During transcription-coupled DNA repair (TCR), RNA polymerase II (Pol II) transitions from a transcriptionally active state to an arrested state that allows for removal of DNA lesions. This... Show moreDuring transcription-coupled DNA repair (TCR), RNA polymerase II (Pol II) transitions from a transcriptionally active state to an arrested state that allows for removal of DNA lesions. This transition requires site-specific ubiquitylation of Pol II by the CRL4CSA ubiquitin ligase, a process that is facilitated by ELOF1 in an unknown way. Using cryogenic electron microscopy, biochemical assays and cell biology approaches, we found that ELOF1 serves as an adaptor to stably position UVSSA and CRL4CSA on arrested Pol II, leading to ligase neddylation and activation of Pol II ubiquitylation. In the presence of ELOF1, a transcription factor IIS (TFIIS)-like element in UVSSA gets ordered and extends through the Pol II pore, thus preventing reactivation of Pol II by TFIIS. Our results provide the structural basis for Pol II ubiquitylation and inactivation in TCR. Show less
The cellular response to transcription-blocking DNA lesions involves the stalling of elongating RNA Polymerase II (RNAPIIo) at the lesion as well as a global shutdown of transcription. The stalling... Show moreThe cellular response to transcription-blocking DNA lesions involves the stalling of elongating RNA Polymerase II (RNAPIIo) at the lesion as well as a global shutdown of transcription. The stalling of RNAPIIo at such lesions initiates the transcription-coupled nucleotide excision repair pathway (TCR) to efficiently remove the damage and restore transcription. The TCR proteins, CSB, CSA, and UVSSA, are essential for the repair of transcription-blocking DNA lesions, but how the interplay between these proteins targets the core repair machinery, including the TFIIH complex, to lesion stalled RNAPIIo remains largely unknown.Here, we demonstrate a sequential and highly cooperative assembly of TCR proteins and unveil the mechanism for TFIIH recruitment to DNA damage-stalled RNAPIIo. Importantly, we identified the previously uncharacterized ELOF1 gene as a core TCR factor with an additional role in preventing DNA damage during DNA replication. Show less
Weegen, Y. van der; Lint, K. de; Heuvel, D. van den; Nakazawa, Y.; Mevissen, T.E.T.; Schie, J.J.M. van; ... ; Luijsterburg, M.S. 2021
Two side-by-side papers report that the transcription elongation factor ELOF1 drives transcription-coupled repair and prevents replication stress.Cells employ transcription-coupled repair (TCR) to... Show moreTwo side-by-side papers report that the transcription elongation factor ELOF1 drives transcription-coupled repair and prevents replication stress.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 of a single lysine (K1268) by the CRL4(CSA) ubiquitin ligase. How CRL4(CSA) is specifically directed towards K1268 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 K1268, revealing ELOF1 as a specificity factor that binds and positions CRL4(CSA) for optimal RNAPII ubiquitylation. Drug-genetic interaction screening also revealed a CSB-independent pathway in which ELOF1 prevents R-loops in active genes and protects cells against DNA replication stress. Our study offers key insights into the molecular mechanisms of TCR and provides a genetic framework of the interplay between transcriptional stress responses and DNA replication. Show less
Heuvel, D. van den; Weegen, Y. van der; Boer, D.E.C.; Ogi, T.; Luijsterburg, M.S. 2021
DNA lesions pose a major obstacle during gene transcription by RNA polymerase II (RNAPII) enzymes. The transcription-coupled DNA repair (TCR) pathway eliminates such DNA lesions. Inherited defects... Show moreDNA lesions pose a major obstacle during gene transcription by RNA polymerase II (RNAPII) enzymes. The transcription-coupled DNA repair (TCR) pathway eliminates such DNA lesions. Inherited defects in TCR cause severe clinical syndromes, including Cockayne syndrome (CS). The molecular mechanism of TCR and the molecular origin of CS have long remained enigmatic. Here we explore new advances in our understanding of how TCR complexes assemble through cooperative interactions between repair factors stimulated by RNAPII ubiquitylation. Mounting evidence suggests that RNAPII ubiquitylation activates TCR complex assembly during repair and, in parallel, promotes processing and degradation of RNAPII to prevent prolonged stalling. The fate of stalled RNAPII is therefore emerging as a crucial link between TCR and associated human diseases. Show less
Heuvel, D. van den; Spruijt, C.G.; Gonzalez Prieto, R.; Kragten, A.; Paulsen, M.T.; Zhou, D.; ... ; Luijsterburg, M.S. 2021
Bulky DNA lesions in transcribed strands block RNA polymerase II (RNAPII) elongation and induce a genome-wide transcriptional arrest. The transcription-coupled repair (TCR) pathway efficiently... Show moreBulky DNA lesions in transcribed strands block RNA polymerase II (RNAPII) elongation and induce a genome-wide transcriptional arrest. The transcription-coupled repair (TCR) pathway efficiently removes transcription-blocking DNA lesions, but how transcription is restored in the genome following DNA repair remains unresolved. Here, we find that the TCR-specific CSB protein loads the PAF1 complex (PAF1C) onto RNAPII in promoter-proximal regions in response to DNA damage. Although dispensable for TCR-mediated repair, PAF1C is essential for transcription recovery after UV irradiation. 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. The transcription-coupled repair pathway removes transcription-blocking DNA lesions, but how transcription is restored following DNA repair is not clear. Here the authors reveal that the PAF1 complex, while dispensable for the repair process, restores transcription after DNA damage. Show less
Weegen, Y. van der; Golan-Berman, H.; Mevissen, T.E.T.; Apelt, K.; Gonzalez-Prieto, R.; Goedhart, J.; ... ; Luijsterburg, M.S. 2020
The response to DNA damage-stalled RNA polymerase II (RNAPIIo) involves the assembly of the transcription-coupled repair (TCR) complex on actively transcribed strands. The function of the TCR... Show moreThe response to DNA damage-stalled RNA polymerase II (RNAPIIo) involves the assembly of the transcription-coupled repair (TCR) complex on actively transcribed strands. The function of the TCR proteins CSB, CSA and UVSSA and the manner in which the core DNA repair complex, including transcription factor IIH (TFIIH), is recruited are largely unknown. Here, we define the assembly mechanism of the TCR complex in human isogenic knockout cells. We show that TCR is initiated by RNAPIIo-bound CSB, which recruits CSA through a newly identified CSA-interaction motif (CIM). Once recruited, CSA facilitates the association of UVSSA with stalled RNAPIIo. Importantly, we find that UVSSA is the key factor that recruits the TFIIH complex in a manner that is stimulated by CSB and CSA. Together these findings identify a sequential and highly cooperative assembly mechanism of TCR proteins and reveal the mechanism for TFIIH recruitment to DNA damage-stalled RNAPIIo to initiate repair. The response to DNA damage-stalled RNA polymerase II leads to the assembly of the transcription-coupled repair (TCR) complex on actively transcribed strands. Here, the authors reveal the complex assembly mechanism of the TCR complex in human cells. Show less
Liebelt, F.; Schimmel, J.; Verlaan-de Vries, M.; Klemann, E.; Royen, M.E. van; Weegen, Y. van der; ... ; Vertegaal, A.C.O. 2020
Cockayne Syndrome (CS) is a severe neurodegenerative and premature aging autosomal-recessive disease, caused by inherited defects in the CSA and CSB genes, leading to defects in transcription... Show moreCockayne Syndrome (CS) is a severe neurodegenerative and premature aging autosomal-recessive disease, caused by inherited defects in the CSA and CSB genes, leading to defects in transcription-coupled nucleotide excision repair (TC-NER) and consequently hypersensitivity to ultraviolet (UV) irradiation. TC-NER is initiated by lesion-stalled RNA polymerase II, which stabilizes the interaction with the SNF2/SWI2 ATPase CSB to facilitate recruitment of the CSA E3 Cullin ubiquitin ligase complex. However, the precise biochemical connections between CSA and CSB are unknown. The small ubiquitinlike modifier SUMO is important in the DNA damage response. We found that CSB, among an extensive set of other target proteins, is the most dynamically SUMOylated substrate in response to UV irradiation. Inhibiting SUMOylation reduced the accumulation of CSB at local sites of UV irradiation and reduced recovery of RNA synthesis. Interestingly, CSA is required for the efficient clearance of SUMOylated CSB. However, subsequent proteomic analysis of CSA-dependent ubiquitinated substrates revealed that CSA does not ubiquitinate CSB in a UV-dependent manner. Surprisingly, we found that CSA is required for the ubiquitination of the largest subunit of RNA polymerase II, RPB1. Combined, our results indicate that the CSA, CSB, RNA polymerase II triad is coordinated by ubiquitin and SUMO in response to UV irradiation. Furthermore, our work provides a resource of SUMO targets regulated in response to UV or ionizing radiation. Show less
Pines, A.; Dijk, M.; Makowski, M.; Meulenbroek, E.M.; Vrouwe, M.G.; Weegen, Y. van der; ... ; Attikum, H. van 2018
