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

Abstract

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.

RTF2 controls replication repriming and ribonucleotide excision at the replisome

“…Its best-understood function is in the ribonucleotide excision repair (RER) pathway, where RNase H2 removes single DNA-embedded ribonucleotides from the genome, reducing replication stress 8 , 13 . It has also been proposed to degrade toxic RNA: DNA hybrids behind stalled forks to promote replication restart 17 . Notably, the S. cerevisiae homolog of RNase H2 can degrade RNA primers and may contribute to Okazaki fragment maturation during DNA replication 18 – 22 .…”