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1.
Int J Mol Sci ; 22(11)2021 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-34198827

RESUMO

The objective of this study was to investigate molecular mechanisms underlying the ability of carnosic acid to attenuate an early increase in reactive oxygen species (ROS) levels during MDI-induced adipocyte differentiation. The levels of superoxide anion and ROS were determined using dihydroethidium (DHE) and 2'-7'-dichlorofluorescin diacetate (DCFH-DA), respectively. Both superoxide anion and ROS levels peaked on the second day of differentiation. They were suppressed by carnosic acid. Carnosic acid attenuates the translation of NADPH (nicotinamide adenine dinucleotide phosphate) oxidase 4 (Nox4), p47phox, and p22phox, and the phosphorylation of nuclear factor-kappa B (NF-κB) and NF-κB inhibitor (IkBa). The translocation of NF-κB into the nucleus was also decreased by carnosic acid. In addition, carnosic acid increased the translation of heme oxygenase-1 (HO-1), γ-glutamylcysteine synthetase (γ-GCSc), and glutathione S-transferase (GST) and both the translation and nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf2). Taken together, these results indicate that carnosic acid could down-regulate ROS level in an early stage of MPI-induced adipocyte differentiation by attenuating ROS generation through suppression of NF-κB-mediated translation of Nox4 enzyme and increasing ROS neutralization through induction of Nrf2-mediated translation of phase II antioxidant enzymes such as HO-1, γ-GCS, and GST, leading to its anti-adipogenetic effect.


Assuntos
ATPases Associadas a Diversas Atividades Celulares/genética , Abietanos/farmacologia , DNA Helicases/genética , Heme Oxigenase-1/genética , Proteínas de Membrana/genética , NADPH Oxidase 4/genética , Inibidor de NF-kappaB alfa/genética , Células 3T3-L1 , Adipócitos/efeitos dos fármacos , Adipócitos/metabolismo , Animais , Antioxidantes/farmacologia , Diferenciação Celular/efeitos dos fármacos , Grupo dos Citocromos b/genética , Etídio/análogos & derivados , Etídio/farmacologia , Fluoresceínas/farmacologia , Glutationa Transferase/genética , Camundongos , NADPH Oxidases/genética , Biossíntese de Proteínas/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo
2.
Mol Cell ; 81(14): 2989-3006.e9, 2021 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-34197737

RESUMO

Stalled DNA replication fork restart after stress as orchestrated by ATR kinase, BLM helicase, and structure-specific nucleases enables replication, cell survival, and genome stability. Here we unveil human exonuclease V (EXO5) as an ATR-regulated DNA structure-specific nuclease and BLM partner for replication fork restart. We find that elevated EXO5 in tumors correlates with increased mutation loads and poor patient survival, suggesting that EXO5 upregulation has oncogenic potential. Structural, mechanistic, and mutational analyses of EXO5 and EXO5-DNA complexes reveal a single-stranded DNA binding channel with an adjacent ATR phosphorylation motif (T88Q89) that regulates EXO5 nuclease activity and BLM binding identified by mass spectrometric analysis. EXO5 phospho-mimetic mutant rescues the restart defect from EXO5 depletion that decreases fork progression, DNA damage repair, and cell survival. EXO5 depletion furthermore rescues survival of FANCA-deficient cells and indicates EXO5 functions epistatically with SMARCAL1 and BLM. Thus, an EXO5 axis connects ATR and BLM in directing replication fork restart.


Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/genética , Replicação do DNA/genética , DNA/genética , Exonucleases/genética , Instabilidade Genômica/genética , RecQ Helicases/genética , Linhagem Celular , Linhagem Celular Tumoral , Dano ao DNA/genética , DNA Helicases/genética , Análise Mutacional de DNA/métodos , Reparo do DNA/genética , Proteínas de Ligação a DNA/genética , Células HEK293 , Células HeLa , Humanos , Mutação/genética , Oncogenes/genética , Fosforilação/genética , Regulação para Cima/genética
3.
Nat Commun ; 12(1): 3520, 2021 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-34112784

RESUMO

The Immunodeficiency Centromeric Instability Facial Anomalies (ICF) 4 syndrome is caused by mutations in LSH/HELLS, a chromatin remodeler promoting incorporation of histone variant macroH2A. Here, we demonstrate that LSH depletion results in degradation of nascent DNA at stalled replication forks and the generation of genomic instability. The protection of stalled forks is mediated by macroH2A, whose knockdown mimics LSH depletion and whose overexpression rescues nascent DNA degradation. LSH or macroH2A deficiency leads to an impairment of RAD51 loading, a factor that prevents MRE11 and EXO1 mediated nascent DNA degradation. The defect in RAD51 loading is linked to a disbalance of BRCA1 and 53BP1 accumulation at stalled forks. This is associated with perturbed histone modifications, including abnormal H4K20 methylation that is critical for BRCA1 enrichment and 53BP1 exclusion. Altogether, our results illuminate the mechanism underlying a human syndrome and reveal a critical role of LSH mediated chromatin remodeling in genomic stability.


Assuntos
DNA Helicases/metabolismo , Replicação do DNA , Instabilidade Genômica , Histonas/metabolismo , Rad51 Recombinase/metabolismo , Animais , Proteína BRCA1/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/genética , Montagem e Desmontagem da Cromatina/genética , Sequenciamento de Cromatina por Imunoprecipitação , DNA Helicases/deficiência , DNA Helicases/genética , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/metabolismo , Replicação do DNA/genética , Epigênese Genética , Exodesoxirribonucleases/genética , Exodesoxirribonucleases/metabolismo , Instabilidade Genômica/genética , Histonas/deficiência , Histonas/genética , Humanos , Proteína Homóloga a MRE11/genética , Proteína Homóloga a MRE11/metabolismo , Metilação , Camundongos , RNA Interferente Pequeno , Rad51 Recombinase/genética , Regulação para Cima
4.
Nat Commun ; 12(1): 3338, 2021 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-34099686

RESUMO

The versatile nucleotide excision repair (NER) pathway initiates as the XPC-RAD23B-CETN2 complex first recognizes DNA lesions from the genomic DNA and recruits the general transcription factor complex, TFIIH, for subsequent lesion verification. Here, we present a cryo-EM structure of an NER initiation complex containing Rad4-Rad23-Rad33 (yeast homologue of XPC-RAD23B-CETN2) and 7-subunit coreTFIIH assembled on a carcinogen-DNA adduct lesion at 3.9-9.2 Å resolution. A ~30-bp DNA duplex could be mapped as it straddles between Rad4 and the Ssl2 (XPB) subunit of TFIIH on the 3' and 5' side of the lesion, respectively. The simultaneous binding with Rad4 and TFIIH was permitted by an unwinding of DNA at the lesion. Translocation coupled with torque generation by Ssl2 and Rad4 would extend the DNA unwinding at the lesion and deliver the damaged strand to Rad3 (XPD) in an open form suitable for subsequent lesion scanning and verification.


Assuntos
Microscopia Crioeletrônica , Dano ao DNA , Reparo do DNA , Proteínas de Ligação a DNA/química , DNA/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismo , Fator de Transcrição TFIIH/química , Adutos de DNA/metabolismo , DNA Helicases/química , DNA Helicases/genética , Proteínas de Ligação a DNA/genética , Proteínas de Saccharomyces cerevisiae/genética , Fator de Transcrição TFIIH/genética
5.
Nat Commun ; 12(1): 3686, 2021 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-34140498

RESUMO

Tumour hypoxia is associated with poor patient prognosis and therapy resistance. A unique transcriptional response is initiated by hypoxia which includes the rapid activation of numerous transcription factors in a background of reduced global transcription. Here, we show that the biological response to hypoxia includes the accumulation of R-loops and the induction of the RNA/DNA helicase SETX. In the absence of hypoxia-induced SETX, R-loop levels increase, DNA damage accumulates, and DNA replication rates decrease. Therefore, suggesting that, SETX plays a role in protecting cells from DNA damage induced during transcription in hypoxia. Importantly, we propose that the mechanism of SETX induction in hypoxia is reliant on the PERK/ATF4 arm of the unfolded protein response. These data not only highlight the unique cellular response to hypoxia, which includes both a replication stress-dependent DNA damage response and an unfolded protein response but uncover a novel link between these two distinct pathways.


Assuntos
Hipóxia Celular , Dano ao DNA/genética , DNA Helicases/metabolismo , Regulação da Expressão Gênica/genética , Enzimas Multifuncionais/metabolismo , Estruturas R-Loop/genética , RNA Helicases/metabolismo , Resposta a Proteínas não Dobradas/genética , Fator 4 Ativador da Transcrição/genética , Fator 4 Ativador da Transcrição/metabolismo , Morte Celular/efeitos dos fármacos , Morte Celular/genética , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Imunoprecipitação da Cromatina , DNA Helicases/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Enzimas Multifuncionais/genética , Inibidores da Síntese de Ácido Nucleico/farmacologia , Oxigênio/farmacologia , Estruturas R-Loop/efeitos dos fármacos , RNA Helicases/genética , RNA-Seq , Resposta a Proteínas não Dobradas/efeitos dos fármacos , Regulação para Cima , Zinostatina/farmacologia , eIF-2 Quinase/metabolismo
6.
Nat Commun ; 12(1): 3856, 2021 06 22.
Artigo em Inglês | MEDLINE | ID: mdl-34158470

RESUMO

The MRN complex (MRX in Saccharomyces cerevisiae, made of Mre11, Rad50 and Nbs1/Xrs2) initiates double-stranded DNA break repair and activates the Tel1/ATM kinase in the DNA damage response. Telomeres counter both outcomes at chromosome ends, partly by keeping MRN-ATM in check. We show that MRX is disabled by telomeric protein Rif2 through an N-terminal motif (MIN, MRN/X-inhibitory motif). MIN executes suppression of Tel1, DNA end-resection and non-homologous end joining by binding the Rad50 N-terminal region. Our data suggest that MIN promotes a transition within MRX that is not conductive for endonuclease activity, DNA-end tethering or Tel1 kinase activation, highlighting an Achilles' heel in MRN, which we propose is also exploited by the RIF2 paralog ORC4 (Origin Recognition Complex 4) in Kluyveromyces lactis and the Schizosaccharomyces pombe telomeric factor Taz1, which is evolutionarily unrelated to Orc4/Rif2. This raises the possibility that analogous mechanisms might be deployed in other eukaryotes as well.


Assuntos
Motivos de Aminoácidos , DNA Helicases/metabolismo , Endodesoxirribonucleases/metabolismo , Exodesoxirribonucleases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Telômero/metabolismo , Sequência de Aminoácidos , Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades , DNA Helicases/genética , DNA Fúngico/genética , DNA Fúngico/metabolismo , Endodesoxirribonucleases/genética , Exodesoxirribonucleases/genética , Instabilidade Genômica , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Complexo de Reconhecimento de Origem/genética , Complexo de Reconhecimento de Origem/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Homologia de Sequência de Aminoácidos , Telômero/genética , Proteínas de Ligação a Telômeros/genética , Proteínas de Ligação a Telômeros/metabolismo
7.
Nat Commun ; 12(1): 3887, 2021 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-34162889

RESUMO

ATRX is a tumor suppressor that has been associated with protection from DNA replication stress, purportedly through resolution of difficult-to-replicate G-quadruplex (G4) DNA structures. While several studies demonstrate that loss of ATRX sensitizes cells to chemical stabilizers of G4 structures, the molecular function of ATRX at G4 regions during replication remains unknown. Here, we demonstrate that ATRX associates with a number of the MCM replication complex subunits and that loss of ATRX leads to G4 structure accumulation at newly synthesized DNA. We show that both the helicase domain of ATRX and its H3.3 chaperone function are required to protect cells from G4-induced replicative stress. Furthermore, these activities are upstream of heterochromatin formation mediated by the histone methyltransferase, ESET, which is the critical molecular event that protects cells from G4-mediated stress. In support, tumors carrying mutations in either ATRX or ESET show increased mutation burden at G4-enriched DNA sequences. Overall, our study provides new insights into mechanisms by which ATRX promotes genome stability with important implications for understanding impacts of its loss on human disease.


Assuntos
Replicação do DNA/genética , DNA/genética , Quadruplex G , Heterocromatina/genética , Proteína Nuclear Ligada ao X/genética , Células Cultivadas , Sequenciamento de Cromatina por Imunoprecipitação/métodos , DNA/química , DNA/metabolismo , DNA Helicases/genética , DNA Helicases/metabolismo , Instabilidade Genômica/genética , Células HeLa , Histonas/genética , Histonas/metabolismo , Humanos , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Mutação , Conformação de Ácido Nucleico , Proteína Nuclear Ligada ao X/metabolismo
8.
Turk Neurosurg ; 31(4): 587-593, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34169999

RESUMO

AIM: To elucidate the association of the MTHFR, MTRR, and RAD54L gene variations with meningioma in Turkish cohort. MATERIAL AND METHODS: DNAs were isolated from 87 retrospective meningioma samples. The MTHFR, MTRR, and RAD54L gene hotspot regions were amplified with specific primers via polymerase chain reaction (PCR), and next-generation sequencing (NGS) was performed. All the detected variations and single-nucleotide polymorphisms (SNPs) were listed and compared with healthy control frequencies in different genomic databases. The histopathological characteristics of meningiomas and genomic variations were compared. Pearson?s chi-squared test was used to detect the statistical differences of SNPs, and correlation analysis was conducted. RESULTS: rs1801131, rs1801133, and rs4846051 on MTHFR, rs1801394 on MTRR, and rs1048771 on RAD54L gene frequencies were found to be significantly altered in the overall cohort of 87 patients with meningioma. The frequency of rs18011031 is 0.09 in the meningioma cohort, which is significantly correlated with WHO tumor grades (p = 0.038). The frequency of rs18011033 is 0.29 in the meningioma cohort, which is significantly correlated with WHO tumor grades (p = 0.045). Furthermore, the frequency of rs4846051 is 0.18 in the meningioma cohort, which is significantly correlated with WHO tumor grades (p = 0.023) and also with low Ki67 proliferation index (p = 0.00455). The frequency of rs1801394 is 0.15 and significantly associated with high Ki67 proliferation index in the meningioma cohort (p = 0.0144). The frequency of rs1048771 is 0.09 in the meningioma cohort and is significantly associated with the non-necrotic histopathological form of the tumor (p = 0.05). CONCLUSION: We reported a significant association between the genetic alterations of folate metabolism (MTHFR, MTRR) and DNA repair mechanism (RAD54L) genes with the histopathological characteristics of meningioma. Five significant SNPs on these genes and four significant correlations of SNPs with histopathological characteristics were identified. This is a preliminary promising study conducted to establish the genetic marker analysis for meningioma diagnosis and prognosis for folate metabolism and DNA repair genes in Turkish cohort.


Assuntos
DNA Helicases/genética , Proteínas de Ligação a DNA/genética , Ferredoxina-NADP Redutase/genética , Neoplasias Meníngeas/genética , Meningioma/genética , Metilenotetra-Hidrofolato Redutase (NADPH2)/genética , Adulto , Idoso , Estudos de Casos e Controles , Estudos de Coortes , Feminino , Frequência do Gene , Estudos de Associação Genética , Genótipo , Humanos , Masculino , Neoplasias Meníngeas/epidemiologia , Meningioma/epidemiologia , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único , Estudos Retrospectivos , Turquia/epidemiologia
9.
Mol Cell ; 81(14): 3007-3017.e5, 2021 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-34107305

RESUMO

RAD51 facilitates replication fork reversal and protects reversed forks from nuclease degradation. Although potentially a useful replication stress response mechanism, unregulated fork reversal can cause genome instability. Here we show that RADX, a single-strand DNA binding protein that binds to and destabilizes RAD51 nucleofilaments, can either inhibit or promote fork reversal depending on replication stress levels. RADX inhibits fork reversal at elongating forks, thereby preventing fork slowing and collapse. Paradoxically, in the presence of persistent replication stress, RADX localizes to stalled forks to generate reversed fork structures. Consequently, inactivating RADX prevents fork-reversal-dependent telomere dysfunction in the absence of RTEL1 and blocks nascent strand degradation when fork protection factors are inactivated. Addition of RADX increases SMARCAL1-dependent fork reversal in conditions in which pre-binding RAD51 to a model fork substrate is inhibitory. Thus, RADX directly interacts with RAD51 and single-strand DNA to confine fork reversal to persistently stalled forks.


Assuntos
Replicação do DNA/genética , Proteínas de Ligação a DNA/genética , Instabilidade Genômica/genética , Origem de Replicação/genética , Linhagem Celular , Linhagem Celular Tumoral , Quebras de DNA de Cadeia Dupla , DNA Helicases/genética , Reparo do DNA/genética , DNA de Cadeia Simples/genética , Células HEK293 , Células HeLa , Humanos , Ligação Proteica/genética , Rad51 Recombinase/genética
10.
Mol Cell ; 81(11): 2332-2348.e9, 2021 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-33974912

RESUMO

Meningioma-1 (MN1) overexpression in AML is associated with poor prognosis, and forced expression of MN1 induces leukemia in mice. We sought to determine how MN1 causes AML. We found that overexpression of MN1 can be induced by translocations that result in hijacking of a downstream enhancer. Structure predictions revealed that the entire MN1 coding frame is disordered. We identified the myeloid progenitor-specific BAF complex as the key interaction partner of MN1. MN1 over-stabilizes BAF on enhancer chromatin, a function directly linked to the presence of a long polyQ-stretch within MN1. BAF over-stabilization at binding sites of transcription factors regulating a hematopoietic stem/progenitor program prevents the developmentally appropriate decommissioning of these enhancers and results in impaired myeloid differentiation and leukemia. Beyond AML, our data detail how the overexpression of a polyQ protein, in the absence of any coding sequence mutation, can be sufficient to cause malignant transformation.


Assuntos
Carcinogênese/genética , DNA Helicases/genética , Proteínas Intrinsicamente Desordenadas/genética , Leucemia Mieloide Aguda/genética , Proteínas Nucleares/genética , Transativadores/genética , Fatores de Transcrição/genética , Proteínas Supressoras de Tumor/genética , Animais , Sequência de Bases , Carcinogênese/metabolismo , Carcinogênese/patologia , Linhagem Celular Tumoral , Cromatina/genética , Cromatina/metabolismo , Cromatina/patologia , DNA Helicases/metabolismo , Elementos Facilitadores Genéticos , Feminino , Regulação Leucêmica da Expressão Gênica , Redes Reguladoras de Genes , Humanos , Proteínas Intrinsicamente Desordenadas/metabolismo , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/mortalidade , Leucemia Mieloide Aguda/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas Nucleares/metabolismo , Peptídeos/genética , Peptídeos/metabolismo , Mapeamento de Interação de Proteínas , Estabilidade Proteica , Transporte Proteico , Transdução de Sinais , Análise de Sobrevida , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/metabolismo
11.
Hum Genet ; 140(7): 1109-1120, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33944996

RESUMO

Located in the critical 1p36 microdeletion region, the chromodomain helicase DNA-binding protein 5 (CHD5) gene encodes a subunit of the nucleosome remodeling and deacetylation (NuRD) complex required for neuronal development. Pathogenic variants in six of nine chromodomain (CHD) genes cause autosomal dominant neurodevelopmental disorders, while CHD5-related disorders are still unknown. Thanks to GeneMatcher and international collaborations, we assembled a cohort of 16 unrelated individuals harboring heterozygous CHD5 variants, all identified by exome sequencing. Twelve patients had de novo CHD5 variants, including ten missense and two splice site variants. Three familial cases had nonsense or missense variants segregating with speech delay, learning disabilities, and/or craniosynostosis. One patient carried a frameshift variant of unknown inheritance due to unavailability of the father. The most common clinical features included language deficits (81%), behavioral symptoms (69%), intellectual disability (64%), epilepsy (62%), and motor delay (56%). Epilepsy types were variable, with West syndrome observed in three patients, generalized tonic-clonic seizures in two, and other subtypes observed in one individual each. Our findings suggest that, in line with other CHD-related disorders, heterozygous CHD5 variants are associated with a variable neurodevelopmental syndrome that includes intellectual disability with speech delay, epilepsy, and behavioral problems as main features.


Assuntos
DNA Helicases/genética , Deficiência Intelectual/genética , Mutação de Sentido Incorreto , Proteínas do Tecido Nervoso/genética , Transtornos do Neurodesenvolvimento/genética , Adolescente , Domínio Catalítico , Criança , Pré-Escolar , Estudos de Coortes , Epilepsia/genética , Feminino , Genes Dominantes , Humanos , Deficiência Intelectual/fisiopatologia , Masculino , Transtornos do Neurodesenvolvimento/fisiopatologia , Linhagem , Adulto Jovem
12.
Cancer Sci ; 112(7): 2781-2791, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33960594

RESUMO

The prevalence of neuroendocrine prostate cancer (NEPC) arising from adenocarcinoma (AC) upon potent androgen receptor (AR) pathway inhibition is increasing. Deeper understanding of NEPC biology and development of novel therapeutic agents are needed. However, research is hindered by the paucity of research models, especially cell lines developed from NEPC patients. We established a novel NEPC cell line, KUCaP13, from tissue of a patient initially diagnosed with AC which later recurred as NEPC. The cell line has been maintained permanently in vitro under regular cell culture conditions and is amenable to gene engineering with lentivirus. KUCaP13 cells lack the expression of AR and overexpress NEPC-associated genes, including SOX2, EZH2, AURKA, PEG10, POU3F2, ENO2, and FOXA2. Importantly, the cell line maintains the homozygous deletion of CHD1, which was confirmed in the primary AC of the index patient. Loss of heterozygosity of TP53 and PTEN, and an allelic loss of RB1 with a transcriptomic signature compatible with Rb pathway aberration were revealed. Knockdown of PEG10 using shRNA significantly suppressed growth in vivo. Introduction of luciferase allowed serial monitoring of cells implanted orthotopically or in the renal subcapsule. Although H3K27me was reduced by EZH2 inhibition, reversion to AC was not observed. KUCaP13 is the first patient-derived, treatment-related NEPC cell line with triple loss of tumor suppressors critical for NEPC development through lineage plasticity. It could be valuable in research to deepen the understanding of NEPC.


Assuntos
Adenocarcinoma/patologia , Carcinoma Neuroendócrino/patologia , Linhagem Celular Tumoral/patologia , Neoplasias da Próstata/patologia , Animais , Proteínas Reguladoras de Apoptose/genética , Carcinoma Neuroendócrino/genética , Carcinoma Neuroendócrino/secundário , Linhagem Celular Tumoral/metabolismo , DNA Helicases/genética , Proteínas de Ligação a DNA/genética , Ensaios de Seleção de Medicamentos Antitumorais , Proteína Potenciadora do Homólogo 2 de Zeste/antagonistas & inibidores , Deleção de Genes , Expressão Gênica , Genes Neoplásicos , Genes do Retinoblastoma , Genes Supressores de Tumor , Genes p53 , Engenharia Genética , Xenoenxertos , Homozigoto , Humanos , Cariotipagem , Perda de Heterozigosidade , Masculino , Camundongos SCID , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/patologia , Transplante de Neoplasias , PTEN Fosfo-Hidrolase/genética , Neoplasias Penianas/genética , Neoplasias Penianas/secundário , Neoplasias da Próstata/genética , Proteínas de Ligação a RNA/genética , Receptores Androgênicos
13.
Nucleic Acids Res ; 49(10): 5671-5683, 2021 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-34048583

RESUMO

Telomeres are copied and reassembled each cell division cycle through a multistep process called telomere replication. Most telomeric DNA is duplicated semiconservatively during this process, but replication forks frequently pause or stall at telomeres in yeast, mouse and human cells, potentially causing chronic telomere shortening or loss in a single cell cycle. We have investigated the cause of this effect by examining the replication of telomeric templates in vitro. Using a reconstituted assay for eukaryotic DNA replication in which a complete eukaryotic replisome is assembled and activated with purified proteins, we show that budding yeast telomeric DNA is efficiently duplicated in vitro unless the telomere binding protein Rap1 is present. Rap1 acts as a roadblock that prevents replisome progression and leading strand synthesis, but also potently inhibits lagging strand telomere replication behind the fork. Both defects can be mitigated by the Pif1 helicase. Our results suggest that GC-rich sequences do not inhibit DNA replication per se, and that in the absence of accessory factors, telomere binding proteins can inhibit multiple, distinct steps in the replication process.


Assuntos
Replicação do DNA/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomycetales/metabolismo , Proteínas de Ligação a Telômeros/metabolismo , Telômero/metabolismo , Fatores de Transcrição/metabolismo , Composição de Bases/genética , DNA Helicases/genética , DNA Helicases/metabolismo , Expressão Gênica , Técnicas In Vitro , Proteínas Recombinantes , Proteínas de Saccharomyces cerevisiae/genética , Saccharomycetales/genética , Telômero/genética , Proteínas de Ligação a Telômeros/genética , Fatores de Transcrição/genética
14.
Vet Microbiol ; 257: 109074, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33940460

RESUMO

Porcine epidemic diarrhea virus (PEDV) is a reemerging Alphacoronavirus that causes lethal diarrhea in piglets. Coronavirus nonstructural protein 13 (nsp13) encodes helicase, which plays pivotal roles during viral replication by unwinding viral RNA. However, the biochemical characterization of PEDV nsp13 remains largely unknown. In this study, PEDV nsp13 was expressed in Escherichia coli and purified. The recombinant nsp13 possessed ATPase and helicase activities for binding and unwinding dsDNA/RNA substrates with 5'-overhangs, and Mg2+ and Mn2+ were critical for its ATPase and helicase activities. PEDV nsp13 also unwound dsDNA into ssDNA in the pH from 6.0-9.0, and used energy from all nucleoside triphosphates and deoxynucleoside triphosphates. Site-directed mutagenesis demonstrated that Lys289 (K289) of PEDV nsp13 was essential for its ATPase and helicase activities. These results provide new insights into the biochemical properties of PEDV nsp13, which is a potential target for developing antiviral drugs.


Assuntos
Adenosina Trifosfatases/metabolismo , DNA Helicases/metabolismo , Vírus da Diarreia Epidêmica Suína/enzimologia , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo , Adenosina Trifosfatases/genética , Trifosfato de Adenosina/metabolismo , Animais , Chlorocebus aethiops , Infecções por Coronavirus/virologia , DNA Helicases/genética , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/genética , RNA Viral/genética , RNA Viral/metabolismo , Suínos , Doenças dos Suínos/virologia , Células Vero
15.
Nat Commun ; 12(1): 3016, 2021 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-34021146

RESUMO

Telomere repeat containing RNAs (TERRAs) are a family of long non-coding RNAs transcribed from the subtelomeric regions of eukaryotic chromosomes. TERRA transcripts can form R-loops at chromosome ends; however the importance of these structures or the regulation of TERRA expression and retention in telomeric R-loops remain unclear. Here, we show that the RTEL1 (Regulator of Telomere Length 1) helicase influences the abundance and localization of TERRA in human cells. Depletion of RTEL1 leads to increased levels of TERRA RNA while reducing TERRA-containing R loops at telomeres. In vitro, RTEL1 shows a strong preference for binding G-quadruplex structures which form in TERRA. This binding is mediated by the C-terminal region of RTEL1, and is independent of the RTEL1 helicase domain. RTEL1 binding to TERRA appears to be essential for cell viability, underscoring the importance of this function. Degradation of TERRA-containing R-loops by overexpression of RNAse H1 partially recapitulates the increased TERRA levels and telomeric instability associated with RTEL1 deficiency. Collectively, these data suggest that regulation of TERRA is a key function of the RTEL1 helicase, and that loss of that function may contribute to the disease phenotypes of patients with RTEL1 mutations.


Assuntos
DNA Helicases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Fatores de Transcrição/metabolismo , DNA Helicases/química , DNA Helicases/genética , Proteínas de Ligação a DNA/genética , Técnicas de Inativação de Genes , Células HEK293 , Humanos , Domínios Proteicos , Estruturas R-Loop , RNA , Ribonuclease H , Alinhamento de Sequência , Telômero , Fatores de Transcrição/genética
16.
Nat Commun ; 12(1): 3014, 2021 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-34021162

RESUMO

Members of the chromodomain-helicase-DNA binding (CHD) protein family are chromatin remodelers implicated in human pathologies, with CHD6 being one of its least studied members. We discovered a de novo CHD6 missense mutation in a patient clinically presenting the rare Hallermann-Streiff syndrome (HSS). We used genome editing to generate isogenic iPSC lines and model HSS in relevant cell types. By combining genomics with functional in vivo and in vitro assays, we show that CHD6 binds a cohort of autophagy and stress response genes across cell types. The HSS mutation affects CHD6 protein folding and impairs its ability to recruit co-remodelers in response to DNA damage or autophagy stimulation. This leads to accumulation of DNA damage burden and senescence-like phenotypes. We therefore uncovered a molecular mechanism explaining HSS onset via chromatin control of autophagic flux and genotoxic stress surveillance.


Assuntos
Autofagia/fisiologia , Dano ao DNA , DNA Helicases/genética , DNA Helicases/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Autofagia/genética , Cromatina , Montagem e Desmontagem da Cromatina/genética , Proteínas de Ligação a DNA/metabolismo , Epigenômica , Edição de Genes , Expressão Gênica , Síndrome de Hallermann/genética , Humanos , Mutação , Fenótipo
17.
Neurol India ; 69(2): 362-366, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33904453

RESUMO

Background: Cockayne syndrome is an autosomal recessive disorder caused by biallelic mutations in ERCC6 or ERCC8 genes. Aims: To study the clinical and mutation spectrum of Cockayne syndrome. Setting and Design: Medical Genetics Outpatient Department of Sanjay Gandhi Post Graduate Institute of Medical Sciences, Lucknow. This was a prospective study from 2007 to 2015. Materials and Methods: Clinical details were recorded, and sequencing of ERCC6 and ERCC8 were performed. Results and Conclusions: Of the six families, one family had a homozygous mutation in ERCC8 and the other five families had homozygous mutations in ERCC6. Novel variants in ERCC6 were identified in four families. Phenotypic features may vary from severe to mild, and a strong clinical suspicion is needed for diagnosis during infancy or early childhood. Hence, molecular diagnosis is needed for confirmation of diagnosis in a child with a suspicion of Cockayne syndrome. Prenatal diagnosis can be provided only if molecular diagnosis is established in the proband.


Assuntos
Síndrome de Cockayne , DNA Helicases/genética , Enzimas Reparadoras do DNA/genética , Proteínas de Ligação a Poli-ADP-Ribose/genética , Fatores de Transcrição , Criança , Pré-Escolar , Síndrome de Cockayne/diagnóstico , Síndrome de Cockayne/genética , Feminino , Humanos , Índia , Mutação , Gravidez , Estudos Prospectivos , Fatores de Transcrição/genética
18.
Methods Mol Biol ; 2281: 81-91, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33847953

RESUMO

Understanding protein-protein interactions is key to unraveling protein function in vivo. Here we describe a dual/triple-plasmid system that enables co-expression of two, or three, recombinant proteins harboring different affinity tags in the same Escherichia coli cell. This novel protein expression system provides a platform to understand protein-protein interactions and enables researchers to study protein complex formation and in vivo localization.


Assuntos
DNA Helicases/química , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/genética , Sítios de Ligação , DNA Helicases/genética , Replicação do DNA , DNA Bacteriano/metabolismo , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/genética , Eletroforese em Gel de Poliacrilamida , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Plasmídeos/genética , Plasmídeos/metabolismo , Ligação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo
19.
Methods Mol Biol ; 2281: 313-322, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33847968

RESUMO

Defects in mitochondrial DNA (mtDNA) maintenance may lead to disturbances in mitochondrial homeostasis and energy production in eukaryotic cells, causing diseases. During mtDNA replication, the mitochondrial single-stranded DNA-binding protein (mtSSB) stabilizes and protects the exposed single-stranded mtDNA from nucleolysis; perhaps more importantly, it appears to coordinate the actions of both the replicative mtDNA helicase Twinkle and DNA polymerase gamma at the replication fork. Here, we describe a helicase stimulation protocol to test in vitro the functional interaction between mtSSB and variant forms of Twinkle. We show for the first time that the C-terminal tail of Twinkle is important for such an interaction, and that it negatively regulates helicase unwinding activity in a salt-dependent manner.


Assuntos
DNA Helicases/química , DNA Helicases/metabolismo , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas Mitocondriais/química , Proteínas Mitocondriais/metabolismo , Mutação , Sítios de Ligação , DNA Helicases/genética , Replicação do DNA , DNA Mitocondrial/química , DNA Mitocondrial/metabolismo , DNA de Cadeia Simples/química , Proteínas de Ligação a DNA/química , Humanos , Proteínas Mitocondriais/genética , Modelos Moleculares , Ligação Proteica , Conformação Proteica
20.
Mol Cell ; 81(11): 2428-2444.e6, 2021 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-33882298

RESUMO

Repair pathway "choice" at stalled mammalian replication forks is an important determinant of genome stability; however, the underlying mechanisms are poorly understood. FANCM encodes a multi-domain scaffolding and motor protein that interacts with several distinct repair protein complexes at stalled forks. Here, we use defined mutations engineered within endogenous Fancm in mouse embryonic stem cells to study how Fancm regulates stalled fork repair. We find that distinct FANCM repair functions are enacted by molecularly separable scaffolding domains. These findings define FANCM as a key mediator of repair pathway choice at stalled replication forks and reveal its molecular mechanism. Notably, mutations that inactivate FANCM ATPase function disable all its repair functions and "trap" FANCM at stalled forks. We find that Brca1 hypomorphic mutants are synthetic lethal with Fancm null or Fancm ATPase-defective mutants. The ATPase function of FANCM may therefore represent a promising "druggable" target for therapy of BRCA1-linked cancer.


Assuntos
Proteína BRCA1/genética , DNA Helicases/genética , Reparo do DNA , Replicação do DNA , Células-Tronco Embrionárias Murinas/metabolismo , Mutações Sintéticas Letais , Animais , Proteína BRCA1/metabolismo , Ciclo Celular/genética , Linhagem Celular , Células Clonais , DNA Helicases/metabolismo , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/genética , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Humanos , Camundongos , Células-Tronco Embrionárias Murinas/citologia , Ubiquitinação
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