RNase H2 degrades toxic RNA:DNA hybrids behind stalled forks to promote replication restart

R-loops represent a major source of replication stress but the mechanism by which these structures impede fork progression remains unclear. To address this question, we monitored fork progression, arrest and restart in S. cerevisiae cells lacking RNase H1 and H2, two enzymes responsible for degrading RNA:DNA hybrids. We found that while RNase H-deficient cells could replicate normally their chromosomes under unchallenged growth conditions, their replication was impaired when exposed to hydroxyurea (HU) or methyl methanesulfonate (MMS). Indeed, these cells exhibited increased levels of RNA:DNA hybrids at stalled forks and were unable to generate RPA-coated single-stranded (ssDNA), an important postreplicative step in resuming replication. Similar impairments in nascent DNA resection at HU-arrested forks were observed in human cells lacking RNase H2. However, the addition of triptolide, an inhibitor of transcription that induces RNA polymerase degradation, fully restored fork resection. Taken together, these data indicate that RNA:DNA hybrids not only act as barriers to replication forks, but also interfere with postreplicative fork repair mechanisms if not promptly degraded by RNase H.R环（R-loops）是引发复制应激的主要诱因之一，但其阻碍复制叉行进的具体分子机制尚未阐明。为解答这一科学问题，我们对缺失核糖核酸酶H1和H2（RNase H1 and H2）的酿酒酵母（S. cerevisiae）细胞的复制叉行进、停滞及重启过程进行了监测——这两种酶的生理功能为降解RNA-DNA杂交链（RNA:DNA hybrids）。实验结果显示，在无外界胁迫的常规培养条件下，核糖核酸酶缺陷型细胞的染色体复制可正常进行；但当细胞暴露于羟基脲（hydroxyurea, HU）或甲基甲烷磺酸酯（methyl methanesulfonate, MMS）时，其复制过程会出现显著损伤。具体表现为：此类细胞在停滞的复制叉处积累了更高水平的RNA-DNA杂交链，且无法完成复制蛋白A包被的单链DNA（ssDNA）的生成——这是重启复制过程中关键的复制后步骤。在缺失核糖核酸酶H2的人类细胞中，同样观察到羟基脲诱导的停滞复制叉处新生DNA链切除功能受损的现象。而加入雷公藤内酯（triptolide，一种可诱导RNA聚合酶降解的转录抑制剂）后，复制叉的切除功能得到了完全恢复。综合上述实验数据，我们得出结论：RNA-DNA杂交链不仅会作为物理屏障阻碍复制叉行进，若未被核糖核酸酶H及时降解，还会干扰复制后叉修复机制的正常运行。