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DDA1, a novel factor in transcription-coupled repair, modulates CRL4CSA dynamics at DNA damage-stalled RNA polymerase II

ABSTRACT: Transcription-blocking DNA lesions are specifically targeted by transcription-coupled nucleotide excision repair (TC-NER), which removes a broad spectrum of DNA lesions to preserve transcriptional output and thereby cellular homeostasis to counteract aging. TC-NER is initiated by the stalling of RNA polymerase II at DNA lesions, which triggers the assembly of the TC-NER-specific proteins CSA, CSB and UVSSA. CSA, a WD40-repeat containing protein, is the substrate receptor subunit of a cullin-RING ubiquitin ligase complex composed of DDB1, CUL4A/B and RBX1 (CRL4CSA). Although ubiquitination of several TC-NER proteins by CRL4CSA has been reported, it is still unknown how this complex is regulated. To unravel the dynamic molecular interactions and the regulation of this complex, we applied a single-step protein-complex isolation coupled to mass spectrometry analysis and identified DDA1 as a CSA interacting protein. Cryo-EM analysis showed that DDA1 is an integral component of the CRL4CSA complex. Functional analysis revealed that DDA1 coordinates ubiquitination dynamics during TC-NER and is required for efficient turnover and progression of this process.

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Alex Pines

LAB HEAD: Alex Pines

PROVIDER: PXD051638 | Pride | 2024-05-02

REPOSITORIES: Pride

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			Action	DRS
	20221005_Fusion1_SA_KK_TC_NER_trypsin_2.raw	Raw
	20221006_Fusion1_SA_KK_TC_NER_trypsin_AspN_3.raw	Raw
	20221006_Fusion1_SA_KK_TC_NER_trypsin_Elastase_4.raw	Raw
	20221006_Fusion1_SA_KK_TC_NER_trypsin_GluC_5.raw	Raw
	20221006_Fusion1_SA_KK_TC_NER_trypsin_ProAla_6.raw	Raw

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DDA1, a novel factor in transcription-coupled repair, modulates CRL4CSA dynamics at DNA damage-stalled RNA polymerase II

Project description:Transcription–blocking lesions are specifically targeted by transcription-coupled nucleotide excision repair (TC-NER), which removes a broad spectrum of DNA lesions to preserve transcriptional output and thereby cellular homeostasis to counteract aging. TC-NER is initiated by the physical stalling of RNA polymerase II by a DNA damage, which triggers the assembly of TC-NER-specific proteins, including CSA, a WD40 domain containing protein, that forms together with DDB1, CUL4A/B and RBX1, a cullin-RING ubiquitin ligase complex (CRL4CSA). Although ubiquitination of several TC-NER proteins by CSA has been reported, it is still unknown how this complex functions in TC-NER progression. To unravel the molecular mechanism underlying TC-NER, we applied a single-step protein-complex isolation coupled to mass spectrometry analysis and identified DDA1 as a CSA interacting protein. Cryo-EM analysis showed that DDA1 is an integral component of the CRL4CSA complex. Functional analysis revealed that DDA1 coordinates the step-wise ubiquitination during TC-NER and is required for efficient progression of this process by controlling the dynamic turnover of CRL4CSA complex.

2024-05-31 | PXD045415 | Pride

STK19 drives transcription-coupled nucleotide excision repair by stimulating Pol II ubiquitylation and TFIIH recruitment

Project description:DNA damage forms a major obstacle for gene transcription by RNA polymerase II (Pol II). Transcription-coupled nucleotide excision repair (TC-NER) efficiently eliminates transcription-blocking lesions (TBLs), thereby safeguarding accurate transcription, preserving correct cellular function and counteracting aging. TC-NER initiation involves the recognition of lesion-stalled Pol II by CSB, which recruits the CRL4CSA E3 ubiquitin ligase complex and UVSSA. TBL-induced Pol II ubiquitylation at lysine K1268 by CRL4CSA serves as a critical TC-NER checkpoint, governing Pol II stability and initiating TBL excision by TFIIH recruitment. However, the precise regulatory mechanisms of the CRL4CSA E3 ligase activity and TFIIH recruitment remains elusive. Here, we reveal Serine/Threonine Kinase 19 (STK19) as a novel TC-NER factor. Cryo-EM studies revealed that STK19 is an integral part of the Pol II-TC‐NER complex, bridging CSA with UVSSA, RPB1 and downstream DNA. Live-cell imaging and interaction proteomics shows that STK19 stimulates TC-NER complex stability and proper CRL4CSA complex assembly, resulting in Pol II (K1268) ubiquitylation and subsequent UVSSA and TFIIH recruitment. These findings underscore the crucial role of STK19 as a core component of the TC-NER machinery and its key involvement to the cellular responses to genomic injuries that interfere with transcription.

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TRiC controls transcription resumption after UV damage by regulating Cockayne Syndrome protein A

Project description:Transcription-blocking DNA lesions are removed by transcription-coupled nucleotide excision repair (TC-NER) to preserve cell viability. TC-NER is triggered by the stalling of RNA polymerase II at DNA lesions, leading to the recruitment of TC-NER-specific factors such as the CSA-DDB1-CUL4A-RBX1 cullin-RING ubiquitin ligase complex (CRLCSA). Despite its vital role in TC-NER, little is known about the regulation of the CRLCSA complex during TC-NER. Using conventional and cross-linking immunoprecipitations coupled to mass spectrometry, we uncover a stable interaction between CSA and the TRiC chaperonin. TRiC’s binding to CSA ensures its stability and DDB1-dependent assembly into the CRLCSA complex. Consequently, loss of TRiC leads to mislocalization and depletion of CSA, as well as impaired transcription recovery following UV damage, suggesting defects in TC-NER. Furthermore, Cockayne syndrome (CS)-causing mutations in CSA lead to increased TRiC binding and a failure to compose the CRLCSA complex. Thus, we uncover CSA as a novel TRiC substrate and reveal a role for TRiC in regulating CSA-dependent TC-NER and the development of CS.

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Transcription-coupled DNA-Protein Crosslink repair by CSB and CRL4(CSA)-mediated degradation

Project description:DNA-protein crosslinks (DPCs), arise from enzymatic intermediates, endogenous metabolism or exogenous chemicals like chemotherapeutics. DPCs are highly cytotoxic as they will impede DNA transacting processes such as replication, which is counteracted by proteolysis-mediated DPC removal by the protease SPRTN or the proteasome. However, how DPCs affect transcription and how transcription-blocking DPCs are repaired remains largely unknown. Here, we show that DPCs severely inhibit RNA polymerase II (Pol II)-mediated transcription, resulting in the recruitment of the transcription-coupled nucleotide excision repair (TC-NER) factors CSA and CSB. Interestingly, CSA and CSB are indispensable for transcription-coupled DPC (TC-DPC) repair, while the downstream TC-NER factors UVSSA and XPA are not, indicative of a non-canonical TC-NER mechanism. TC-DPC repair functions independent of SPRTN, but is mediated by the activities of the ubiquitin ligase CRL4CSA and the proteasome. Thus, DPCs are preferentially repaired in genes by a dedicated transcription-coupled repair mechanism, which is crucial to warrant correct transcription.

2024-02-09 | PXD041679 | Pride

Posttranslational modifications regulate cellular responses upon UV-induced DNA damage

Project description:SUMOylation is a posttranslational protein modification which is characterized by the covalent attachment of a small 11kDa protein, called Small Ubiquitin-like MOdifier (SUMO). SUMOylation plays a pivotal role in a multitude of cellular pathways including cellular responses upon DNA damage. Here, we identified multiple proteins which are SUMOylated in U2OS cells in response to ultraviolet light (UV) irradiation and ionizing radiation (IR). We show that the SUMOylation response upon UV irradiation was more pronounced compared to the response upon IR. The major SUMOylation target upon UV-irradiation was the transcription-coupled nucleotide excision repair (TC-NER) protein, Cockayne Syndrome B (CSB). This protein plays an important role in the repair of UV-induced lesions in actively transcribed genes. In a second proteomic approach we identified SUMOylation-dependent and independent protein interactors of the N-terminus of CSB. Here, we uncovered that the affinity of multiple RNA polymerase-associated proteins towards CSB is influenced by SUMOylation. Finally, we set out to identify ubiquitination events upon UV-irradiation which are influenced by the CSA-ubiquitin ligase complex, which is also involved in TC-NER and is closely connected to CSB, because mutations in either CSA or CSB result in the same phenotype, Cockayne syndrome. We found that RPB1, the major subunit of RNA polymerase II, was ubiquitinated in a CSA-dependent manner upon UV which finally led to its degradation.

2019-10-11 | PXD010609 | Pride

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.

2023-11-24 | PXD042388 | Pride

Cockayne syndrome group A and ferrochelatase finely tune ribosomal gene transcription and its response to UV irradiation

Project description:CSA and CSB proteins are key players in transcription-coupled nucleotide excision repair (TC-NER) pathway that removes UV-induced DNA lesions from the transcribed strands of expressed genes. Additionally, CS proteins play relevant but still elusive roles in other cellular pathways whose alteration may explain neurodegeneration and progeroid features in Cockayne syndrome (CS). Here we identify a CSA-dependent chromatin-associated protein complex that modulates rRNA transcription. Besides RNA polymerase I (RNAP1) and specific ribosomal proteins (RPs), the CSA complex includes ferrochelatase (FECH), a well-known mitochondrial enzyme whose deficiency causes erythropoietic protoporphyria (EPP). Impairment of either CSA or FECH functionality leads to reduced RNAP1 occupancy on rDNA promoter that is associated to reduced transcription when CSA is affected and abnormal ribosomal transcript accumulation after FECH silencing. Accordingly, primary fibroblasts from CS and EPP fibroblasts show opposed amount of total rRNAs. After cell irradiation with UV light, CSA triggers the dissociation of the CSA-FECH-RNAP1-RPs complex from the chromatin while it stabilizes its binding to FECH. Besides disclosing a function for FECH within nucleoli, this study sheds light on the still unknown mechanisms through which CSA modulates rRNA transcription.

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Elf1 promotes TC-NER in yeast by using its C-terminal domain to bind TFIIH

Project description:Transcription coupled-nucleotide excision repair (TC-NER) repairs DNA lesions that stall RNA polymerase II (Pol II) transcription. Here, we show that the C-terminal domain (CTD) of elongation factor-1 (Elf1) plays a critical role in TC-NER in yeast. Analysis of genome-wide repair of UV-induced cyclobutane pyrimidine dimers (CPDs) using CPD-seq indicates that the Elf1 CTD is required for efficient Rad26-dependent and Rad26-independent TC-NER across the yeast genome. The Elf1-CTD is also important for TC-NER in rad16∆ cells deficient in GG-NER. Finally, we show that a mutant in the Elf1-CTD (elf1-Y99A) that disrupts binding to a subunit of TFIIH affects Rad26-independent repair in a rad26∆ mutant background.

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