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. To investigate the profil of origin firing in rnh1delta rnh201delta cells, we measured DNA Copy Number across the genome of S. cerevisiae wild-type strains and rnh1delta rnh201delta cells that were synchronised in G1 and released into S-phase + 0.2M hydroxyurea.

R环（R-loops）是复制应激的主要来源之一，但其阻碍复制叉行进的具体机制尚未阐明。为解答这一科学问题，我们对缺失核糖核酸酶H1（RNase H1）与H2（RNase H2）——这两种负责降解RNA:DNA杂交体的酶——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。研究发现，在无外界胁迫的培养条件下，核糖核酸酶H缺陷型细胞的染色体复制可正常进行，但当暴露于羟基脲（HU）或甲磺酸甲酯（MMS）时，其复制过程会出现显著受损。此类细胞在停滞的复制叉处的RNA:DNA杂交体水平显著升高，且无法完成覆有复制蛋白A（RPA）的单链DNA（ssDNA）形成这一重启复制所需的关键复制后步骤。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切除过程存在类似缺陷。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，复制叉的切除过程可完全恢复。综合上述实验数据，我们认为RNA:DNA杂交体不仅会作为复制叉的行进障碍，若未被核糖核酸酶H及时降解，还会干扰复制后复制叉的修复机制。为探究rnh1Δ rnh201Δ突变细胞的复制起始位点激活模式，我们对同步化于G1期并被释放进入添加了0.2M羟基脲的S期的酿酒酵母野生型菌株与rnh1Δ rnh201Δ突变株的全基因组DNA拷贝数进行了检测。