNA replication and mutation prevention. Here, we discover the pervasive existence of replication fork-associated RNA-DNA hybrids (RF-RDs) in transcriptionally active regions of human cells. These hybrids function as protective barriers, preventing DNA2-mediated nascent DNA degradation and replication fork collapse under replication stress. We also identify DDX39A as a RAD51-associated protein that binds to stalled forks and resolves RF-RDs, facilitating proper DNA2-mediated DNA resection and replication fork restart. Excessive dissolution of RF-RDs causes replication fork collapse and genomic instability, while insufficient dissolution of RF-RDs under replication stress increases fork stability, resulting in chemoresistance that can be reversed by eliminating RF-RDs. In summary, we elucidated the prevalence of RF-RDs at replication forks within transcriptionally active regions, revealed their pivotal role in safeguarding replication fork stability, and proposed that targeting RF-RDs holds promise for augmenting chemotherapeutic efficacy.
Keywords: DDX39A; DNA damage; DNA2; Rad51; chemoresistance; fork restart; genomic stability; replication stress.
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