Replication Fork-Associated RNA-DNA Hybrids Safeguard Genomic Stability in Transcriptionally Active Regions [EdU_seq]. Replication Fork-Associated RNA-DNA Hybrids Safeguard Genomic Stability in Transcriptionally Active Regions [EdU_seq]

Safeguarding replication fork stability in transcriptionally active regions, which require high DNA replication accuracy, is crucial for precise DNA replication and prevention of mutations. However, how cells ensure the stability of replication forks in these regions remains a critical challenge. Here, we discovered the pervasive existence of replication forks-associated RNA-DNA hybrids (RF-RDs) within transcriptionally active regions, where they act as a protective barrier against DNA2-mediated nascent DNA degradation and prevent replication fork collapse upon replication stress. Subsequently, the RNA helicase DDX39A dismantles 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. Overall design: EdU-seq to analyze the genomic location of replication fork with or without fork stress treatment in DDX39A knockdown or wild-type cells.

在转录活跃区域维持复制叉（replication fork）稳定性至关重要，这类区域需要高精度的DNA复制，这对精准的DNA复制以及预防突变均具有关键意义。然而，细胞如何保障这类区域内复制叉的稳定性，仍是一项亟待解决的重大挑战。本研究发现，转录活跃区域内普遍存在与复制叉相关的RNA-DNA杂交体（RNA-DNA hybrids, RF-RDs），它们可作为保护性屏障抵御DNA2介导的新生DNA降解，并在复制应激（replication stress）状态下阻止复制叉崩解。随后，RNA解旋酶DDX39A可拆解此类RF-RDs，促进DNA2介导的正常DNA切除与复制叉重启。若RF-RDs被过度拆解，会引发复制叉崩解与基因组不稳定（genomic instability）；而在复制应激状态下RF-RDs拆解不足则会增强叉体稳定性，最终导致化疗耐药性，且该耐药性可通过消除RF-RDs得以逆转。综上，本研究阐明了转录活跃区域内复制叉处RF-RDs的普遍存在性，揭示了其在维持复制叉稳定性中的核心作用，并提出靶向RF-RDs有望增强化疗疗效。整体实验设计：通过EdU-seq技术，分析DDX39A敲低细胞与野生型（wild-type）细胞在有无复制应激处理条件下的复制叉基因组定位情况。