Institut de Génétique Humaine, Université de Montpellier, CNRS, Equipe labélisée Ligue contre le Cancer, Montpellier, France.
Université Paris Cité, CNRS, Institut Jacques Monod, Paris, France.
EMBO J. 2023 Dec 1;42(23):e113104. doi: 10.15252/embj.2022113104. Epub 2023 Oct 19.
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 Saccharomyces 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 their chromosomes normally under unchallenged growth conditions, their replication was impaired when exposed to hydroxyurea (HU) or methyl methanesulfonate (MMS). Treated cells exhibited increased levels of RNA:DNA hybrids at stalled forks and were unable to generate RPA-coated single-stranded (ssDNA), an important postreplicative intermediate in resuming replication. Similar impairments in nascent DNA resection and ssDNA formation at HU-arrested forks were observed in human cells lacking RNase H2. However, fork resection was fully restored by addition of triptolide, an inhibitor of transcription that induces RNA polymerase degradation. 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 环是复制压力的主要来源，但这些结构阻碍叉进展的机制仍不清楚。为了解决这个问题，我们在缺乏 RNase H1 和 H2 的酿酒酵母细胞中监测了叉的进展、停滞和重新启动，这两种酶负责降解 RNA:DNA 杂交体。我们发现，虽然 RNase H 缺陷细胞在不受挑战的生长条件下可以正常复制它们的染色体，但当它们暴露于羟基脲 (HU) 或甲磺酸甲酯 (MMS) 时，它们的复制受到了损害。处理过的细胞在停滞的叉处表现出更高水平的 RNA:DNA 杂交体，并且无法产生 RPA 包裹的单链 (ssDNA)，这是在复制恢复过程中的一个重要的复制后中间体。在缺乏 RNase H2 的人细胞中，在 HU 停滞的叉处也观察到新生 DNA 切除和 ssDNA 形成的类似损伤。然而，添加三萜内酯（一种诱导 RNA 聚合酶降解的转录抑制剂）可以完全恢复叉切除。总之，这些数据表明，RNA:DNA 杂交体不仅作为复制叉的障碍，而且如果不能被 RNase H 及时降解，还会干扰复制后叉修复机制。
