RNase H2 degrades toxic RNA:DNA hybrids behind stalled forks to promote replication restart
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA891245 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR21930549.fastq.gz | Fastqsanger.gz | |||
| SRR21930550.fastq.gz | Fastqsanger.gz | |||
| SRR25372896.fastq.gz | Fastqsanger.gz | |||
| SRR25372897.fastq.gz | Fastqsanger.gz | |||
| SRR25372898.fastq.gz | Fastqsanger.gz |
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Project description: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.
2023-10-14 | GSE215896 | GEO
Project description:The accurate processing of stalled forks by the DNA2 nuclease is pivotal for replication fork restart, as excessive degradation poses a threat to genomic stability. However, the regulation of DNA2 activity at stalled forks remains elusive. Here, we demonstrate that, upon replication stress, RNA polymerase II (RNAPII) is recruited to stalled forks, actively promoting the transient formation of RNA-DNA hybrids. Furthermore, we provide evidence that DDX39A, functioning as an RNA-DNA resolver, unwinds these hybrids, allowing DNA2 access to stalled forks. This orchestrated process facilitates controlled DNA2-dependent stalled fork processing and restart. Nevertheless, premature removal of RNA-DNA hybrids at stalled forks leads to DNA2-dependent excessive degradation of nascent DNA. Finally, we reveal that loss of DDX39A enhances the protection of stalled forks in BRCA1/2-deficient cells, consequently conferring chemoresistance within this specific cellular context. Our results suggest that the dynamic regulation of RNA-DNA hybrid formation at stalled forks by RNAPII and DDX39A precisely governs the timing of DNA2 activation, contributing to stalled fork processing and restart, ultimately promoting genome stability.
2024-12-19 | GSE252171 | GEO
Project description:The accurate processing of stalled forks by the DNA2 nuclease is pivotal for replication fork restart, as excessive degradation poses a threat to genomic stability. However, the regulation of DNA2 activity at stalled forks remains elusive. Here, we demonstrate that, upon replication stress, RNA polymerase II (RNAPII) is recruited to stalled forks, actively promoting the transient formation of RNA-DNA hybrids. Furthermore, we provide evidence that DDX39A, functioning as an RNA-DNA resolver, unwinds these hybrids, allowing DNA2 access to stalled forks. This orchestrated process facilitates controlled DNA2-dependent stalled fork processing and restart. Nevertheless, premature removal of RNA-DNA hybrids at stalled forks leads to DNA2-dependent excessive degradation of nascent DNA. Finally, we reveal that loss of DDX39A enhances the protection of stalled forks in BRCA1/2-deficient cells, consequently conferring chemoresistance within this specific cellular context. Our results suggest that the dynamic regulation of RNA-DNA hybrid formation at stalled forks by RNAPII and DDX39A precisely governs the timing of DNA2 activation, contributing to stalled fork processing and restart, ultimately promoting genome stability.
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Project description: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.
| S-EPMC10690446 | biostudies-literature
Project description: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 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.
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Project description:The Bloom syndrome helicase BLM interacts with topoisomerase IIIa (TOP3A), RMI1 and RMI2 to form the BTR complex, which dissolves double Holliday junctions to produce non-crossover products during homologous recombination (HR). BLM also promotes DNA-end resection and stalled replication fork restart. However, it is still unclear how the activities of the BTR complex are regulated in cells. Here, we identify multiple highly conserved short linear peptide motifs within the BTR complex that interact cooperatively with RPA. Furthermore, we demonstrate that RPA-binding is essential for stable recruitment of BLM to DNA damage sites and for stalled fork restart, but not for its roles in HR. Disruption of RPA-binding thus constitutes a novel separation-of-function mutation of the BTR complex, and suggest a model in which BTR contains the intrinsic ability to measure levels of RPA-ssDNA at replication forks, which controls its recruitment and activation in response to replication stress.
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Project description:SAMHD1 degrades nascent DNA at stalled Forks to promote replication restart
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Project description:The progression of replication forks (RFs) can be challenged by obstacles of endogenous or exogenous origin. Stalled forks need to be readily stabilized and restarted in order to prevent genomic instability. Replication fork restart requires the recruitment of multiple enzymes and involves different DNA transactions. The MRX (Mre11-Rad50-Xrs2) complex plays a central role in this process. It has been implicated in the nucleolytic degradation of nascent DNA and in the loading of cohesin at stalled forks. However, little is known on how these functions are regulated. Here we show that MRX structural features are predominant on the nuclease activity of Mre11 for DNA resection at stalled replication forks. This results raise the question of the mechanisms by which MRX promotes nascent strand resection. At DNA double strand breaks (DSB) MRX promotes the binding of the chromatin remodeler RSC, from which the activity is required for 5’ end resection. Interestingly, MRX mutants exhibit increased nucleosome occupancy at stalled replication forks. This result suggest that a dynamic chromatin structure promoted by MRX could be required for the processing of stalled replication forks. Strinkingly the absence of two histones modifiers Gcn5 and Set1 recapitulates the phenotype of MRX mutants both on chromatin structure and nascent strand resection at stalled forks even though they are dispensable for its recruitment. Since nucleosomes also represent obstacles for the loading of SMC complexes on DNA, it is coherent that we observed that cohesin is no longer recruited at stalled forks in gcn5 and set1, as in MRX mutants. Together our data suggest that the regulation of chromatin structure at stalled replication forks is essential for their processing and to promote genome stability.
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Project description:Oncoproteins of the MYC family drive the development of numerous human tumors. In unperturbed cells, MYC proteins bind to virtually all active promoters and control transcription by RNA Polymerase II (RNAPII). MYC proteins can also coordinate transcription with DNA replication and promote the repair of transcription-associated DNA damage, but how they exert these mechanistically diverse functions is unknown. Here we show that MYC dissociates from many of its binding sites in active promoters and forms multimeric, often sphere-like structures in response to perturbation of transcription elongation, mRNA splicing or inhibition of the proteasome. Multimerization is accompanied by a global change in the MYC interactome towards proteins involved in transcription termination and RNA processing. MYC multimers accumulate on chromatin immediately adjacent to stalled replication forks and surround FANCD2, ATR and BRCA1 proteins, which are located at stalled forks. MYC multimerization is triggered in a HUWE1 and ubiquitylation-dependent manner. At active promoters, MYC multimers block antisense transcription and stabilize FANCD2 association with chromatin. This limits DNA double-strand break formation during S-phase and allows rapid recovery from replication stress, suggesting that multimerization of MYC enables tumor cells to proliferate under stressful conditions
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