RESUMO
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 apply a single-step protein-complex isolation coupled to mass spectrometry analysis and identified DDA1 as a CSA interacting protein. Cryo-EM analysis shows that DDA1 is an integral component of the CRL4CSA complex. Functional analysis reveals that DDA1 coordinates ubiquitination dynamics during TC-NER and is required for efficient turnover and progression of this process.
Assuntos
Reparo do DNA , Proteínas de Ligação a DNA , Transcrição Gênica , Ubiquitinação , Humanos , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas Culina/metabolismo , Proteínas Culina/genética , Enzimas Reparadoras do DNA/metabolismo , Enzimas Reparadoras do DNA/genética , Microscopia Crioeletrônica , Células HEK293 , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genética , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/genética , Dano ao DNA , RNA Polimerase II/metabolismo , Ligação Proteica , Reparo por Excisão , Proteínas de Transporte , DNA Helicases , Fatores de Transcrição , Receptores de Interleucina-17RESUMO
DNA-protein crosslinks (DPCs) arise from enzymatic intermediates, metabolism or chemicals like chemotherapeutics. DPCs are highly cytotoxic as they impede DNA-based processes such as replication, which is counteracted through proteolysis-mediated DPC removal by spartan (SPRTN) or the proteasome. However, whether DPCs affect transcription and how transcription-blocking DPCs are repaired remains largely unknown. Here we show that DPCs severely impede RNA polymerase II-mediated transcription and are preferentially repaired in active genes by transcription-coupled DPC (TC-DPC) repair. TC-DPC repair is initiated by recruiting the transcription-coupled nucleotide excision repair (TC-NER) factors CSB and CSA to DPC-stalled RNA polymerase II. CSA and CSB are indispensable for TC-DPC repair; however, the downstream TC-NER factors UVSSA and XPA are not, a result indicative of a non-canonical TC-NER mechanism. TC-DPC repair functions independently of SPRTN but is mediated by the ubiquitin ligase CRL4CSA and the proteasome. Thus, DPCs in genes are preferentially repaired in a transcription-coupled manner to facilitate unperturbed transcription.
Assuntos
DNA Helicases , Enzimas Reparadoras do DNA , Reparo do DNA , Proteínas de Ligação a Poli-ADP-Ribose , Proteólise , RNA Polimerase II , Transcrição Gênica , Humanos , Proteínas de Transporte , DNA/metabolismo , DNA/genética , Dano ao DNA , DNA Helicases/metabolismo , DNA Helicases/genética , Enzimas Reparadoras do DNA/metabolismo , Enzimas Reparadoras do DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Células HEK293 , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Receptores de Interleucina-17 , RNA Polimerase II/metabolismo , RNA Polimerase II/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genéticaRESUMO
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.
RESUMO
The brittle hair syndrome Trichothiodystrophy (TTD) is characterized by variable clinical features, including photosensitivity, ichthyosis, growth retardation, microcephaly, intellectual disability, hypogonadism, and anaemia. TTD-associated mutations typically cause unstable mutant proteins involved in various steps of gene expression, severely reducing steady-state mutant protein levels. However, to date, no such link to instability of gene-expression factors for TTD-associated mutations in MPLKIP/TTDN1 has been established. Here, we present seven additional TTD individuals with MPLKIP mutations from five consanguineous families, with a newly identified MPLKIP variant in one family. By mass spectrometry-based interaction proteomics, we demonstrate that MPLKIP interacts with core splicing factors and the lariat debranching protein DBR1. MPLKIP-deficient primary fibroblasts have reduced steady-state DBR1 protein levels. Using Human Skin Equivalents (HSEs), we observed impaired keratinocyte differentiation associated with compromised splicing and eventually, an imbalanced proteome affecting skin development and, interestingly, also the immune system. Our data show that MPLKIP, through its DBR1 stabilizing role, is implicated in mRNA splicing, which is of particular importance in highly differentiated tissue.
Assuntos
Síndromes de Tricotiodistrofia , Humanos , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Consanguinidade , Mutação , Fenótipo , Splicing de RNA , Síndromes de Tricotiodistrofia/genética , Síndromes de Tricotiodistrofia/metabolismoRESUMO
Transcription-blocking lesions are specifically targeted by transcription-coupled nucleotide excision repair (TC-NER), which prevents DNA damage-induced cellular toxicity and maintains proper transcriptional processes. TC-NER is initiated by the stalling of RNA polymerase II (RNAPII), which triggers the assembly of TC-NER-specific proteins, namely CSB, CSA and UVSSA, which collectively control and drive TC-NER progression. Previous research has revealed molecular functions for these proteins, however, exact mechanisms governing the initiation and regulation of TC-NER, particularly at low UV doses have remained elusive, partly due to technical constraints. In this study, we employ knock-in cell lines designed to target the endogenous CSB gene locus with mClover, a GFP variant. Through live cell imaging, we uncover the intricate molecular dynamics of CSB in response to physiologically relevant UV doses. We showed that the DNA damage-induced association of CSB with chromatin is tightly regulated by the CSA-containing ubiquitin-ligase CRL complex (CRL4CSA). Combining the CSB-mClover knock-in cell line with SILAC-based GFP-mediated complex isolation and mass-spectrometry-based proteomics, revealed novel putative CSB interactors as well as discernible variations in complex composition during distinct stages of TC-NER progression. Our work not only provides molecular insight into TC-NER, but also illustrates the versatility of endogenously tagging fluorescent and affinity tags.
Assuntos
Dano ao DNA , Reparo do DNA , Linhagem Celular , Cromatina , Espectrometria de MassasRESUMO
The SWI/SNF family of ATP-dependent chromatin remodeling complexes is implicated in multiple DNA damage response mechanisms and frequently mutated in cancer. The BAF, PBAF and ncBAF complexes are three major types of SWI/SNF complexes that are functionally distinguished by their exclusive subunits. Accumulating evidence suggests that double-strand breaks (DSBs) in transcriptionally active DNA are preferentially repaired by a dedicated homologous recombination pathway. We show that different BAF, PBAF and ncBAF subunits promote homologous recombination and are rapidly recruited to DSBs in a transcription-dependent manner. The PBAF and ncBAF complexes promote RNA polymerase II eviction near DNA damage to rapidly initiate transcriptional silencing, while the BAF complex helps to maintain this transcriptional silencing. Furthermore, ARID1A-containing BAF complexes promote RNaseH1 and RAD52 recruitment to facilitate R-loop resolution and DNA repair. Our results highlight how multiple SWI/SNF complexes perform different functions to enable DNA repair in the context of actively transcribed genes.
Assuntos
Proteínas Cromossômicas não Histona , Estruturas R-Loop , Montagem e Desmontagem da Cromatina/genética , Proteínas Cromossômicas não Histona/metabolismo , DNA , Reparo do DNA/genética , Recombinação Homóloga/genética , HumanosRESUMO
UV-DDB, consisting of subunits DDB1 and DDB2, recognizes UV-induced photoproducts during global genome nucleotide excision repair (GG-NER). We recently demonstrated a noncanonical role of UV-DDB in stimulating base excision repair (BER) which raised several questions about the timing of UV-DDB arrival at 8-oxoguanine (8-oxoG), and the dependency of UV-DDB on the recruitment of downstream BER and NER proteins. Using two different approaches to introduce 8-oxoG in cells, we show that DDB2 is recruited to 8-oxoG immediately after damage and colocalizes with 8-oxoG glycosylase (OGG1) at sites of repair. 8-oxoG removal and OGG1 recruitment is significantly reduced in the absence of DDB2. NER proteins, XPA and XPC, also accumulate at 8-oxoG. While XPC recruitment is dependent on DDB2, XPA recruitment is DDB2-independent and transcription-coupled. Finally, DDB2 accumulation at 8-oxoG induces local chromatin unfolding. We propose that DDB2-mediated chromatin decompaction facilitates the recruitment of downstream BER proteins to 8-oxoG lesions.
Assuntos
Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Guanina/análogos & derivados , Linhagem Celular Tumoral , Cromatina/metabolismo , Montagem e Desmontagem da Cromatina , Dano ao DNA/efeitos da radiação , DNA Glicosilases/metabolismo , Proteínas de Ligação a DNA/genética , Técnicas de Silenciamento de Genes , Técnicas de Inativação de Genes , Guanina/metabolismo , Guanina/efeitos da radiação , Células HEK293 , Humanos , Raios Ultravioleta/efeitos adversos , Proteína de Xeroderma Pigmentoso Grupo A/genética , Proteína de Xeroderma Pigmentoso Grupo A/metabolismoRESUMO
Damage to cellular macromolecules and organelles by chemical exposure evokes activation of various stress response pathways. To what extent different chemical stressors activate common and stressor-specific pathways is largely unknown. Here, we used quantitative phosphoproteomics to compare the signaling events induced by four stressors with different modes of action: the DNA damaging agent: cisplatin (CDDP), the topoisomerase II inhibitor: etoposide (ETO), the pro-oxidant: diethyl maleate (DEM) and the immunosuppressant: cyclosporine A (CsA) administered at an equitoxic dose to mouse embryonic stem cells. We observed major differences between the stressors in the number and identity of responsive phosphosites and the amplitude of phosphorylation. Kinase motif and pathway analyses indicated that the DNA damage response (DDR) activation by CDDP occurs predominantly through the replication-stress-related Atr kinase, whereas ETO triggers the DDR through Atr as well as the DNA double-strand-break-associated Atm kinase. CsA shares with ETO activation of CK2 kinase. Congruent with their known modes of action, CsA-mediated signaling is related to down-regulation of pathways that control hematopoietic differentiation and immunity, whereas oxidative stress is the most prominent initiator of DEM-modulated stress signaling. This study shows that even at equitoxic doses, different stressors induce distinctive and complex phosphorylation signaling cascades.
Assuntos
Proteoma/metabolismo , Animais , Proteínas Mutadas de Ataxia Telangiectasia , Diferenciação Celular , Cisplatino/toxicidade , Quebras de DNA de Cadeia Dupla , Etoposídeo/toxicidade , Humanos , Camundongos , Estresse Oxidativo , Fosforilação , Transdução de Sinais , Inibidores da Topoisomerase IIRESUMO
The effectiveness of many DNA-damaging chemotherapeutic drugs depends on their ability to form monoadducts, intrastrand crosslinks and/or interstrand crosslinks (ICLs) that interfere with transcription and replication. The ERCC1-XPF endonuclease plays a critical role in removal of these lesions by incising DNA either as part of nucleotide excision repair (NER) or interstrand crosslink repair (ICLR). Engagement of ERCC1-XPF in NER is well characterized and is facilitated by binding to the XPA protein. However, ERCC1-XPF recruitment to ICLs is less well understood. Moreover, specific mutations in XPF have been found to disrupt its function in ICLR but not in NER, but whether this involves differences in lesion targeting is unknown. Here, we imaged GFP-tagged ERCC1, XPF and ICLR-defective XPF mutants to investigate how in human cells ERCC1-XPF is localized to different types of psoralen-induced DNA lesions, repaired by either NER or ICLR. Our results confirm its dependence on XPA in NER and furthermore show that its engagement in ICLR is dependent on FANCD2. Interestingly, we find that two ICLR-defective XPF mutants (R689S and S786F) are less well recruited to ICLs. These studies highlight the differential mechanisms that regulate ERCC1-XPF activity in DNA repair.
Assuntos
Proteínas de Ligação a DNA/genética , Endonucleases/genética , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/genética , Proteína de Xeroderma Pigmentoso Grupo A/genética , Linhagem Celular , DNA/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , Ficusina/farmacologia , Humanos , Mutação/efeitos dos fármacosRESUMO
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-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 ubiquitin-like 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.
Assuntos
DNA Helicases/genética , Enzimas Reparadoras do DNA/genética , Reparo do DNA , Proteínas de Ligação a Poli-ADP-Ribose/genética , Processamento de Proteína Pós-Traducional , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genética , Fatores de Transcrição/genética , Transcrição Gênica , Ubiquitina/genética , Linhagem Celular Transformada , Linhagem Celular Tumoral , DNA Helicases/metabolismo , Enzimas Reparadoras do DNA/metabolismo , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Células Epiteliais/efeitos da radiação , Fibroblastos/citologia , Fibroblastos/metabolismo , Fibroblastos/efeitos da radiação , Regulação da Expressão Gênica , Redes Reguladoras de Genes , Humanos , Osteoblastos/citologia , Osteoblastos/metabolismo , Osteoblastos/efeitos da radiação , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Sumoilação , Fatores de Transcrição/metabolismo , Ubiquitina/metabolismo , Ubiquitinação , Raios UltravioletaRESUMO
Transcription-coupled nucleotide excision repair (TC-NER) is a dedicated DNA repair pathway that removes transcription-blocking DNA lesions (TBLs). TC-NER is initiated by the recognition of lesion-stalled RNA Polymerase II by the joint action of the TC-NER factors Cockayne Syndrome protein A (CSA), Cockayne Syndrome protein B (CSB) and UV-Stimulated Scaffold Protein A (UVSSA). However, the exact recruitment mechanism of these factors toward TBLs remains elusive. Here, we study the recruitment mechanism of UVSSA using live-cell imaging and show that UVSSA accumulates at TBLs independent of CSA and CSB. Furthermore, using UVSSA deletion mutants, we could separate the CSA interaction function of UVSSA from its DNA damage recruitment activity, which is mediated by the UVSSA VHS and DUF2043 domains, respectively. Quantitative interaction proteomics showed that the Spt16 subunit of the histone chaperone FACT interacts with UVSSA, which is mediated by the DUF2043 domain. Spt16 is recruited to TBLs, independently of UVSSA, to stimulate UVSSA recruitment and TC-NER-mediated repair. Spt16 specifically affects UVSSA, as Spt16 depletion did not affect CSB recruitment, highlighting that different chromatin-modulating factors regulate different reaction steps of the highly orchestrated TC-NER pathway.
Assuntos
Proteínas de Transporte/genética , Proteínas de Ciclo Celular/genética , Reparo do DNA , Proteínas de Ligação a DNA/genética , DNA/genética , Proteínas de Grupo de Alta Mobilidade/genética , RNA Polimerase II/genética , Fatores de Transcrição/genética , Transcrição Gênica , Fatores de Elongação da Transcrição/genética , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Transformada , Linhagem Celular Tumoral , Cromatina/metabolismo , Cromatina/ultraestrutura , DNA/metabolismo , Quebras de DNA de Cadeia Dupla , DNA Helicases/genética , DNA Helicases/metabolismo , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Fibroblastos/metabolismo , Fibroblastos/ultraestrutura , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Proteínas de Grupo de Alta Mobilidade/metabolismo , Humanos , Imagem Óptica , Osteoblastos/metabolismo , Osteoblastos/ultraestrutura , Proteínas de Ligação a Poli-ADP-Ribose/genética , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Ligação Proteica , Domínios Proteicos , Transporte Proteico , RNA Polimerase II/metabolismo , Fatores de Transcrição/metabolismo , Fatores de Elongação da Transcrição/metabolismoRESUMO
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 TRiC substrate and reveal that TRiC regulates CSA-dependent TC-NER and the development of CS.
Assuntos
Chaperonina com TCP-1/metabolismo , Dano ao DNA/efeitos da radiação , Enzimas Reparadoras do DNA/metabolismo , Fatores de Transcrição/metabolismo , Raios Ultravioleta , Western Blotting , Linhagem Celular Tumoral , Sobrevivência Celular/genética , Sobrevivência Celular/efeitos da radiação , Chaperonina com TCP-1/genética , Síndrome de Cockayne/genética , Dano ao DNA/genética , Enzimas Reparadoras do DNA/genética , Humanos , Imunoprecipitação , Espectrometria de Massas , Microscopia de Fluorescência , Mutação/genética , Interferência de RNA , Fatores de Transcrição/genética , Transcrição Gênica/genética , Transcrição Gênica/efeitos da radiaçãoRESUMO
Urinary 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) is a widely measured biomarker of oxidative stress. It has been commonly assumed to be a product of DNA repair, and therefore reflective of DNA oxidation. However, the source of urinary 8-oxodGuo is not understood, although potential confounding contributions from cell turnover and diet have been ruled out. Clearly it is critical to understand the precise biological origins of this important biomarker, so that the target molecule that is oxidised can be identified, and the significance of its excretion can be interpreted fully. In the present study we aimed to assess the contributions of nucleotide excision repair (NER), by both the global genome NER (GG-NER) and transcription-coupled NER (TC-NER) pathways, and sanitisation of the dGTP pool (e.g. via the activity of the MTH1 protein), on the production of 8-oxodGuo, using selected genetically-modified mice. In xeroderma pigmentosum A (XPA) mice, in which GG-NER and TC-NER are both defective, the urinary 8-oxodGuo data were unequivocal in ruling out a contribution from NER. In line with the XPA data, the production of urinary 8-oxodGuo was not affected in the xeroderma pigmentosum C mice, specifically excluding a role of the GG-NER pathway. The bulk of the literature supports the mechanism that the NER proteins are responsible for removing damage to the transcribed strand of DNA via TC-NER, and on this basis we also examined Cockayne Syndrome mice, which have a functional loss of TC-NER. These mice showed no difference in urinary 8-oxodGuo excretion, compared to wild type, demonstrating that TC-NER does not contribute to urinary 8-oxodGuo levels. These findings call into question whether genomic DNA is the primary source of urinary 8-oxodGuo, which would largely exclude it as a biomarker of DNA oxidation. The urinary 8-oxodGuo levels from the MTH1 mice (both knock-out and hMTH1-Tg) were not significantly different to the wild-type mice. We suggest that these findings are due to redundancy in the process, and that other enzymes substitute for the lack of MTH1, however the present study cannot determine whether or not the 2'-deoxyribonucleotide pool is the source of urinary 8-oxodGuo. On the basis of the above, urinary 8-oxodGuo is most accurately defined as a non-invasive biomarker of oxidative stress, derived from oxidatively generated damage to 2'-deoxyguanosine.
Assuntos
Síndrome de Cockayne/urina , Desoxiguanosina/análogos & derivados , Desoxiguanosina/metabolismo , Estresse Oxidativo , Xeroderma Pigmentoso/urina , 8-Hidroxi-2'-Desoxiguanosina , Animais , Biomarcadores/urina , Síndrome de Cockayne/genética , Síndrome de Cockayne/patologia , DNA/metabolismo , Dano ao DNA , Reparo do DNA , Nucleotídeos de Desoxiguanina/metabolismo , Desoxiguanosina/urina , Modelos Animais de Doenças , Feminino , Expressão Gênica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Monoéster Fosfórico Hidrolases/deficiência , Monoéster Fosfórico Hidrolases/genética , Xeroderma Pigmentoso/genética , Xeroderma Pigmentoso/patologia , Proteína de Xeroderma Pigmentoso Grupo A/genética , Proteína de Xeroderma Pigmentoso Grupo A/metabolismoRESUMO
The response to DNA double-strand breaks (DSBs) requires alterations in chromatin structure to promote the assembly of repair complexes on broken chromosomes. Non-homologous end-joining (NHEJ) is the dominant DSB repair pathway in human cells, but our understanding of how it operates in chromatin is limited. Here, we define a mechanism that plays a crucial role in regulating NHEJ in chromatin. This mechanism is initiated by DNA damage-associated poly(ADP-ribose) polymerase 1 (PARP1), which recruits the chromatin remodeler CHD2 through a poly(ADP-ribose)-binding domain. CHD2 in turn triggers rapid chromatin expansion and the deposition of histone variant H3.3 at sites of DNA damage. Importantly, we find that PARP1, CHD2, and H3.3 regulate the assembly of NHEJ complexes at broken chromosomes to promote efficient DNA repair. Together, these findings reveal a PARP1-dependent process that couples ATP-dependent chromatin remodeling with histone variant deposition at DSBs to facilitate NHEJ and safeguard genomic stability.
Assuntos
Cromatina/genética , Reparo do DNA por Junção de Extremidades , Proteínas de Ligação a DNA/metabolismo , Histonas/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Linhagem Celular Tumoral , Montagem e Desmontagem da Cromatina , Quebras de DNA de Cadeia Dupla , Instabilidade Genômica , Células HEK293 , Humanos , Poli(ADP-Ribose) Polimerase-1RESUMO
DNA damage response signaling is crucial for genome maintenance in all organisms and is corrupted in cancer. In an RNA interference (RNAi) screen for (de)ubiquitinases and sumoylases modulating the apoptotic response of embryonic stem (ES) cells to DNA damage, we identified the E3 ubiquitin ligase/ISGylase, ariadne homologue 1 (ARIH1). Silencing ARIH1 sensitized ES and cancer cells to genotoxic compounds and ionizing radiation, irrespective of their p53 or caspase-3 status. Expression of wild-type but not ubiquitinase-defective ARIH1 constructs prevented sensitization caused by ARIH1 knockdown. ARIH1 protein abundance increased after DNA damage through attenuation of proteasomal degradation that required ATM signaling. Accumulated ARIH1 associated with 4EHP, and in turn, this competitive inhibitor of the eukaryotic translation initiation factor 4E (eIF4E) underwent increased nondegradative ubiquitination upon DNA damage. Genotoxic stress led to an enrichment of ARIH1 in perinuclear, ribosome-containing regions and triggered 4EHP association with the mRNA 5' cap as well as mRNA translation arrest in an ARIH1-dependent manner. Finally, restoration of DNA damage-induced translation arrest in ARIH1-depleted cells by means of an eIF2 inhibitor was sufficient to reinstate resistance to genotoxic stress. These findings identify ARIH1 as a potent mediator of DNA damage-induced translation arrest that protects stem and cancer cells against genotoxic stress.
Assuntos
Proteínas de Transporte/metabolismo , Dano ao DNA , Fator de Iniciação 4E em Eucariotos/metabolismo , Proteínas de Ligação ao Cap de RNA/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Proteínas de Transporte/genética , Caspase 3/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Células-Tronco Embrionárias/metabolismo , Humanos , Camundongos , Biossíntese de Proteínas , Interferência de RNA , RNA Mensageiro/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Ubiquitina-Proteína Ligases/genética , UbiquitinaçãoRESUMO
Nucleotide excision repair (NER) is a key component of the DNA damage response (DDR) and it is essential to safeguard genome integrity against genotoxic insults. The regulation of NER is primarily mediated by protein post-translational modifications (PTMs). The NER machinery removes a wide spectrum of DNA helix distorting lesions, including those induced by solar radiation, through two sub-pathways: global genome nucleotide excision repair (GG-NER) and transcription coupled nucleotide excision repair (TC-NER). Severe clinical consequences associated with inherited NER defects, including premature ageing, neurodegeneration and extreme cancer-susceptibility, underscore the biological relevance of NER. In the last two decades most of the core NER machinery has been elaborately described, shifting attention to molecular mechanisms that either facilitate NER in the context of chromatin or promote the timely and accurate interplay between NER factors and various post-translational modifications. In this review, we summarize and discuss the latest findings in NER. In particular, we focus on emerging factors and novel molecular mechanisms by which NER is regulated.
Assuntos
Reparo do DNA/genética , Processamento de Proteína Pós-Traducional , Transcrição Gênica/genética , Animais , HumanosRESUMO
Distinct types of DNA damage elicit signaling and repair pathways that counteract the adverse effect of DNA lesions to maintain genome stability. The negatively charged polymer poly(ADP-ribose), which is catalyzed by poly(ADP-ribose) polymerase (PARP) enzymes, is a post-translational modification that serves as a chromatin-based platform for the recruitment of a variety of repair factors and chromatin-remodeling enzymes. Recent work implicates PARP3 in the efficient joining of DNA double-strand breaks during non-homologous end-joining (NHEJ), whereas PARP1 modulates the repair of UV-induced DNA lesions. Here we discuss emerging roles of PARP enzymes in mechanistically distinct DNA repair pathways and highlight unresolved issues and questions for future research.
Assuntos
Dano ao DNA , Reparo do DNA , Poli(ADP-Ribose) Polimerases/metabolismo , Raios UltravioletaRESUMO
In pluripotent stem cells, DNA damage triggers loss of pluripotency and apoptosis as a safeguard to exclude damaged DNA from the lineage. An intricate DNA damage response (DDR) signaling network ensures that the response is proportional to the severity of the damage. We combined an RNA interference screen targeting all kinases, phosphatases, and transcription factors with global transcriptomics and phosphoproteomics to map the DDR in mouse embryonic stem cells treated with the DNA cross-linker cisplatin. Networks derived from canonical pathways shared in all three data sets were implicated in DNA damage repair, cell cycle and survival, and differentiation. Experimental probing of these networks identified a mode of DNA damage-induced Wnt signaling that limited apoptosis. Silencing or deleting the p53 gene demonstrated that genotoxic stress elicited Wnt signaling in a p53-independent manner. Instead, this response occurred through reduced abundance of Csnk1a1 (CK1α), a kinase that inhibits ß-catenin. Together, our findings reveal a balance between p53-mediated elimination of stem cells (through loss of pluripotency and apoptosis) and Wnt signaling that attenuates this response to tune the outcome of the DDR.
Assuntos
Caseína Quinase I/metabolismo , Dano ao DNA , Células-Tronco Embrionárias/enzimologia , Células-Tronco Pluripotentes/enzimologia , Biologia de Sistemas , Via de Sinalização Wnt , Animais , Apoptose/genética , Caseína Quinase I/genética , Linhagem Celular , Células-Tronco Embrionárias/citologia , Camundongos , Células-Tronco Pluripotentes/citologia , Interferência de RNA , Transcriptoma/genética , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
A hyperpolarized 129Xe contrast agent composed of many cryptophane-A molecular cages assembled on an M13 bacteriophage has been demonstrated. Saturation of xenon bound in the large number of cryptophane cages is transferred to the pool of aqueous-solvated xenon via chemical exchange, resulting in efficient generation of hyperCEST contrast. No significant loss of contrast per cryptophane cage was observed for the multivalent phage when compared with unscaffolded cryptophane. Detection of this phage-based hyperCEST agent is reported at concentrations as low as 230 fM, representing the current lower limit for NMR/MRI-based contrast agents.
Assuntos
Bacteriófagos/química , Técnicas Biossensoriais/métodos , Portadores de Fármacos/química , Espectroscopia de Ressonância Magnética/métodos , Imagem Molecular/métodos , Compostos Policíclicos/química , Isótopos de Xenônio/análise , Algoritmos , Meios de Contraste/análise , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Isótopos de Xenônio/químicaRESUMO
The WD40-repeat protein DDB2 is essential for efficient recognition and subsequent removal of ultraviolet (UV)-induced DNA lesions by nucleotide excision repair (NER). However, how DDB2 promotes NER in chromatin is poorly understood. Here, we identify poly(ADP-ribose) polymerase 1 (PARP1) as a novel DDB2-associated factor. We demonstrate that DDB2 facilitated poly(ADP-ribosyl)ation of UV-damaged chromatin through the activity of PARP1, resulting in the recruitment of the chromatin-remodeling enzyme ALC1. Depletion of ALC1 rendered cells sensitive to UV and impaired repair of UV-induced DNA lesions. Additionally, DDB2 itself was targeted by poly(ADP-ribosyl)ation, resulting in increased protein stability and a prolonged chromatin retention time. Our in vitro and in vivo data support a model in which poly(ADP-ribosyl)ation of DDB2 suppresses DDB2 ubiquitylation and outline a molecular mechanism for PARP1-mediated regulation of NER through DDB2 stabilization and recruitment of the chromatin remodeler ALC1.