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1.
Mol Cell ; 71(6): 897-910.e8, 2018 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-30122534

RESUMO

Chromatin ubiquitination by the ubiquitin ligase RNF168 is critical to regulate the DNA damage response (DDR). DDR deficiencies lead to cancer-prone syndromes, but whether this reflects DNA repair defects is still elusive. We identified key factors of the RNF168 pathway as essential mediators of efficient DNA replication in unperturbed S phase. We found that loss of RNF168 leads to reduced replication fork progression and to reversed fork accumulation, particularly evident at repetitive sequences stalling replication. Slow fork progression depends on MRE11-dependent degradation of reversed forks, implicating RNF168 in reversed fork protection and restart. Consistent with regular nucleosomal organization of reversed forks, the replication function of RNF168 requires H2A ubiquitination. As this novel function is shared with the key DDR players ATM, γH2A.X, RNF8, and 53BP1, we propose that double-stranded ends at reversed forks engage classical DDR factors, suggesting an alternative function of this pathway in preventing genome instability and human disease.


Assuntos
Dano ao DNA/fisiologia , Reparo do DNA/fisiologia , Histonas/metabolismo , Linhagem Celular , Quebras de DNA de Cadeia Dupla , Replicação do DNA/fisiologia , Proteínas de Ligação a DNA/metabolismo , Humanos , Fase S/fisiologia , Transdução de Sinais , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação/fisiologia
2.
Mol Cell ; 67(5): 882-890.e5, 2017 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-28886337

RESUMO

DNA damage tolerance during eukaryotic replication is orchestrated by PCNA ubiquitination. While monoubiquitination activates mutagenic translesion synthesis, polyubiquitination activates an error-free pathway, elusive in mammals, enabling damage bypass by template switching. Fork reversal is driven in vitro by multiple enzymes, including the DNA translocase ZRANB3, shown to bind polyubiquitinated PCNA. However, whether this interaction promotes fork remodeling and template switching in vivo was unknown. Here we show that damage-induced fork reversal in mammalian cells requires PCNA ubiquitination, UBC13, and K63-linked polyubiquitin chains, previously involved in error-free damage tolerance. Fork reversal in vivo also requires ZRANB3 translocase activity and its interaction with polyubiquitinated PCNA, pinpointing ZRANB3 as a key effector of error-free DNA damage tolerance. Mutations affecting fork reversal also induced unrestrained fork progression and chromosomal breakage, suggesting fork remodeling as a global fork slowing and protection mechanism. Targeting these fork protection systems represents a promising strategy to potentiate cancer chemotherapy.


Assuntos
Dano ao DNA , DNA Helicases/metabolismo , Replicação do DNA , DNA de Neoplasias/biossíntese , Neoplasias/enzimologia , Poliubiquitina/metabolismo , Antígeno Nuclear de Célula em Proliferação/metabolismo , Origem de Replicação , Animais , Sistemas CRISPR-Cas , DNA Helicases/genética , DNA de Neoplasias/genética , DNA de Neoplasias/ultraestrutura , Células HCT116 , Células HEK293 , Humanos , Cinética , Camundongos , Mutação , Neoplasias/genética , Neoplasias/ultraestrutura , Antígeno Nuclear de Célula em Proliferação/genética , Interferência de RNA , Transfecção , Enzimas de Conjugação de Ubiquitina/genética , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitinação
3.
Mol Cell ; 57(5): 812-823, 2015 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-25661486

RESUMO

Chromosomal replication is entwined with DNA damage tolerance (DDT) and chromatin structure establishment via elusive mechanisms. Here we examined how specific replication conditions affecting replisome architecture and repriming impact on DDT. We show that Saccharomyces cerevisiae Polα/Primase/Ctf4 mutants, proficient in bulk DNA replication, are defective in recombination-mediated damage-bypass by template switching (TS) and have reduced sister chromatid cohesion. The decrease in error-free DDT is accompanied by increased usage of mutagenic DDT, fork reversal, and higher rates of genome rearrangements mediated by faulty strand annealing. Notably, the DDT defects of Polα/Primase/Ctf4 mutants are not the consequence of increased sister chromatid distance, but are instead caused by altered single-stranded DNA metabolism and abnormal replication fork topology. We propose that error-free TS is driven by timely replicative helicase-coupled re-priming. Defects in this event impact on replication fork architecture and sister chromatid proximity, and represent a frequent source of chromosome lesions upon replication dysfunctions.


Assuntos
Cromátides/genética , Dano ao DNA , DNA Polimerase I/metabolismo , DNA Primase/metabolismo , Replicação do DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , DNA Polimerase I/genética , DNA Primase/genética , Reparo do DNA/genética , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , DNA de Cadeia Simples/ultraestrutura , Proteínas de Ligação a DNA/genética , Pontos de Checagem da Fase G2 do Ciclo Celular/genética , Microscopia Eletrônica , Modelos Genéticos , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Mutação , Recombinação Genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Transdução de Sinais/genética , Fatores de Tempo
4.
Proc Natl Acad Sci U S A ; 110(1): 141-6, 2013 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-23248313

RESUMO

Synthetic biology has significantly advanced the design of genetic devices that can reprogram cellular activities and provide novel treatment strategies for future gene- and cell-based therapies. However, many metabolic disorders are functionally linked while developing distinct diseases that are difficult to treat using a classic one-drug-one-disease intervention scheme. For example, hypertension, hyperglycemia, obesity, and dyslipidemia are interdependent pathologies that are collectively known as the metabolic syndrome, the prime epidemic of the 21st century. We have designed a unique therapeutic strategy in which the clinically licensed antihypertensive drug guanabenz (Wytensin) activates a synthetic signal cascade that stimulates the secretion of metabolically active peptides GLP-1 and leptin. Therefore, the signal transduction of a chimeric trace-amine-associated receptor 1 (cTAAR1) was functionally rewired via cAMP and cAMP-dependent phosphokinase A (PKA)-mediated activation of the cAMP-response element binding protein (CREB1) to transcription of synthetic promoters containing CREB1-specific cAMP response elements. Based on this designer signaling cascade, it was possible to use guanabenz to dose-dependently control expression of GLP-1-Fc(mIgG)-Leptin, a bifunctional therapeutic peptide hormone that combines the glucagon-like peptide 1 (GLP-1) and leptin via an IgG-Fc linker. In mice developing symptoms of the metabolic syndrome, this three-in-one treatment strategy was able to simultaneously attenuate hypertension and hyperglycemia as well as obesity and dyslipidemia. Using a clinically licensed drug to coordinate expression of therapeutic transgenes combines drug- and gene-based therapies for coordinated treatment of functionally related metabolic disorders.


Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Guanabenzo/farmacologia , Leptina/metabolismo , Síndrome Metabólica/tratamento farmacológico , Modelos Moleculares , Transdução de Sinais/efeitos dos fármacos , Animais , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Relação Dose-Resposta a Droga , Desenho de Fármacos , Camundongos , Receptores Acoplados a Proteínas G/metabolismo , Transgenes/genética
5.
Nat Struct Mol Biol ; 30(3): 348-359, 2023 03.
Artigo em Inglês | MEDLINE | ID: mdl-36864174

RESUMO

Transcription-replication collisions (TRCs) are crucial determinants of genome instability. R-loops were linked to head-on TRCs and proposed to obstruct replication fork progression. The underlying mechanisms, however, remained elusive due to the lack of direct visualization and of non-ambiguous research tools. Here, we ascertained the stability of estrogen-induced R-loops on the human genome, visualized them directly by electron microscopy (EM), and measured R-loop frequency and size at the single-molecule level. Combining EM and immuno-labeling on locus-specific head-on TRCs in bacteria, we observed the frequent accumulation of DNA:RNA hybrids behind replication forks. These post-replicative structures are linked to fork slowing and reversal across conflict regions and are distinct from physiological DNA:RNA hybrids at Okazaki fragments. Comet assays on nascent DNA revealed a marked delay in nascent DNA maturation in multiple conditions previously linked to R-loop accumulation. Altogether, our findings suggest that TRC-associated replication interference entails transactions that follow initial R-loop bypass by the replication fork.


Assuntos
Replicação do DNA , RNA , Humanos , DNA/química , Proteínas de Ligação a DNA/metabolismo , Cromossomos/metabolismo , Instabilidade Genômica
6.
Nat Struct Mol Biol ; 21(10): 884-92, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25195051

RESUMO

Template switching (TS) mediates damage bypass via a recombination-related mechanism involving PCNA polyubiquitination and polymerase δ-dependent DNA synthesis. Using two-dimensional gel electrophoresis and EM, here we characterize TS intermediates arising in Saccharomyces cerevisiae at a defined chromosome locus, identifying five major families of intermediates. Single-stranded DNA gaps of 150-200 nt, and not DNA ends, initiate TS by strand invasion. This causes reannealing of the parental strands and exposure of the nondamaged newly synthesized chromatid, which serves as a replication template for the other blocked nascent strand. Structures resembling double Holliday junctions, postulated to be central double-strand break-repair intermediates but so far visualized only in meiosis, mediate late stages of TS before being processed to hemicatenanes. Our results reveal the DNA transitions accounting for recombination-mediated DNA-damage tolerance in mitotic cells and replication under conditions of genotoxic stress.


Assuntos
Dano ao DNA , Reparo do DNA , Replicação do DNA/genética , Recombinação Genética , Saccharomyces cerevisiae/genética , Moldes Genéticos , Cromátides , DNA Polimerase III/genética , DNA Cruciforme , DNA Fúngico/biossíntese , DNA Fúngico/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Ubiquitinação
7.
Nat Commun ; 5: 5392, 2014 Nov 11.
Artigo em Inglês | MEDLINE | ID: mdl-25386727

RESUMO

Synthetic devices for traceless remote control of gene expression may provide new treatment opportunities in future gene- and cell-based therapies. Here we report the design of a synthetic mind-controlled gene switch that enables human brain activities and mental states to wirelessly programme the transgene expression in human cells. An electroencephalography (EEG)-based brain-computer interface (BCI) processing mental state-specific brain waves programs an inductively linked wireless-powered optogenetic implant containing designer cells engineered for near-infrared (NIR) light-adjustable expression of the human glycoprotein SEAP (secreted alkaline phosphatase). The synthetic optogenetic signalling pathway interfacing the BCI with target gene expression consists of an engineered NIR light-activated bacterial diguanylate cyclase (DGCL) producing the orthogonal second messenger cyclic diguanosine monophosphate (c-di-GMP), which triggers the stimulator of interferon genes (STING)-dependent induction of synthetic interferon-ß promoters. Humans generating different mental states (biofeedback control, concentration, meditation) can differentially control SEAP production of the designer cells in culture and of subcutaneous wireless-powered optogenetic implants in mice.


Assuntos
Interfaces Cérebro-Computador , Expressão Gênica , Implantes Experimentais , Optogenética/métodos , Transgenes , Fosfatase Alcalina/biossíntese , Animais , GMP Cíclico/análogos & derivados , GMP Cíclico/metabolismo , Eletroencefalografia , Feminino , Humanos , Camundongos , Transdução de Sinais , Transcrição Gênica , Tecnologia sem Fio
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