Cells employ global genome nucleotide excision repair (GGR) to eliminate a broad spectrum of DNA lesions, including those induced by UV light. The lesion-recognition factor XPC initiates repair of... Show moreCells employ global genome nucleotide excision repair (GGR) to eliminate a broad spectrum of DNA lesions, including those induced by UV light. The lesion-recognition factor XPC initiates repair of helix-destabilizing DNA lesions, but binds poorly to lesions such as CPDs that do not destabilize DNA. How difficult-to-repair lesions are detected in chromatin is unknown. Here, we identify the poly-(ADP-ribose) polymerases PARP1 and PARP2 as constitutive interactors of XPC. Their interaction results in the XPC-stimulated synthesis of poly-(ADP-ribose) (PAR) by PARP1 at UV lesions, which in turn enables the recruitment and activation of the PAR-regulated chromatin remodeler ALC1. PARP2, on the other hand, modulates the retention of ALC1 at DNA damage sites. Notably, ALC1 mediates chromatin expansion at UV-induced DNA lesions, leading to the timely clearing of CPD lesions. Thus, we reveal how chromatin containing difficult-to-repair DNA lesions is primed for repair, providing insight into mechanisms of chromatin plasticity during GGR.Cells employ global genome nucleotide excision repair to repair a broad spectrum of genomic DNA lesions. Here, the authors reveal how chromatin is primed for repair, providing insight into mechanisms of chromatin plasticity during DNA repair. Show less
The integrity and proper expression of genomes are safeguarded by DNA and RNA surveillance pathways. While many RNA surveillance factors have additional functions in the nucleus, little is known... Show moreThe integrity and proper expression of genomes are safeguarded by DNA and RNA surveillance pathways. While many RNA surveillance factors have additional functions in the nucleus, little is known about the incidence and physiological impact of converging RNA and DNA signals. Here, using genetic screens and genome-wide analyses, we identified unforeseen SMG-1-dependent crosstalk between RNA surveillance and DNA repair in living animals. Defects in RNA processing, due to viable THO complex or PNN-1 mutations, induce a shift in DNA repair in dividing and non-dividing tissues. Loss of SMG-1, an ATM/ATR-like kinase central to RNA surveillance by nonsense-mediated decay (NMD), restores DNA repair and radio-resistance in THO-deficient animals. Mechanistically, we find SMG-1 and its downstream target SMG-2/UPF1, but not NMD per se, to suppress DNA repair by non-homologous end-joining in favour of single strand annealing. We postulate that moonlighting proteins create short-circuits in vivo, allowing aberrant RNA to redirect DNA repair. Show less
Goossens, R.; Tihaya, M.S.; Heuvel, A. van den; Tabot-Ndip, K.; Willemsen, I.M.; Tapscott, S.J.; ... ; Maarel, S.M. van der 2021
Structural Maintenance of Chromosomes Hinge Domain Containing 1 (SMCHD1) is a chromatin repressor, which is mutated in > 95% of Facioscapulohumeral dystrophy (FSHD) type 2 cases. In FSHD2,... Show moreStructural Maintenance of Chromosomes Hinge Domain Containing 1 (SMCHD1) is a chromatin repressor, which is mutated in > 95% of Facioscapulohumeral dystrophy (FSHD) type 2 cases. In FSHD2, SMCHD1 mutations ultimately result in the presence of the cleavage stage transcription factor DUX4 in muscle cells due to a failure in epigenetic repression of the D4Z4 macrosatellite repeat on chromosome 4q, which contains the DUX4 locus. While binding of SMCHD1 to D4Z4 and its necessity to maintain a repressive D4Z4 chromatin structure in somatic cells are well documented, it is unclear how SMCHD1 is recruited to D4Z4, and how it exerts its repressive properties on chromatin. Here, we employ a quantitative proteomics approach to identify and characterize novel SMCHD1 interacting proteins, and assess their functionality in D4Z4 repression. We identify 28 robust SMCHD1 nuclear interactors, of which 12 are present in D4Z4 chromatin of myocytes. We demonstrate that loss of one of these SMCHD1 interacting proteins, RuvB-like 1 (RUVBL1), further derepresses DUX4 in FSHD myocytes. We also confirm the interaction of SMCHD1 with EZH inhibitory protein (EZHIP), a protein which prevents global H3K27me3 deposition by the Polycomb repressive complex PRC2, providing novel insights into the potential function of SMCHD1 in the repression of DUX4 in the early stages of embryogenesis. The SMCHD1 interactome outlined herein can thus provide further direction into research on the potential function of SMCHD1 at genomic loci where SMCHD1 is known to act, such as D4Z4 repeats, the inactive X chromosome, autosomal gene clusters, imprinted loci and telomeres. Show less
Singh, J.K.; Smith, R.; Rother, M.B.; Groot, A.J.L. de; Wiegant, W.W.; Vreeken, K.; ... ; Attikum, H. van 2021
Classical non-homologous end-joining (cNHEJ) is the dominant pathway used by human cells to repair DNA double-strand breaks (DSBs) and maintain genome stability. Here the authors show that PARP1... Show moreClassical non-homologous end-joining (cNHEJ) is the dominant pathway used by human cells to repair DNA double-strand breaks (DSBs) and maintain genome stability. Here the authors show that PARP1-driven chromatin expansion allows the recruitment of ZNF384, which in turn recruits Ku70/Ku80 to facilitate cNHEJ.DNA double-strand breaks (DSBs) are among the most deleterious types of DNA damage as they can lead to mutations and chromosomal rearrangements, which underlie cancer development. Classical non-homologous end-joining (cNHEJ) is the dominant pathway for DSB repair in human cells, involving the DNA-binding proteins XRCC6 (Ku70) and XRCC5 (Ku80). Other DNA-binding proteins such as Zinc Finger (ZnF) domain-containing proteins have also been implicated in DNA repair, but their role in cNHEJ remained elusive. Here we show that ZNF384, a member of the C2H2 family of ZnF proteins, binds DNA ends in vitro and is recruited to DSBs in vivo. ZNF384 recruitment requires the poly(ADP-ribosyl) polymerase 1 (PARP1)-dependent expansion of damaged chromatin, followed by binding of its C2H2 motifs to the exposed DNA. Moreover, ZNF384 interacts with Ku70/Ku80 via its N-terminus, thereby promoting Ku70/Ku80 assembly and the accrual of downstream cNHEJ factors, including APLF and XRCC4/LIG4, for efficient repair at DSBs. Altogether, our data suggest that ZNF384 acts as a 'Ku-adaptor' that binds damaged DNA and Ku70/Ku80 to facilitate the build-up of a cNHEJ repairosome, highlighting a role for ZNF384 in DSB repair and genome maintenance. Show less
Cabello-Lobato, M.J.; Gonzalez-Garrido, C.; Cano-Linares, M.I.; Wong, R.P.; Yanez-Vilchez, A.; Morillo-Huesca, M.; ... ; Prado, F. 2021
The minichromosome maintenance (MCM) helicase physically interacts with the recombination proteins Rad51 and Rad52 from yeast to human cells. We show, in Saccharomyces cerevisiae, that these... Show moreThe minichromosome maintenance (MCM) helicase physically interacts with the recombination proteins Rad51 and Rad52 from yeast to human cells. We show, in Saccharomyces cerevisiae, that these interactions occur within a nuclease-insoluble scaffold enriched in replication/repair factors. Rad51 accumulates in a MCM- and DNA-binding-independent manner and interacts with MCMhelicases located outside of the replication origins and forks. MCM, Rad51, and Rad52 accumulate in this scaffold in G1 and are released during the S phase. In the presence of replication-blocking lesions, Cdc7 prevents their release from the scaffold, thus maintaining the interactions. We identify a rad51 mutant that is impaired in its ability to bind to MCM but not to the scaffold. This mutant is proficient in recombination but partially defective in single-stranded DNA (ssDNA) gap filling and replication fork progression through damaged DNA. Therefore, cells accumulate MCM/Rad51/Rad52 complexes at specific nuclear scaffolds in G1 to assist stressed forks through non-recombinogenic functions. 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
Apelt, K.; White, S.M.; Kim, H.S.; Yeo, J.E.; Kragten, A.; Wondergem, A.P.; ... ; Luijsterburg, M.S. 2021
ERCC1-XPF is a multifunctional endonuclease involved in nucleotide excision repair (NER), interstrand cross-link (ICL) repair, and DNA double-strand break (DSB) repair. Only two patients with bi... Show moreERCC1-XPF is a multifunctional endonuclease involved in nucleotide excision repair (NER), interstrand cross-link (ICL) repair, and DNA double-strand break (DSB) repair. Only two patients with bi-allelic ERCC1 mutations have been reported, both of whom had features of Cockayne syndrome and died in infancy. Here, we describe two siblings with bi-allelic ERCC1 mutations in their teenage years. Genomic sequencing identified a deletion and a missense variant (R156W) within ERCC1 that disrupts a salt bridge below the XPA-binding pocket. Patient-derived fibroblasts and knock-in epithelial cells carrying the R156W substitution show dramatically reduced protein levels of ERCC1 and XPF. Moreover, mutant ERCC1 weakly interacts with NER and ICL repair proteins, resulting in diminished recruitment to DNA damage. Consequently, patient cells show strongly reduced NER activity and increased chromosome breakage induced by DNA cross-linkers, while DSB repair was relatively normal. We report a new case of ERCC1 deficiency that severely affects NER and considerably impacts ICL repair, which together result in a unique phenotype combining short stature, photosensitivity, and progressive liver and kidney dysfunction. Show less
Koedoot, E.; Steijn, E. van; Vermeer, M.; Gonzalez Prieto, R.; Vertegaal, A.C.O.; Martens, J.W.M.; ... ; Water, B. van de 2021
BackgroundTriple negative breast cancer (TNBC) is an aggressive subtype of breast cancer with limited therapeutic opportunities. Recently, splicing factors have gained attention as potential... Show moreBackgroundTriple negative breast cancer (TNBC) is an aggressive subtype of breast cancer with limited therapeutic opportunities. Recently, splicing factors have gained attention as potential targets for cancer treatment. Here we systematically evaluated the role of RNA splicing factors in TNBC cell proliferation.MethodsIn this study, we performed an RNAi screen targeting 244 individual splicing factors to systematically evaluate their role in TNBC cell proliferation. For top candidates, mechanistic insight was gained using amongst others western blot, PCR, FACS, molecular imaging and cloning. Pulldown followed by mass spectrometry were used to determine protein-protein interactions and patient-derived RNA sequencing data was used relate splicing factor expression levels to proliferation markers.ResultsWe identified nine splicing factors, including SNRPD2, SNRPD3 and NHP2L1, of which depletion inhibited proliferation in two TNBC cell lines by deregulation of sister chromatid cohesion (SCC) via increased sororin intron 1 retention and down-regulation of SMC1, MAU2 and ESPL1. Protein-protein interaction analysis of SNRPD2, SNRPD3 and NHP2L1 identified that seven out of the nine identified splicing factors belong to the same spliceosome complex including novel component SUN2 that was also critical for efficient sororin splicing. Finally, sororin transcript levels are highly correlated to various proliferation markers in BC patients.ConclusionWe systematically determined splicing factors that control proliferation of breast cancer cells through a mechanism that involves effective sororin splicing and thereby appropriate sister chromatid cohesion. Moreover, we identified SUN2 as an important new spliceosome complex interacting protein that is critical in this process. We anticipate that deregulating sororin levels through targeting of the relevant splicing factors might be a potential strategy to treat TNBC. Show less
Apelt, K.; White, S.M.; Kim, H.S.; Yeo, J.E.; Kragten, A.; Wondergem, A.P.; ... ; Luijsterburg, M.S. 2021
ERCC1-XPF is a multifunctional endonuclease involved in nucleotide excision repair (NER), interstrand cross-link (ICL) repair, and DNA double-strand break (DSB) repair. Only two patients with bi... Show moreERCC1-XPF is a multifunctional endonuclease involved in nucleotide excision repair (NER), interstrand cross-link (ICL) repair, and DNA double-strand break (DSB) repair. Only two patients with bi-allelic ERCC1 mutations have been reported, both of whom had features of Cockayne syndrome and died in infancy. Here, we describe two siblings with bi-allelic ERCC1 mutations in their teenage years. Genomic sequencing identified a deletion and a missense variant (R156W) within ERCC1 that disrupts a salt bridge below the XPA-binding pocket. Patient-derived fibroblasts and knock-in epithelial cells carrying the R156W substitution show dramatically reduced protein levels of ERCC1 and XPF. Moreover, mutant ERCC1 weakly interacts with NER and ICL repair proteins, resulting in diminished recruitment to DNA damage. Consequently, patient cells show strongly reduced NER activity and increased chromosome breakage induced by DNA cross-linkers, while DSB repair was relatively normal. We report a new case of ERCC1 deficiency that severely affects NER and considerably impacts ICL repair, which together result in a unique phenotype combining short stature, photosensitivity, and progressive liver and kidney dysfunction. 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
In contrast to our extensive knowledge on covalent small ubiquitin-like modifier (SUMO) target proteins, we are limited in our understanding of non-covalent SUMO-binding proteins. We identify... Show moreIn contrast to our extensive knowledge on covalent small ubiquitin-like modifier (SUMO) target proteins, we are limited in our understanding of non-covalent SUMO-binding proteins. We identify interactors of different SUMO isoforms-monomeric SUMO1, monomeric SUMO2, or linear trimeric SUMO2 chains-using a mass spectrometry-based proteomics approach. We identify 379 proteins that bind to different SUMO isoforms, mainly in a preferential manner. Interestingly, XRCC4 is the only DNA repair protein in our screen with a preference for SUMO2 trimers over mono-SUMO2, as well as the only protein in our screen that belongs to the non-homologous end joining (NHEJ) DNA double-strand break repair pathway. A SUMO interaction motif (SIM) in XRCC4 regulates its recruitment to sites of DNA damage and phosphorylation of S320 by DNA-PKcs. Our data highlight the importance of non-covalent and covalent sumoylation for DNA double-strand break repair via the NHEJ pathway and provide a resource of SUMO isoform interactors. Show less
Cano-Linares, M.I.; Yanez-Vilches, A.; Garcia-Rodriguez, N.; Barrientos-Moreno, M.; Gonzalez Prieto, R.; San-Segundo, P.; ... ; Prado, F. 2021
DNA damage tolerance relies on homologous recombination (HR) and translesion synthesis (TLS) mechanisms to fill in the ssDNA gaps generated during passing of the replication fork over DNA lesions... Show moreDNA damage tolerance relies on homologous recombination (HR) and translesion synthesis (TLS) mechanisms to fill in the ssDNA gaps generated during passing of the replication fork over DNA lesions in the template. Whereas TLS requires specialized polymerases able to incorporate a dNTP opposite the lesion and is error-prone, HR uses the sister chromatid and is mostly error-free. We report that the HR protein Rad52-but not Rad51 and Rad57-acts in concert with the TLS machinery (Rad6/Rad18-mediated PCNA ubiquitylation and polymerases Rev1/Pol zeta) to repair MMS and UV light-induced ssDNA gaps through a non-recombinogenic mechanism, as inferred from the different phenotypes displayed in the absence of Rad52 and Rad54 (essential for MMS- and UV-induced HR); accordingly, Rad52 is required for efficient DNA damage-induced mutagenesis. In addition, Rad52, Rad51, and Rad57, but not Rad54, facilitate Rad6/Rad18 binding to chromatin and subsequent DNA damage-induced PCNA ubiquitylation. Therefore, Rad52 facilitates the tolerance process not only by HR but also by TLS through Rad51/Rad57-dependent and -independent processes, providing a novel role for the recombination proteins in maintaining genome integrity. 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
Liu, S.J.; Gonzalez Prieto, R.; Zhang, M.D.; Geurink, P.P.; Kooij, R.; Iyengar, P.V.; ... ; Dijke, P. ten 2020
Purpose: Therapies directed to specific molecular targets are still unmet for patients with triple-negative breast cancer (TNBC). Deubiquitinases (DUB) are emerging drug targets. The identification... Show morePurpose: Therapies directed to specific molecular targets are still unmet for patients with triple-negative breast cancer (TNBC). Deubiquitinases (DUB) are emerging drug targets. The identification of highly active DUBs in TNBC may lead to novel therapies.Experimental Design: Using DUB activity probes, we profiled global DUB activities in 52 breast cancer cell lines and 52 patients' tumor tissues. To validate our findings in vivo, we employed both zebrafish and murine breast cancer xenograft models. Cellular and molecular mechanisms were elucidated using in vivo and in vitro biochemical methods. A specific inhibitor was synthesized, and its biochemical and biological functions were assessed in a range of assays. Finally, we used patient sera samples to investigate clinical correlations.Results: Two DUB activity profiling approaches identified UCHL1 as being highly active in TNBC cell lines and aggressive tumors. Functionally, UCHL1 promoted metastasis in zebrafish and murine breast cancer xenograft models. Mechanistically, UCHL1 facilitates TGFb signaling-induced metastasis by protecting TGFb type I receptor and SMAD2 from ubiquitination. We found that these responses are potently suppressed by the specific UCHL1 inhibitor, 6RK73. Furthermore, UCHL1 levels were significantly increased in sera of patients with TNBC, and highly enriched in sera exosomes as well as TNBC cell-conditioned media. UCHL1-enriched exosomes stimulated breast cancer migration and extravasation, suggesting that UCHL1 may act in a paracrine manner to promote tumor progression.Conclusions: Our DUB activity profiling identified UCHL1 as a candidate oncoprotein that promotes TGFb-induced breast cancer metastasis and may provide a potential target for TNBC treatment. Show less
Salas-Lloret, D.; Agabitini, G.; Gonzalez Prieto, R. 2019
Protein modification by Ubiquitin or Ubiquitin-like modifiers is mediated by an enzyme cascade composed of E1, E2, and E3 enzymes. E1s, or ubiquitin-activating enzymes, perform ubiquitin activation... Show moreProtein modification by Ubiquitin or Ubiquitin-like modifiers is mediated by an enzyme cascade composed of E1, E2, and E3 enzymes. E1s, or ubiquitin-activating enzymes, perform ubiquitin activation. Next, ubiquitin is transferred to ubiquitin-conjugating enzymes or E2s. Finally, ubiquitin ligases or E3s catalyze the transfer of ubiquitin to the acceptor proteins. E3 enzymes are responsible for determining the substrate specificity. Determining which E3 enzyme maps to which substrate is a major challenge that is greatly facilitated by the TULIP2 methodology. TULIP2 methodology is fast, precise, and cost-effective. Compared to the previous TULIP methodology protocol, TULIP2 methodology achieves a more than 50-fold improvement in the purification yield and two orders of magnitude improvement in the signal-to-background ratio after label free quantification by mass spectrometry analysis. The method includes the generation of TULIP2 cell lines, subsequent purification of TULIP2 conjugates, preparation, and analysis of samples by mass spectrometry. Show less
