STK19 accelerates the clearance of lesion-stalled RNAPII to facilitate transcription-coupled DNA repair [BrU-seq]
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA1105856 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR28827938_1.fastq.gz | Fastqsanger.gz | |||
| SRR28827938_2.fastq.gz | Fastqsanger.gz | |||
| SRR28827939_1.fastq.gz | Fastqsanger.gz | |||
| SRR28827939_2.fastq.gz | Fastqsanger.gz | |||
| SRR28827940_1.fastq.gz | Fastqsanger.gz |
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Project description:Transcription-coupled DNA repair (TCR) removes bulky DNA lesions impeding RNA polymerase II (RNAPII) transcription. Recent studies have outlined the stepwise assembly of TCR factors CSB, CSA, UVSSA, and TFIIH around lesion-stalled RNAPII. However, the mechanism and factors required for the transition to downstream repair steps, including RNAPII removal to provide repair proteins access to the DNA lesion, remain unclear. Here, we identify STK19 as a new TCR factor facilitating this transition. Loss of STK19 does not impact initial TCR complex assembly or RNAPII ubiquitylation but delays lesion-stalled RNAPII clearance, thereby interfering with the downstream repair reaction. Cryo-EM and mutational analysis reveal that STK19 associates with the TCR complex, positioning itself between RNAPII, UVSSA, and CSA. The structural insights and molecular modeling suggest that STK19 positions the ATPase subunits of TFIIH onto DNA in front of RNAPII. Together, these findings provide new insights into the factors and mechanisms required for TCR.
2024-09-02 | GSE266083 | GEO
Project description:Transcription-coupled DNA repair (TCR) removes bulky DNA lesions impeding RNA polymerase II (RNAPII) transcription. Recent studies have outlined the stepwise assembly of TCR factors CSB, CSA, UVSSA, and TFIIH around lesion-stalled RNAPII. However, the mechanism and factors required for the transition to downstream repair steps, including RNAPII removal to provide repair proteins access to the DNA lesion, remain unclear. Here, we identify STK19 as a new TCR factor facilitating this transition. Loss of STK19 does not impact initial TCR complex assembly or RNAPII ubiquitylation but delays lesion-stalled RNAPII clearance, thereby interfering with the downstream repair reaction. Cryo-EM and mutational analysis reveal that STK19 associates with the TCR complex, positioning itself between RNAPII, UVSSA, and CSA. The structural insights and molecular modeling suggest that STK19 positions the ATPase subunits of TFIIH onto DNA in front of RNAPII. Together, these findings provide new insights into the factors and mechanisms required for TCR.
2024-09-02 | GSE266085 | GEO
Project description:Transcription-coupled DNA repair (TCR) removes bulky DNA lesions impeding RNA polymerase II (RNAPII) transcription. Recent studies have outlined the stepwise assembly of TCR factors CSB, CSA, UVSSA, and TFIIH around lesion-stalled RNAPII. However, the mechanism and factors required for the transition to downstream repair steps, including RNAPII removal to provide repair proteins access to the DNA lesion, remain unclear. Here, we identify STK19 as a new TCR factor facilitating this transition. Loss of STK19 does not impact initial TCR complex assembly or RNAPII ubiquitylation but delays lesion-stalled RNAPII clearance, thereby interfering with the downstream repair reaction. Cryo-EM and mutational analysis reveal that STK19 associates with the TCR complex, positioning itself between RNAPII, UVSSA, and CSA. The structural insights and molecular modelling suggest that STK19 positions the ATPase subunits of TFIIH onto DNA in front of RNAPII. Together, these findings provide new insights into the factors and mechanisms required for TCR.
2024-11-26 | GSE272790 | GEO
Project description:STK19 accelerates the clearance of lesion-stalled RNAPII to facilitate transcription-coupled DNA repair
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Project description:STK19 accelerates the clearance of lesion-stalled RNAPII to facilitate transcription-coupled DNA repair
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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 | GSE252172 | 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:Deregulation of RNA Polymerase II (RNAPII) by oncogenic signaling leads to collisions of RNAPII with DNA synthesis machinery (transcription-replication conflicts, TRCs). TRCs can result in DNA damage and underlie genomic instability in tumor cells. Here we provide evidence that elongating RNAPII promotes activation of the ATM kinase at TRCs to drive DNA repair. We show the ATPase Wrnip1 that binds and protects stalled replication forks, associates with RNAPII and limits ATM activation. Wrnip1 binding to elongating RNAPII requires catalytic activity of the ubiquitin ligase Huwe1. Mutation of Huwe1 promotes the transfer of Wrnip1 onto replisome and induces TRCs stimulating ATM activation on RNAPII. This mechanism is evoked early upon replicative stress to induce Wrnip1 translocation and ATM signaling at TRCs. Thus, although primarily considered as genotoxic events, TRCs can provide a mechanism to maintain genome stability under replicative stress.
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Project description:Endogenous aldehydes induce inter-strand crosslinks (ICL) and DNA-protein crosslinks (DPC). While DNA-repair and aldehyde-clearance systems cope with cellular toxicity, deficiencies in these mechanisms cause genome-instability disorders. The FA-pathway, defective in Fanconi anemia (FA), specifically removes ICL. In contrast, SPRTN, compromised in Ruijs-Aalfs syndrome, eliminates DPC during replication. However, AMeDS patients lacking aldehyde-detoxification display combined features of FA and Cockayne syndrome, associated with transcription-coupled repair (TCR) deficiency, suggesting a novel repair mechanism for aldehyde-induced DNA lesions in active genes. In this report, we demonstrate efficient resolution of aldehyde-induced transcription-blocking lesions by TCR. Mass-spectrometry and DPC-seq identify the TCR complex and additional factors involved in DPC removal and formaldehyde-induced damage tolerance. Notably, TFIIS-dependent cleavage of stalled-RNAPII transcripts exclusively protects against formaldehyde-induced damage. A mouse-model lacking both aldehyde-clearance and TCR pathways confirms endogenous DPC accumulation in transcribed regions. These findings highlight the importance of DPC removal in preventing transcription-roadblocks and contribute to understanding disorders related to aldehyde clearance and TCR deficiencies.
2024-02-12 | PXD044310 | Pride
Project description:Transcripts produced by RNA polymerase II (RNAPII) are fundamental for cellular responses to environmental changes. It is therefore no surprise that there exist multiple avenues for the regulation of this process. To explore the regulation mediated by RNAPII-interacting proteins, we used an siRNA-based screen to systematically evaluate their influence on RNA synthesis. We identified several proteins that strongly affected RNAPII activity. We evaluated one of the top hits, SCAF1, using an auxin-inducible degradation system and sequencing approaches. In agreement with our screen results, acute depletion of SCAF1 decreased RNA synthesis, and showed an increase of Serine-2 phosphorylated-RNAPII (pS2-RNAPII). We found that the accumulation of pS2-RNAPII within the gene body occurred at GC-rich regions and was indicative of stalled RNAPII complexes. The accumulation of stalled RNAPII complexes was accompanied by reduced recruitment of initiating RNAPII, explaining the observed global decrease in transcriptional output. Furthermore, upon SCAF1 depletion, RNAPII complexes showed increased association with components of the proteasomal-degradation machinery. We concluded that in cells lacking SCAF1, RNAPII undergoes a rather interrupted passage, resulting in intervention by the proteasomal-degradation machinery to clear stalled RNAPII. While cells survive the compromised transcription caused by absence of SCAF1, further inhibition of proteasomal-degradation machinery, is synthetically lethal.
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