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1.
bioRxiv ; 2024 Jul 22.
Artigo em Inglês | MEDLINE | ID: mdl-39091731

RESUMO

Transcription-coupled DNA repair (TCR) removes bulky DNA lesions impeding RNA polymerase II (RNAPII) transcription. Recent studies have outlined the stepwise assembly of TCR factors CSB, CSA, UVSSA, and TFIIH around lesion-stalled RNAPII. However, the mechanism and factors required for the transition to downstream repair steps, including RNAPII removal to provide repair proteins access to the DNA lesion, remain unclear. Here, we identify STK19 as a new TCR factor facilitating this transition. Loss of STK19 does not impact initial TCR complex assembly or RNAPII ubiquitylation but delays lesion-stalled RNAPII clearance, thereby interfering with the downstream repair reaction. Cryo-EM and mutational analysis reveal that STK19 associates with the TCR complex, positioning itself between RNAPII, UVSSA, and CSA. The structural insights and molecular modeling suggest that STK19 positions the ATPase subunits of TFIIH onto DNA in front of RNAPII. Together, these findings provide new insights into the factors and mechanisms required for TCR.

2.
Nat Cell Biol ; 26(5): 797-810, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38600235

RESUMO

Covalent DNA-protein cross-links (DPCs) are toxic DNA lesions that block replication and require repair by multiple pathways. Whether transcription blockage contributes to the toxicity of DPCs and how cells respond when RNA polymerases stall at DPCs is unknown. Here we find that DPC formation arrests transcription and induces ubiquitylation and degradation of RNA polymerase II. Using genetic screens and a method for the genome-wide mapping of DNA-protein adducts, DPC sequencing, we discover that Cockayne syndrome (CS) proteins CSB and CSA provide resistance to DPC-inducing agents by promoting DPC repair in actively transcribed genes. Consequently, CSB- or CSA-deficient cells fail to efficiently restart transcription after induction of DPCs. In contrast, nucleotide excision repair factors that act downstream of CSB and CSA at ultraviolet light-induced DNA lesions are dispensable. Our study describes a transcription-coupled DPC repair pathway and suggests that defects in this pathway may contribute to the unique neurological features of CS.


Assuntos
Síndrome de Cockayne , DNA Helicases , Enzimas Reparadoras do DNA , Reparo do DNA , Proteínas de Ligação a Poli-ADP-Ribose , RNA Polimerase II , Humanos , Síndrome de Cockayne/genética , Síndrome de Cockayne/metabolismo , Síndrome de Cockayne/patologia , Adutos de DNA/metabolismo , Adutos de DNA/genética , Dano ao DNA , DNA Helicases/metabolismo , DNA Helicases/genética , Enzimas Reparadoras do DNA/metabolismo , Enzimas Reparadoras do DNA/genética , Reparo por Excisão , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/genética , Receptores de Interleucina-17 , RNA Polimerase II/metabolismo , RNA Polimerase II/genética , Fatores de Transcrição , Transcrição Gênica , Ubiquitinação , Raios Ultravioleta
3.
Nat Struct Mol Biol ; 31(3): 536-547, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38316879

RESUMO

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 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.


Assuntos
RNA Polimerase II , Transcrição Gênica , RNA Polimerase II/metabolismo , Reparo por Excisão , Reparo do DNA , DNA/metabolismo , Ubiquitinação , Ligases , Receptores de Antígenos de Linfócitos T
4.
Proc Natl Acad Sci U S A ; 120(11): e2208860120, 2023 03 14.
Artigo em Inglês | MEDLINE | ID: mdl-36893274

RESUMO

XPA is a central scaffold protein that coordinates the assembly of repair complexes in the global genome (GG-NER) and transcription-coupled nucleotide excision repair (TC-NER) subpathways. Inactivating mutations in XPA cause xeroderma pigmentosum (XP), which is characterized by extreme UV sensitivity and a highly elevated skin cancer risk. Here, we describe two Dutch siblings in their late forties carrying a homozygous H244R substitution in the C-terminus of XPA. They present with mild cutaneous manifestations of XP without skin cancer but suffer from marked neurological features, including cerebellar ataxia. We show that the mutant XPA protein has a severely weakened interaction with the transcription factor IIH (TFIIH) complex leading to an impaired association of the mutant XPA and the downstream endonuclease ERCC1-XPF with NER complexes. Despite these defects, the patient-derived fibroblasts and reconstituted knockout cells carrying the XPA-H244R substitution show intermediate UV sensitivity and considerable levels of residual GG-NER (~50%), in line with the intrinsic properties and activities of the purified protein. By contrast, XPA-H244R cells are exquisitely sensitive to transcription-blocking DNA damage, show no detectable recovery of transcription after UV irradiation, and display a severe deficiency in TC-NER-associated unscheduled DNA synthesis. Our characterization of a new case of XPA deficiency that interferes with TFIIH binding and primarily affects the transcription-coupled subpathway of nucleotide excision repair, provides an explanation of the dominant neurological features in these patients, and reveals a specific role for the C-terminus of XPA in TC-NER.


Assuntos
Neoplasias Cutâneas , Xeroderma Pigmentoso , Humanos , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Alelos , Proteína de Xeroderma Pigmentoso Grupo A/genética , Proteína de Xeroderma Pigmentoso Grupo A/metabolismo , Reparo do DNA/genética , Dano ao DNA/genética , Xeroderma Pigmentoso/genética , Xeroderma Pigmentoso/metabolismo , Neoplasias Cutâneas/genética , Fator de Transcrição TFIIH/genética , Fator de Transcrição TFIIH/metabolismo
5.
Bio Protoc ; 13(3)2023 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-36816995

RESUMO

Nucleotide excision repair (NER) removes a wide variety of structurally unrelated lesions from the genome, including UV-induced photolesions such as 6-4 pyrimidine-pyrimidone photoproducts (6-4PPs) and cyclobutane pyrimidine dimers (CPDs). NER removes lesions by excising a short stretch of single-stranded DNA containing the damaged DNA, leaving a single-stranded gap that is resynthesized in a process called unscheduled DNA synthesis (UDS). Measuring UDS after UV irradiation in non-dividing cells provides a measure of the overall NER activity, of which approximately 90% is carried out by the global genome repair (GGR) sub pathway. Here, we present a protocol for the microscopy-based analysis and quantification of UDS as a measurement for GGR activity. Following local UV-C irradiation, serum-starved human cells are supplemented with the thymidine analogue 5-ethynyl-2'-deoxyuridine (EdU), which is incorporated into repair patches following NER-dependent dual incision. The incorporated nucleotide analogue is coupled to a fluorophore using Click-iT chemistry, followed by immunodetection of CPD photolesions to simultaneously visualize both signals by fluorescence microscopy. Accompanying this protocol is a custom-built ImageJ plug-in to analyze and quantify UDS signals at sites of CPD-marked local damage. The local UDS assay enables an effective and sensitive fluorescence-based quantification of GGR activity in single cells with application in basic research to better understand the regulatory mechanism in NER, as well as in diagnostics to characterize fibroblasts from individuals with NER-deficiency disorder. Graphical abstract.

6.
Nat Commun ; 13(1): 4762, 2022 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-35963869

RESUMO

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 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.


Assuntos
Cromatina , Inibidores de Poli(ADP-Ribose) Polimerases , Cromatina/genética , DNA/genética , DNA/metabolismo , Dano ao DNA , Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Poli Adenosina Difosfato Ribose/metabolismo
7.
DNA Repair (Amst) ; 106: 103192, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34358806

RESUMO

Transcription-blocking DNA lesions (TBLs) in genomic DNA are triggered by a wide variety of DNA-damaging agents. Such lesions cause stalling of elongating RNA polymerase II (RNA Pol II) enzymes and fully block transcription when unresolved. The toxic impact of DNA damage on transcription progression is commonly referred to as transcription stress. In response to RNA Pol II stalling, cells activate and employ transcription-coupled repair (TCR) machineries to repair cytotoxic TBLs and resume transcription. Increasing evidence indicates that the modification and processing of stalled RNA Pol II is an integral component of the cellular response to and the repair of TBLs. If TCR pathways fail, the prolonged stalling of RNA Pol II will impede global replication and transcription as well as block the access of other DNA repair pathways that may act upon the TBL. Consequently, such prolonged stalling will trigger profound genome instability and devastating clinical features. In this review, we will discuss the mechanisms by which various types of TBLs are repaired by distinct TCR pathways and how RNA Pol II processing is regulated during these processes. We will also discuss the clinical consequences of transcription stress and genotype-phenotype correlations of related TCR-deficiency disorders.


Assuntos
Dano ao DNA , Reparo do DNA , Instabilidade Genômica , RNA Polimerase II/metabolismo , Transcrição Gênica , Envelhecimento , Animais , DNA/metabolismo , Eucariotos/enzimologia , Eucariotos/genética , Eucariotos/metabolismo , Humanos
8.
Nat Cell Biol ; 23(6): 595-607, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-34108663

RESUMO

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 CRL4CSA ubiquitin ligase. How CRL4CSA 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 CRL4CSA 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.


Assuntos
Dano ao DNA , Reparo do DNA , Fator 1 de Elongação de Peptídeos/metabolismo , RNA Polimerase II/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , DNA Helicases/genética , DNA Helicases/metabolismo , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/metabolismo , Humanos , Fator 1 de Elongação de Peptídeos/genética , Proteínas de Ligação a Poli-ADP-Ribose/genética , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , RNA Polimerase II/genética , Elongação da Transcrição Genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Ubiquitina-Proteína Ligases/genética
9.
Trends Cell Biol ; 31(5): 359-371, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33685798

RESUMO

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 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.


Assuntos
Reparo do DNA/fisiologia , RNA Polimerase II/metabolismo , Animais , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , DNA/metabolismo , Dano ao DNA/fisiologia , DNA Helicases/metabolismo , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/metabolismo , Humanos , RNA Polimerase II/genética , Ubiquitinação
10.
Nat Commun ; 12(1): 1342, 2021 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-33637760

RESUMO

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 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.


Assuntos
Dano ao DNA/fisiologia , DNA Helicases/metabolismo , Enzimas Reparadoras do DNA/metabolismo , Reparo do DNA/fisiologia , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Fatores de Transcrição/metabolismo , Núcleo Celular , DNA/efeitos da radiação , Humanos , Proteínas de Ligação a Poli-ADP-Ribose/genética , Mapas de Interação de Proteínas , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Fatores de Transcrição/genética , Transcrição Gênica , Raios Ultravioleta
12.
Nat Commun ; 11(1): 2104, 2020 04 30.
Artigo em Inglês | MEDLINE | ID: mdl-32355176

RESUMO

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 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.


Assuntos
Proteínas de Transporte/metabolismo , Dano ao DNA , DNA Helicases/metabolismo , Enzimas Reparadoras do DNA/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , RNA Polimerase II/metabolismo , Fator de Transcrição TFIIH/metabolismo , Fatores de Transcrição/metabolismo , Animais , Linhagem Celular Tumoral , Reparo do DNA , Humanos , Transcrição Gênica , Raios Ultravioleta , Xenopus laevis
13.
Cell ; 180(6): 1228-1244.e24, 2020 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-32142649

RESUMO

Transcription-coupled nucleotide excision repair (TC-NER) is initiated by the stalling of elongating RNA polymerase II (RNAPIIo) at DNA lesions. The ubiquitination of RNAPIIo in response to DNA damage is an evolutionarily conserved event, but its function in mammals is unknown. Here, we identified a single DNA damage-induced ubiquitination site in RNAPII at RPB1-K1268, which regulates transcription recovery and DNA damage resistance. Mechanistically, RPB1-K1268 ubiquitination stimulates the association of the core-TFIIH complex with stalled RNAPIIo through a transfer mechanism that also involves UVSSA-K414 ubiquitination. We developed a strand-specific ChIP-seq method, which revealed RPB1-K1268 ubiquitination is important for repair and the resolution of transcriptional bottlenecks at DNA lesions. Finally, RPB1-K1268R knockin mice displayed a short life-span, premature aging, and neurodegeneration. Our results reveal RNAPII ubiquitination provides a two-tier protection mechanism by activating TC-NER and, in parallel, the processing of DNA damage-stalled RNAPIIo, which together prevent prolonged transcription arrest and protect against neurodegeneration.


Assuntos
Reparo do DNA/fisiologia , RNA Polimerase II/metabolismo , Animais , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , DNA/metabolismo , Dano ao DNA/fisiologia , DNA Helicases/metabolismo , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/metabolismo , Feminino , Células HCT116 , Células HEK293 , Células HeLa , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , RNA Polimerase II/genética , Ubiquitinação
14.
Sci Rep ; 10(1): 4332, 2020 03 09.
Artigo em Inglês | MEDLINE | ID: mdl-32152397

RESUMO

Transcription-coupled repair (TCR) removes DNA lesions from the transcribed strand of active genes. Stalling of RNA polymerase II (RNAPII) at DNA lesions initiates TCR through the recruitment of the CSB and CSA proteins. The full repertoire of proteins required for human TCR - particularly in a chromatin context - remains to be determined. Studies in mice have revealed that the nucleosome-binding protein HMGN1 is required to enhance the repair of UV-induced lesions in transcribed genes. However, whether HMGN1 is required for human TCR remains unaddressed. Here, we show that knockout or knockdown of HMGN1, either alone or in combination with HMGN2, does not render human cells sensitive to UV light or Illudin S-induced transcription-blocking DNA lesions. Moreover, transcription restart after UV irradiation was not impaired in HMGN-deficient cells. In contrast, TCR-deficient cells were highly sensitive to DNA damage and failed to restart transcription. Furthermore, GFP-tagged HMGN1 was not recruited to sites of UV-induced DNA damage under conditions where GFP-CSB readily accumulated. In line with this, HMGN1 did not associate with the TCR complex, nor did TCR proteins require HMGN1 to associate with DNA damage-stalled RNAPII. Together, our findings suggest that HMGN1 and HMGN2 are not required for human TCR.


Assuntos
Reparo do DNA , Proteína HMGN1/genética , Proteína HMGN2/genética , Transcrição Gênica , Linhagem Celular , Dano ao DNA/genética , Dano ao DNA/efeitos da radiação , Técnicas de Inativação de Genes , Proteína HMGN1/metabolismo , Proteína HMGN2/metabolismo , Humanos , Tolerância a Radiação , Telomerase/genética , Telomerase/metabolismo , Transcrição Gênica/efeitos da radiação , Raios Ultravioleta
15.
Pediatr Allergy Immunol ; 28(6): 579-587, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28686349

RESUMO

BACKGROUND: Previous studies have demonstrated that vitamin D affects T-cell function and maturation via the vitamin D receptor. However, no studies in children have been performed on this topic. Because most of the T-cell memory is formed in the first 5 years of life, we aimed to determine the association between serum 25-hydroxyvitamin D (25(OH)D) levels and numbers of circulatory naive, central memory (Tcm), and effector memory (Tem) T lymphocytes in a large population of healthy children. METHODS: Among 3189 children participating in a population-based prospective cohort, we measured 25(OH)D levels and performed detailed immunophenotyping of naive and memory T lymphocytes at a median age of 6.0 years (95% range 5.7-7.9). Detailed lymphocyte subsets were available in 986 children. Multivariable linear regression analyses were performed to determine the association between 25(OH)D and the maturation of T lymphocytes in children adjusted for cord blood 25(OH)D levels, herpes seropositivity, sociodemographic and lifestyle confounders. Furthermore, multivariable logistic regression analyses were performed to determine associations between 25(OH)D and childhood infections. RESULTS: Higher 25(OH)D levels were associated with higher numbers of Tem lymphocytes. Every 10 nmol/L higher 25(OH)D was associated with 2.20% (95% CI 0.54-3.89; P=.009) higher CD4TemRA, 1.50% (95% CI 0.38-2.62; P=.008) higher CD4TemRO, and 1.82% (95% CI 0.11-3.56; P=.037) higher CD8TemRA cell numbers. Generally, stronger associations were observed among boys. 25(OH)D levels were not significantly associated with naive, Tcm cell numbers, herpes seropositivity, or URTIs. CONCLUSIONS: Our results suggest that vitamin D enhances cellular immunity in young children.


Assuntos
Imunidade Celular , Subpopulações de Linfócitos T/metabolismo , Deficiência de Vitamina D/imunologia , Vitamina D/análogos & derivados , Biomarcadores/sangue , Criança , Feminino , Humanos , Imunofenotipagem , Infecções/imunologia , Modelos Lineares , Modelos Logísticos , Masculino , Estudos Prospectivos , Vitamina D/sangue , Vitamina D/imunologia , Deficiência de Vitamina D/sangue , Deficiência de Vitamina D/diagnóstico
16.
J Infect Dis ; 215(6): 883-888, 2017 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-28453846

RESUMO

Persistent infections with cytomegalovirus (CMV) differentially affect the host immune phenotype in middle-aged males and females. Because CMV already impacts on T-cell memory at a young age, we studied whether these effects were modified by sex in 1,079 children with an average age of 6 years. Sex and CMV independently impacted on multiple B-cell and T-cell subsets. However, there was no significant effect of their interaction. Importantly, the effects of sex and CMV were in part explained by age and infection with other herpesviruses. Thus, immune aging is likely to be more complex, with involvement of hormonal changes with age, socioeconomic status, birth characteristics, and pathogen exposure.


Assuntos
Linfócitos B/imunologia , Infecções por Citomegalovirus/imunologia , Imunofenotipagem , Fatores Sexuais , Linfócitos T/imunologia , Fatores Etários , Austrália , Criança , Pré-Escolar , Citomegalovirus , Feminino , Humanos , Memória Imunológica , Modelos Lineares , Masculino , Estudos Prospectivos
17.
Elife ; 62017 02 27.
Artigo em Inglês | MEDLINE | ID: mdl-28240985

RESUMO

DNA double-strand breaks (DSB) elicit a ubiquitylation cascade that controls DNA repair pathway choice. This cascade involves the ubiquitylation of histone H2A by the RNF168 ligase and the subsequent recruitment of RIF1, which suppresses homologous recombination (HR) in G1 cells. The RIF1-dependent suppression is relieved in S/G2 cells, allowing PALB2-driven HR to occur. With the inhibitory impact of RIF1 relieved, it remains unclear how RNF168-induced ubiquitylation influences HR. Here, we uncover that RNF168 links the HR machinery to H2A ubiquitylation in S/G2 cells. We show that PALB2 indirectly recognizes histone ubiquitylation by physically associating with ubiquitin-bound RNF168. This direct interaction is mediated by the newly identified PALB2-interacting domain (PID) in RNF168 and the WD40 domain in PALB2, and drives DNA repair by facilitating the assembly of PALB2-containing HR complexes at DSBs. Our findings demonstrate that RNF168 couples PALB2-dependent HR to H2A ubiquitylation to promote DNA repair and preserve genome integrity.


Assuntos
DNA/metabolismo , Proteína do Grupo de Complementação N da Anemia de Fanconi/metabolismo , Fibroblastos/metabolismo , Histonas/metabolismo , Reparo de DNA por Recombinação , Ubiquitina-Proteína Ligases/metabolismo , Animais , Ciclo Celular/genética , Ciclo Celular/efeitos da radiação , Linhagem Celular Transformada , Linhagem Celular Tumoral , DNA/genética , Quebras de DNA de Cadeia Dupla/efeitos da radiação , Proteína do Grupo de Complementação N da Anemia de Fanconi/genética , Fibroblastos/citologia , Fibroblastos/efeitos da radiação , Células HEK293 , Histonas/genética , Humanos , Lasers de Excimer , Camundongos , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/metabolismo , Células-Tronco Embrionárias Murinas/efeitos da radiação , Osteoblastos/citologia , Osteoblastos/metabolismo , Osteoblastos/efeitos da radiação , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteínas de Ligação a Telômeros/genética , Proteínas de Ligação a Telômeros/metabolismo , Ubiquitina/genética , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitinação , Raios X
18.
Arch Dis Child ; 102(6): 529-534, 2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-28052882

RESUMO

OBJECTIVE: The aim was to identify whether ethnic differences in coeliac disease autoimmunity (CDA) in children at 6 years of age exist, and when present, to evaluate how these differences may be explained by sociodemographic and environmental factors. DESIGN: This study was embedded within a multi-ethnic population-based prospective cohort study. SETTING AND PATIENTS: 4442 six-year-old children born between 2002 and 2006 were included. Information on ethnicity, environmental and lifestyle characteristics was assessed by questionnaires. Ethnicity was categorised into Western (Dutch, European, Indonesian, American, Oceanian) and non-Western (Turkish, Moroccan, Cape Verdean, Antillean, Surinamese). Serum transglutaminase type 2 antibody (TG2A) levels were measured with fluorescence enzyme immunoassay. Serum IgG levels against cytomegalovirus (CMV) were measured by ELISA. MAIN OUTCOME MEASURES: TG2A positivity was defined as TG2A ≥7 U/mL, strong TG2A positivity as TG2A ≥10 upper limit normal (70 U/mL). RESULTS: Of 4442 children, 60 (1.4%) children were TG2A positive, of whom 31 were strong positive. 66% of children were Western, 33% non-Western. Western ethnicity, high socioeconomic position and daycare attendance were positively associated with strong TG2A positivity (odds ratio (OR) 6.85 (1.62 to 28.8) p<0.01, OR 3.70 (1.40 to 9.82) p<0.01, OR 3.90 (1.38 to 11.0) p=0.01 resp.), whereas CMV seropositivity was inversely related to strong TG2A positivity (OR 0.32 (0.12 to 0.84) p=0.02). Together, these factors explained up to 47% (-67 to -17; p=0.02) of the ethnic differences in TG2A positivity between Western and non-Western children. CONCLUSIONS: Ethnic differences in children with CDA are present in childhood. Socioeconomic position, daycare attendance and CMV seropositivity partly explained these differences, which may serve as targets for prevention strategies for CDA.


Assuntos
Doenças Autoimunes/etnologia , Doença Celíaca/etnologia , Anticorpos Antivirais/sangue , Autoanticorpos/sangue , Doenças Autoimunes/imunologia , Autoimunidade , Doença Celíaca/imunologia , Criança , Citomegalovirus/imunologia , Feminino , Proteínas de Ligação ao GTP/imunologia , Humanos , Imunoglobulina G/sangue , Estilo de Vida , Masculino , Países Baixos/epidemiologia , Estudos Prospectivos , Proteína 2 Glutamina gama-Glutamiltransferase , Classe Social , Fatores Socioeconômicos , Transglutaminases/imunologia
19.
J Allergy Clin Immunol ; 139(6): 1923-1934.e17, 2017 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-27913304

RESUMO

BACKGROUND: Numbers of blood leukocyte subsets are highly dynamic in childhood and differ greatly between subjects. Interindividual variation is only partly accounted for by genetic factors. OBJECTIVE: We sought to determine which nongenetic factors affect the dynamics of innate leukocytes and naive and memory lymphocyte subsets. METHODS: We performed 6-color flow cytometry and linear mixed-effects modeling to define the dynamics of 62 leukocyte subsets from birth to 6 years of age in 1182 children, with 1 to 5 measurements per subject. Subsequently, we defined the effect of prenatal maternal lifestyle-related or immune-mediated determinants, birth characteristics, and bacterial/viral exposure-related determinants on leukocyte subset dynamics. RESULTS: Functionally similar leukocyte populations were grouped by using unbiased hierarchical clustering of patterns of age-related leukocyte dynamics. Innate leukocyte numbers were high at birth and predominantly affected by maternal low education level. Naive lymphocyte counts peaked around 1 year, whereas most memory lymphocyte subsets more gradually increased during the first 4 years of life. Dynamics of CD4+ T cells were predominantly associated with sex, birth characteristics, and persistent infections with cytomegalovirus (CMV) or EBV. CD8+ T cells were predominantly associated with CMV and EBV infections, and T-cell receptor γδ+ T cells were predominantly associated with premature rupture of membranes and CMV infection. B-cell subsets were predominantly associated with sex, breast-feeding, and Helicobacter pylori carriership. CONCLUSIONS: Our study identifies specific dynamic patterns of leukocyte subset numbers, as well as nongenetic determinants that affect these patterns, thereby providing new insights into the shaping of the childhood immune system.


Assuntos
Leucócitos/classificação , Contagem de Células , Criança , Pré-Escolar , Infecções por Citomegalovirus/imunologia , Infecções por Vírus Epstein-Barr/imunologia , Feminino , Humanos , Lactente , Recém-Nascido , Leucócitos/imunologia , Masculino , Saúde Materna , Gravidez
20.
J Leukoc Biol ; 101(4): 949-956, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-27821468

RESUMO

The EBV is known to persist in memory B cells, but it remains unclear how this affects cell numbers and humoral immunity. We here studied EBV persistence in memory B cell subsets and consequences on B cell memory in young children. EBV genome loads were quantified in 6 memory B cell subsets in EBV+ adults. The effects of EBV infection on memory B cell numbers and vaccination responses were studied longitudinally in children within the Generation R population cohort between 14 mo and 6 yr of age. EBV genomes were more numerous in CD27+IgG+, CD27+IgA+, and CD27-IgA+ memory B cells than in IgM-only, natural effector, and CD27-IgG+ B cells. The blood counts of IgM-only, CD27+IgA+, CD27-IgG+, and CD27+IgG+ memory B cells were significantly lower in EBV+ children than in uninfected controls at 14 mo of age-the age when these cells peak in numbers. At 6 yr, all of these memory B cell counts had normalized, as had plasma IgG levels to previous primary measles and booster tetanus vaccinations. In conclusion, EBV persists predominantly in Ig class-switched memory B cells, even when derived from T cell-independent responses (CD27-IgA+), and EBV infection results in a transient depletion of these cells in young children.


Assuntos
Linfócitos B/imunologia , Infecções por Vírus Epstein-Barr/imunologia , Herpesvirus Humano 4/imunologia , Imunoglobulina A/imunologia , Imunoglobulina G/imunologia , Memória Imunológica , Adolescente , Adulto , Criança , Infecções por Vírus Epstein-Barr/virologia , Feminino , Humanos , Lactente , Contagem de Linfócitos , Depleção Linfocítica , Masculino , Vacinação
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