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ELOF1 is a transcription-coupled DNA repair factor that directs RNA polymerase II ubiquitylation

ABSTRACT: Removal of transcription-blocking DNA lesions requires the binding of transcription-coupled repair (TCR) factors to DNA damage-stalled RNA polymerase II (RNAPII). The full repertoire of factors required for TCR remains to be elucidated. Through genome-wide CRISPR screens and strand-specific ChIP-seq, we identify the RNAPII-associated factor ELOF1 as a new core TCR component. Mechanistically, ELOF1 does not affect the association of TCR components CSB and the CRL4CSA ubiquitin ligase, but instead regulates RNAPII ubiquitylation, and subsequent ubiquitin-dependent recruitment of repair factors. This function requires its constitutive interaction with RNAPII close to the K1268 site, revealing ELOF1 as a specificity factor that positions CRL4CSA for optimal RNAPII ubiquitylation to orchestrate transcription-coupled repair. Finally, ELOF1 operates in a second transcription stress-response pathway, regulated by the deubiquitylating enzyme OTUD5.

ORGANISM(S): Homo sapiens

PROVIDER: GSE149760 | GEO | 2021/04/20

REPOSITORIES: GEO

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ELOF1 is a transcription-coupled DNA repair factor that directs RNA polymerase II ubiquitylation

Project description:Cells employ transcription-coupled repair (TCR) to eliminate transcription-blocking DNA lesions. DNA damage-induced binding of the TCR-specific repair factor CSB to RNA polymerase II (RNAPII) triggers RNAPII ubiquitylation at a single lysine (K1268) by the CRL4CSA ubiquitin ligase. How CRL4CSA is specifically directed toward the K1268 site is unknown. Here, we identify ELOF1 as the missing link that facilitates RNAPII ubiquitylation, a key signal for the assembly of downstream repair factors. This function requires its constitutive interaction with RNAPII close to the K1268 site, revealing ELOF1 as a specificity factor that interacts with and positions CRL4CSA for optimal RNAPII ubiquitylation. Drug-genetic interaction screening also reveals a CSB-independent compensatory pathway in which ELOF1 protects cells against DNA replication stress by preventing DNA damage-induced R-loops. Our study offers key insights into the molecular mechanisms of TCR and provides a genetic framework of the interplay between the transcriptional stress response and DNA replication.

2021-04-21 | PXD024051 | Pride

STK19 accelerates the clearance of lesion-stalled RNAPII to facilitate transcription-coupled DNA repair

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STK19 accelerates the clearance of lesion-stalled RNAPII to facilitate transcription-coupled DNA repair [BrU-seq]

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ELOF1 anchors CRL4CSA to RNA pol II to activate transcription-coupled repair

Project description:Transcription-coupled DNA repair (TCR) removes transcription-blocking DNA lesions through the coordinated assembly of repair factors on arrested RNA polymerase II (Pol II). This process requires the site-specific ubiquitylation of Pol II by the CRL4CSA ubiquitin ligase. Pol II ubiquitylation is facilitated by ELOF1, a recently identified TCR factor. Using cryogenic electron microscopy, biochemical assays, and cell biology, we reveal that ELOF1 functions as an adaptor to correctly dock CRL4CSA onto Pol II and facilitate the recruitment of UVSSA. The ELOF1-CSA-UVSSA interaction positions CRL4CSA on arrested Pol II, leading to its ubiquitylation upon ligase activation by neddylation. ELOF1-mediated repositioning enables a TFIIS-like element in UVSSA to reach the Pol II active site and the pore region to prevent Pol II re-activation. These findings provide the structural and molecular basis underlying the transition of Pol II from a transcriptionally active to an arrested state, primed for DNA repair.

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Project description:The coordinated transcription of genes involves RNA polymerase II enzymes (RNAPII), which pull DNA through their active sites. DNA lesions in transcribed strands block RNAPII elongation and induce a strong transcriptional arrest. The transcription-coupled repair (TCR) pathway ensures the efficient removal of transcription-blocking DNA lesions, but this is not sufficient to overcome this arrest and resume transcription. Through proteomics screens, we find that the TCR-specific CSB protein loads the evolutionary conserved PAF1 complex (PAF1C) onto RNAPII in promoter-proximal regions specifically in response to DNA damage. PAF1C is dispensable for TCR-mediated repair,but is essential to resume RNA synthesis after UV irradiation, suggesting an unexpected uncoupling between DNA repair and transcription restart. Moreover, we find that PAF1C promotes RNAPII pause release in promoter-proximal regions and subsequently acts as a processivity factor that stimulates transcription elongation waves throughout genes. Our findings expose the molecular basis for a non-canonical PAF1C-dependent pathway that restores transcription throughout the human genome after genotoxic stress.

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A CSB-PAF1C axis restores processive transcription elongation after DNA damage repair

Project description:The coordinated transcription of genes involves the regulated release of RNA polymerase II (RNAPII) from promoter-proximal sites into active elongation. DNA lesions in transcribed strands block elongation and induce a strong transcriptional arrest. The transcription-coupled repair (TCR) pathway efficiently removes transcription-blocking DNA lesions, but this is not sufficient to resume transcription. Through proteomics screens, we find that the TCR-specific CSB protein loads the evolutionary conserved PAF1 complex (PAF1C) onto RNAPII in promoter-proximal regions in response to DNA damage. PAF1C is dispensable for TCR-mediated repair, but is essential for recovery of RNA synthesis after UV irradiation, suggesting an uncoupling between DNA repair and transcription recovery. Moreover, we find that PAF1C promotes RNAPII pause release in promoter-proximal regions and subsequently acts as a processivity factor that stimulates transcription elongation throughout genes. Our findings expose the molecular basis for a non-canonical PAF1C-dependent pathway that restores transcription throughout the human genome after genotoxic stress.

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