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1.
Nat Cell Biol ; 21(9): 1068-1077, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31481797

RESUMO

Recent development of innovative tools for live imaging of actin filaments (F-actin) enabled the detection of surprising nuclear structures responding to various stimuli, challenging previous models that actin is substantially monomeric in the nucleus. We review these discoveries, focusing on double-strand break (DSB) repair responses. These studies revealed a remarkable network of nuclear filaments and regulatory mechanisms coordinating chromatin dynamics with repair progression and led to a paradigm shift by uncovering the directed movement of repair sites.


Assuntos
Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Núcleo Celular/metabolismo , Reparo do DNA/fisiologia , Animais , Cromatina/metabolismo , Quebras de DNA de Cadeia Dupla , Humanos
2.
Nat Chem Biol ; 15(10): 992-1000, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31527837

RESUMO

Post-translational modifications of histone variant H2A.Z accompany gene transactivation, but its modifying enzymes still remain elusive. Here, we reveal a hitherto unknown function of human KAT2A (GCN5) as a histone acetyltransferase (HAT) of H2A.Z at the promoters of a set of transactivated genes. Expression of these genes also depends on the DNA repair complex XPC-RAD23-CEN2. We established that XPC-RAD23-CEN2 interacts both with H2A.Z and KAT2A to drive the recruitment of the HAT at promoters and license H2A.Z acetylation. KAT2A selectively acetylates H2A.Z.1 versus H2A.Z.2 in vitro on several well-defined lysines and we unveiled that alanine-14 in H2A.Z.2 is responsible for inhibiting the activity of KAT2A. Notably, the use of a nonacetylable H2A.Z.1 mutant shows that H2A.Z.1ac recruits the epigenetic reader BRD2 to promote RNA polymerase II recruitment. Our studies identify KAT2A as an H2A.Z.1 HAT in mammals and implicate XPC-RAD23-CEN2 as a transcriptional co-activator licensing the reshaping of the promoter epigenetic landscape.


Assuntos
Reparo do DNA/fisiologia , Histona Acetiltransferases/metabolismo , Histonas/metabolismo , Acetilação , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Fibroblastos , Regulação da Expressão Gênica , Humanos , Lisina Acetiltransferase 5
3.
Gene ; 717: 144043, 2019 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-31400407

RESUMO

Genes involved in the repair of DNA damage are emerging as playing important roles during the disease processes caused by pathogenic fungi. However, there are potentially hundreds of genes involved in DNA repair in a fungus and some of those genes can play additional roles within the cell. One such gene is RAD23, required for virulence of the human pathogenic fungus Cryptococcus neoformans, that encodes a protein involved in the nucleotide excision repair (NER) pathway. However, Rad23 is a dual function protein, with a role in either repair of damaged DNA or protein turn over by directing proteins to the proteasome. Here, these two functions of Rad23 were tested by the creation of a series of domain deletion alleles of RAD23 and the assessment of the strains for DNA repair, proteasome functions, and virulence properties. Deletion of the different domains was able to uncouple the two functions of Rad23, and the phenotypes of strains carrying such forms indicated that the role of RAD23 in virulence is due to its function in proteasomal-mediated protein degradation rather than NER.


Assuntos
Cryptococcus neoformans/genética , Cryptococcus neoformans/patogenicidade , Reparo do DNA/fisiologia , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Animais , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Larva/microbiologia , Mariposas/microbiologia , Mutação , Complexo de Endopeptidases do Proteassoma/metabolismo , Domínios Proteicos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Estresse Fisiológico/genética , Virulência
4.
Cell Mol Life Sci ; 76(22): 4511-4524, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31338556

RESUMO

The nucleolus is a sub-nuclear body known primarily for its role in ribosome biogenesis. Increased number and/or size of nucleoli have historically been used by pathologists as a prognostic indicator of cancerous lesions. This increase in nucleolar number and/or size is classically attributed to the increased need for protein synthesis in cancer cells. However, evidences suggest that the nucleolus plays critical roles in many cellular functions in both normal cell biology and disease pathologies, including cancer. As new functions of the nucleolus are elucidated, there is mounting evidence to support the role of the nucleolus in regulating additional cellular functions, particularly response to cellular stressors, maintenance of genome stability, and DNA damage repair, as well as the regulation of gene expression and biogenesis of several ribonucleoproteins. This review highlights the central role of the nucleolus in carcinogenesis and cancer progression and discusses how cancer cells may become "addicted" to nucleolar functions.


Assuntos
Nucléolo Celular/fisiologia , Neoplasias/patologia , Animais , Carcinogênese/patologia , Dano ao DNA/fisiologia , Reparo do DNA/fisiologia , Progressão da Doença , Instabilidade Genômica/fisiologia , Humanos
5.
PLoS Genet ; 15(5): e1008174, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31120885

RESUMO

Proteins of the Fanconi Anemia (FA) complementation group are required for crosslink (CL) repair in humans and their loss leads to severe pathological phenotypes. Here we characterize a homolog of the Fe-S cluster helicase FANCJ in the model plant Arabidopsis, AtFANCJB, and show that it is involved in interstrand CL repair. It acts at a presumably early step in concert with the nuclease FAN1 but independently of the nuclease AtMUS81, and is epistatic to both error-prone and error-free post-replicative repair in Arabidopsis. The simultaneous knock out of FANCJB and the Fe-S cluster helicase RTEL1 leads to induced cell death in root meristems, indicating an important role of the enzymes in replicative DNA repair. Surprisingly, we found that AtFANCJB is involved in safeguarding rDNA stability in plants. In the absence of AtRTEL1 and AtFANCJB, we detected a synergetic reduction to about one third of the original number of 45S rDNA copies. It is tempting to speculate that the detected rDNA instability might be due to deficiencies in G-quadruplex structure resolution and might thus contribute to pathological phenotypes of certain human genetic diseases.


Assuntos
Proteínas de Grupos de Complementação da Anemia de Fanconi/genética , Proteínas de Grupos de Complementação da Anemia de Fanconi/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Fatores de Transcrição de Zíper de Leucina Básica/genética , Dano ao DNA , DNA Helicases/genética , DNA Helicases/metabolismo , Reparo do DNA/fisiologia , Replicação do DNA , DNA Ribossômico/genética , DNA Ribossômico/metabolismo , Anemia de Fanconi/genética , Instabilidade Genômica , Meristema/metabolismo , Mutação , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , RNA Helicases/genética
6.
Nat Protoc ; 14(5): 1489-1508, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30962605

RESUMO

Non-coding RNA (ncRNA) molecules have been shown to play a variety of cellular roles; however, the contributions of different types of RNA to specific phenomena are often hard to dissect. To study the role of RNA in the assembly of DNA damage response (DDR) foci, we developed the RNase A treatment and reconstitution (RATaR) method, in which cells are mildly permeabilized, incubated with recombinant RNase A and subsequently reconstituted with different RNA species, under conditions of RNase A inactivation and inhibition of endogenous transcription. The block of transcription right after RNase A removal represents a key innovation of RATaR, preventing potential contributions of endogenously neo-synthesized transcripts to the phenotypes studied. A critical aspect of this technique is the balance between sufficient permeabilization of membranes to allow enzyme/RNA access into the cell nucleus and cell viability. Here, we present our protocol for RNA-dependent DDR foci disassembly and reassembly using fluorescent DDR RNAs (DDRNAs) in NIH2/4 cells, an engineered NIH3T3-derived cell line. The use of sequence-specific, fluorescent RNA molecules permits the concomitant determination of their subcellular localization and biological functions. We also outline adaptations of RATaR when implemented in different cell lines exposed to various genotoxic treatments, such as γ-radiation, restriction enzymes and telomere deprotection. In all these cases, the entire procedure can be completed within 2 h without the need for special equipment or uncommon skills. We believe this technique will prove useful for investigating the contribution of RNA to a variety of relevant cellular processes.


Assuntos
Dano ao DNA , Reparo do DNA , RNA não Traduzido , Ribonuclease Pancreático/metabolismo , Animais , Dano ao DNA/genética , Dano ao DNA/fisiologia , Reparo do DNA/genética , Reparo do DNA/fisiologia , Técnicas Genéticas , Células HeLa , Humanos , Camundongos , Células NIH 3T3 , RNA/análise , RNA/genética , RNA/metabolismo , RNA não Traduzido/genética , RNA não Traduzido/fisiologia
7.
Nat Commun ; 10(1): 895, 2019 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-30837464

RESUMO

Sleep is essential to all animals with a nervous system. Nevertheless, the core cellular function of sleep is unknown, and there is no conserved molecular marker to define sleep across phylogeny. Time-lapse imaging of chromosomal markers in single cells of live zebrafish revealed that sleep increases chromosome dynamics in individual neurons but not in two other cell types. Manipulation of sleep, chromosome dynamics, neuronal activity, and DNA double-strand breaks (DSBs) showed that chromosome dynamics are low and the number of DSBs accumulates during wakefulness. In turn, sleep increases chromosome dynamics, which are necessary to reduce the amount of DSBs. These results establish chromosome dynamics as a potential marker to define single sleeping cells, and propose that the restorative function of sleep is nuclear maintenance.


Assuntos
Cromossomos/genética , Quebras de DNA de Cadeia Dupla , Reparo do DNA/fisiologia , Neurônios/metabolismo , Sono/fisiologia , Animais , Animais Geneticamente Modificados , Núcleo Celular/genética , Núcleo Celular/metabolismo , Cromossomos/metabolismo , Feminino , Masculino , Microscopia Confocal , Modelos Animais , Neurônios/citologia , Imagem com Lapso de Tempo , Peixe-Zebra
8.
Nat Commun ; 10(1): 1207, 2019 03 14.
Artigo em Inglês | MEDLINE | ID: mdl-30872584

RESUMO

In bacteria, transcription-coupled repair of DNA lesions initiates after the Mfd protein removes RNA polymerases (RNAPs) stalled at the lesions. The bacterial RNA helicase, Rho, is a transcription termination protein that dislodges the elongation complexes. Here, we show that Rho dislodges the stalled RNAPs at DNA lesions. Strains defective in both Rho and Mfd are susceptible to DNA-damaging agents and are inefficient in repairing or propagating UV-damaged DNA. In vitro transcription assays show that Rho dissociates the stalled elongation complexes at the DNA lesions. We conclude that Rho-dependent termination recycles stalled RNAPs, which might facilitate DNA repair and other DNA-dependent processes essential for bacterial cell survival. We surmise that Rho might compete with, or augment, the Mfd function.


Assuntos
Reparo do DNA/fisiologia , RNA Polimerases Dirigidas por DNA/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/fisiologia , Terminação da Transcrição Genética/fisiologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Dano ao DNA/efeitos dos fármacos , Dano ao DNA/efeitos da radiação , DNA Bacteriano/metabolismo , Proteínas de Escherichia coli/genética , Mitomicina/farmacologia , Mutação , Fatores de Alongamento de Peptídeos/genética , Fatores de Alongamento de Peptídeos/metabolismo , RNA Bacteriano/biossíntese , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Raios Ultravioleta/efeitos adversos
9.
BMC Res Notes ; 12(1): 92, 2019 Feb 18.
Artigo em Inglês | MEDLINE | ID: mdl-30777129

RESUMO

OBJECTIVES: Reactive oxygen species (ROS) oxidize guanine residues in DNA to form 7,8-dihydro-oxo-2'-deoxyguanosine (8oxoG) lesions in the genome. Human 8-oxoguanine glycosylase-1 (hOGG1) recognizes and excises this highly mutagenic species when it is base-paired opposite a cytosine. We sought to characterize biochemically several hOGG1 variants that have been found in cancer tissues and cell lines, reasoning that if these variants have reduced repair capabilities, they could lead to an increased chance of mutagenesis and carcinogenesis. RESULTS: We have over-expressed and purified the R46Q, A85S, R154H, and S232T hOGG1 variants and have investigated their repair efficiency and thermostability. The hOGG1 variants showed only minor perturbations in the kinetics of 8oxoG excision relative to wild-type hOGG1. Thermal denaturation monitored by circular dichroism revealed that R46Q hOGG1 had a significantly lower Tm (36.6 °C) compared to the other hOGG1 variants (40.9 °C to 43.2 °C). Prolonged pre-incubation at 37 °C prior to the glycosylase assay dramatically reduces the excision activity of R46Q hOGG1, has a modest effect on wild-type hOGG1, and a negligible effect on A85S, R154H, and S232T hOGG1. The observed thermolability of hOGG1 variants was mostly alleviated by co-incubation with stoichiometric amounts of competitor DNA.


Assuntos
DNA Glicosilases/metabolismo , Reparo do DNA/fisiologia , Guanina/análogos & derivados , Desnaturação de Ácido Nucleico , DNA Glicosilases/genética , Reparo do DNA/genética , Guanina/metabolismo , Humanos , Mutação
10.
Endocrinol. diabetes nutr. (Ed. impr.) ; 66(2): 90-98, feb. 2019. tab
Artigo em Inglês | IBECS | ID: ibc-175800

RESUMO

Introduction: Increasing number of experimental and clinical studies suggest a strong relationship between hyperglycemia, oxidative stress, DNA damage and diabetic nephropathy (DN). Also, epidemiologic studies remark an enhanced risk of cancer with type 2 diabetes. This research aims to assess whether the X-ray cross complementing group 3 (XRCC3) gene T241M polymorphism (rs861539) and X-ray cross complementing group 1 (XRCC1) gene A399G polymorphism (rs25487) are related with predisposition to type 2 diabetes mellitus (T2DM) and to diabetic nephropathy in Turkish population. Materials and methods: Polymerase chain reaction-based restriction fragment length polymorphism (PCR-RFLP) was performed to identify the distribution of genotypes and frequency of alleles of T241M polymorphism of the XRCC3 gene (XRCC3 T241M) and A399G polymorphism of the XRCC1 gene (XRCC1 A399G). The study population included 238 subjects residing in Istanbul, Turkey; 116 with T2DM, 50 with DN and 72 with normal glucose metabolism. Results and conclusion: Polymorphic Gln allele of XRCC1 gene was significantly related with T2DM and DN (OR 3.09, 95% CI 1.14-8.40 and OR 3.29 95% CI 1.23-8.80, respectively) however, there was no statistical association of XRCC3 T241M with T2DM or DN. The results of this study suggest that XRCC1 399Gln polymorphism is related with an increased susceptibility to T2DM and DN in the studied Turkish population


Introducción: Un número creciente de estudios experimentales y clínicos sugiere una sólida relación entre hiperglucemia, estrés oxidativo, daño en el ADN y nefropatía diabética (ND). Además, los estudios epidemiológicos advierten mayor riesgo de cáncer con diabetes de tipo 2. Esta investigación tiene como objetivo evaluar si el polimorfismo del gen T241M del grupo 3 (XRCC3) complementario cruzado de rayos X (rs861539) y el polimorfismo del gen A399G del grupo 1 (XRCC1) complementario cruzado de rayos X (rs25487) están relacionados con la predisposición a la diabetes mellitus de tipo 2 (DM2) y a la nefropatía diabética en la población turca. Materiales y métodos: Se realizó un polimorfismo de longitud de fragmento de restricción basado en la reacción en cadena de la polimerasa (PCR-RFLP) para identificar la distribución de genotipos y la frecuencia de los alelos del polimorfismo T241M del gen XRCC3 (XRCC3 T241M) y el polimorfismo A399G del gen XRCC1(XRCC1 A399G). La población de estudio incluyó a 238 individuos que residían en Estambul, Turquía; 116 de ellos con DM2, 50 con ND y 72 con metabolismo de la glucosa normal. Resultados y conclusión: El alelo Gln polimórfico del gen XRCC1 se relacionó de manera importante con DM2 y ND (OR: 3,09; IC95%: 1,14-8,40 y OR: 3,29; IC95%:1,23-8,80, respectivamente). Sin embargo, no hubo asociación estadística de XRCC3 T241M con DM2 o ND. Los resultados de este estudio sugieren que el polimorfismo XRCC1 399Gln está relacionado con un aumento de la susceptibilidad a la DM2 y a la ND en la población turca estudiada


Assuntos
Humanos , Masculino , Feminino , Adulto Jovem , Adulto , Pessoa de Meia-Idade , Idoso , Idoso de 80 Anos ou mais , Diabetes Mellitus Tipo 2/genética , Neuropatias Diabéticas/genética , Reparo do DNA/fisiologia , Polimorfismo de Fragmento de Restrição/genética , Polimorfismo Genético/genética , Polimorfismo Genético/fisiologia , Turquia , Polimorfismo de Fragmento de Restrição/fisiologia , Diabetes Mellitus Tipo 2/diagnóstico , Nefropatias Diabéticas/diagnóstico
11.
PLoS One ; 14(2): e0212889, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30807606

RESUMO

Pulmonary arterial hypertension (PAH) is characterized by remodeling and narrowing of the pulmonary arteries, which lead to elevation of right ventricular pressure, heart failure, and death. Proliferation of pulmonary artery smooth muscle cells (PASMCs) is thought to be central to the pathogenesis of PAH, although the underlying mechanisms are still being explored. The protein p53 is involved in cell cycle coordination, DNA repair, apoptosis, and cellular senescence, but its role in pulmonary hypertension (PH) is not fully known. We developed a mouse model of hypoxia-induced pulmonary hypertension (PH) and found significant reduction of p53 expression in the lungs. Our in vitro experiments with metabolomic analyses and the Seahorse XF extracellular flux analyzer indicated that suppression of p53 expression in PASMCs led to upregulation of glycolysis and downregulation of mitochondrial respiration, suggesting a proliferative phenotype resembling that of cancer cells. It was previously shown that systemic genetic depletion of p53 in a murine PH model led to more severe lung manifestations. Lack of information about the role of cell-specific p53 signaling promoted us to investigate it in our mouse PH model with the inducible Cre-loxP system. We generated a mouse model with SMC-specific gain or loss of p53 function by crossing Myh11-Cre/ERT2 mice with floxed Mdm4 mice or floxed Trp53 mice. After these animals were exposed to hypoxia for 4 weeks, we conducted hemodynamic and echocardiographic studies. Surprisingly, the severity of PH was similar in both groups of mice and there were no differences between the genotypes. Our findings in these mice indicate that activation or suppression of p53 signaling in SMCs has a minor role in the pathogenesis of PH and suggest that p53 signaling in other cells (endothelial cells, immune cells, or fibroblasts) may be involved in the progression of this condition.


Assuntos
Hipertensão Pulmonar/metabolismo , Miócitos de Músculo Liso/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Animais , Apoptose/genética , Apoptose/fisiologia , Western Blotting , Células Cultivadas , Senescência Celular/genética , Senescência Celular/fisiologia , Reparo do DNA/genética , Reparo do DNA/fisiologia , Ecocardiografia , Humanos , Hipertensão Pulmonar/genética , Hipóxia/metabolismo , Masculino , Metabolômica/métodos , Camundongos , Camundongos Endogâmicos C57BL , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteína Supressora de Tumor p53/genética
12.
Nat Commun ; 10(1): 241, 2019 01 16.
Artigo em Inglês | MEDLINE | ID: mdl-30651562

RESUMO

Cell survival after oxidative DNA damage requires signaling, repair and transcriptional events often enabled by nucleosome displacement, exchange or removal by chromatin remodeling enzymes. Here, we show that Chromodomain Helicase DNA-binding protein 6 (CHD6), distinct to other CHD enzymes, is stabilized during oxidative stress via reduced degradation. CHD6 relocates rapidly to DNA damage in a manner dependent upon oxidative lesions and a conserved N-terminal poly(ADP-ribose)-dependent recruitment motif, with later retention requiring the double chromodomain and central core. CHD6 ablation increases reactive oxygen species persistence and impairs anti-oxidant transcriptional responses, leading to elevated DNA breakage and poly(ADP-ribose) induction that cannot be rescued by catalytic or double chromodomain mutants. Despite no overt epigenetic or DNA repair abnormalities, CHD6 loss leads to impaired cell survival after chronic oxidative stress, abnormal chromatin relaxation, amplified DNA damage signaling and checkpoint hypersensitivity. We suggest that CHD6 is a key regulator of the oxidative DNA damage response.


Assuntos
Montagem e Desmontagem da Cromatina/fisiologia , Dano ao DNA/fisiologia , DNA Helicases/metabolismo , Reparo do DNA/fisiologia , Proteínas do Tecido Nervoso/metabolismo , Estresse Oxidativo/fisiologia , Células A549 , Sobrevivência Celular/fisiologia , Dano ao DNA/efeitos da radiação , DNA Helicases/genética , Pontos de Checagem da Fase G2 do Ciclo Celular/fisiologia , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos da radiação , Células HEK293 , Humanos , Microscopia Intravital , Lasers/efeitos adversos , Proteínas do Tecido Nervoso/genética , RNA Interferente Pequeno/metabolismo , Espécies Reativas de Oxigênio/metabolismo
13.
Nat Chem Biol ; 15(3): 269-275, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30664685

RESUMO

Holliday junction (HJ) resolution by resolving enzymes is essential for chromosome segregation and recombination-mediated DNA repair. HJs undergo two types of structural dynamics that determine the outcome of recombination: conformer exchange between two isoforms and branch migration. However, it is unknown how the preferred branch point and conformer are achieved between enzyme binding and HJ resolution given the extensive binding interactions seen in static crystal structures. Single-molecule fluorescence resonance energy transfer analysis of resolving enzymes from bacteriophages (T7 endonuclease I), bacteria (RuvC), fungi (GEN1) and humans (hMus81-Eme1) showed that both types of HJ dynamics still occur after enzyme binding. These dimeric enzymes use their multivalent interactions to achieve this, going through a partially dissociated intermediate in which the HJ undergoes nearly unencumbered dynamics. This evolutionarily conserved property of HJ resolving enzymes provides previously unappreciated insight on how junction resolution, conformer exchange and branch migration may be coordinated.


Assuntos
DNA Cruciforme/metabolismo , DNA Cruciforme/fisiologia , Resolvases de Junção Holliday/metabolismo , Animais , Proteínas de Arabidopsis , Segregação de Cromossomos/genética , Reparo do DNA/fisiologia , Proteínas de Ligação a DNA/fisiologia , Desoxirribonuclease I , Endodesoxirribonucleases , Endonucleases , Proteínas de Escherichia coli , Transferência Ressonante de Energia de Fluorescência/métodos , Resolvases de Junção Holliday/fisiologia , Humanos , Ligação Proteica , Recombinação Genética/genética , Imagem Individual de Molécula/métodos , Especificidade por Substrato
14.
Methods Mol Biol ; 1917: 3-24, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30610624

RESUMO

Remarkable progress in the development of technologies for sequence-specific modification of primary DNA sequences has enabled the precise engineering of crops with novel characteristics. These programmable sequence-specific modifiers include site-directed nucleases (SDNs) and base editors (BEs). Currently, these genome editing machineries can be targeted to specific chromosomal locations to induce sequence changes. However, the sequence mutation outcomes are often greatly influenced by the type of DNA damage being generated, the status of host DNA repair machinery, and the presence and structure of DNA repair donor molecule. The outcome of sequence modification from repair of DNA double-strand breaks (DSBs) is often uncontrollable, resulting in unpredictable sequence insertions or deletions of various sizes. For base editing, the precision of intended edits is much higher, but the efficiency can vary greatly depending on the type of BE used or the activity of the endogenous DNA repair systems. This article will briefly review the possible DNA repair pathways present in the plant cells commonly used for generating edited variants for genome engineering applications. We will discuss the potential use of DNA repair mechanisms for developing and improving methodologies to enhance genome engineering efficiency and to direct DNA repair processes toward the desired outcomes.


Assuntos
DNA de Plantas/genética , Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades/genética , Reparo do DNA por Junção de Extremidades/fisiologia , Reparo do DNA/genética , Reparo do DNA/fisiologia , Edição de Genes , Engenharia Genética , Genoma de Planta/genética
15.
Int J Mol Sci ; 20(3)2019 Jan 22.
Artigo em Inglês | MEDLINE | ID: mdl-30678294

RESUMO

Chemotherapeutic dosing, is largely based on the tolerance levels of toxicity today. Molecular imaging strategies can be leveraged to quantify DNA cytotoxicity and thereby serve as a theranostic tool to improve the efficacy of treatments. Methoxyamine-modified cyanine-7 (Cy7MX) is a molecular probe which binds to apurinic/apyrimidinic (AP)-sites, inhibiting DNA-repair mechanisms implicated by cytotoxic chemotherapies. Herein, we loaded (Cy7MX) onto polyethylene glycol-coated gold nanoparticles (AuNP) to selectively and stably deliver the molecular probe intravenously to tumors. We optimized the properties of Cy7MX-loaded AuNPs using optical spectroscopy and tested the delivery mechanism and binding affinity using the DLD1 colon cancer cell line in vitro. A 10:1 ratio of Cy7MX-AuNPs demonstrated a strong AP site-specific binding and the cumulative release profile demonstrated 97% release within 12 min from a polar to a nonpolar environment. We further demonstrated targeted delivery using imaging and biodistribution studies in vivo in an xenografted mouse model. This work lays a foundation for the development of real-time molecular imaging techniques that are poised to yield quantitative measures of the efficacy and temporal profile of cytotoxic chemotherapies.


Assuntos
Dano ao DNA/fisiologia , Reparo do DNA/fisiologia , Ouro/química , Nanopartículas Metálicas/química , Nanomedicina Teranóstica/métodos , Linhagem Celular Tumoral , Humanos , Estrutura Molecular
16.
Cell ; 176(1-2): 167-181.e21, 2019 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-30595447

RESUMO

Covalent DNA-protein cross-links (DPCs) impede replication fork progression and threaten genome integrity. Using Xenopus egg extracts, we previously showed that replication fork collision with DPCs causes their proteolysis, followed by translesion DNA synthesis. We show here that when DPC proteolysis is blocked, the replicative DNA helicase CMG (CDC45, MCM2-7, GINS), which travels on the leading strand template, bypasses an intact leading strand DPC. Single-molecule imaging reveals that GINS does not dissociate from CMG during bypass and that CMG slows dramatically after bypass, likely due to uncoupling from the stalled leading strand. The DNA helicase RTEL1 facilitates bypass, apparently by generating single-stranded DNA beyond the DPC. The absence of RTEL1 impairs DPC proteolysis, suggesting that CMG must bypass the DPC to enable proteolysis. Our results suggest a mechanism that prevents inadvertent CMG destruction by DPC proteases, and they reveal CMG's remarkable capacity to overcome obstacles on its translocation strand.


Assuntos
DNA Helicases/metabolismo , DNA Helicases/fisiologia , Reparo do DNA/fisiologia , Animais , Proteínas de Ciclo Celular/metabolismo , DNA/metabolismo , Replicação do DNA , DNA de Cadeia Simples , Proteínas de Ligação a DNA/fisiologia , Feminino , Masculino , Proteólise , Imagem Individual de Molécula/métodos , Xenopus laevis/metabolismo
17.
Cell ; 176(1-2): 127-143.e24, 2019 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-30633903

RESUMO

DNA damage provokes mutations and cancer and results from external carcinogens or endogenous cellular processes. However, the intrinsic instigators of endogenous DNA damage are poorly understood. Here, we identify proteins that promote endogenous DNA damage when overproduced: the DNA "damage-up" proteins (DDPs). We discover a large network of DDPs in Escherichia coli and deconvolute them into six function clusters, demonstrating DDP mechanisms in three: reactive oxygen increase by transmembrane transporters, chromosome loss by replisome binding, and replication stalling by transcription factors. Their 284 human homologs are over-represented among known cancer drivers, and their RNAs in tumors predict heavy mutagenesis and a poor prognosis. Half of the tested human homologs promote DNA damage and mutation when overproduced in human cells, with DNA damage-elevating mechanisms like those in E. coli. Our work identifies networks of DDPs that provoke endogenous DNA damage and may reveal DNA damage-associated functions of many human known and newly implicated cancer-promoting proteins.


Assuntos
Dano ao DNA/genética , Dano ao DNA/fisiologia , Reparo do DNA/fisiologia , Proteínas de Bactérias/metabolismo , Instabilidade Cromossômica/fisiologia , Replicação do DNA/fisiologia , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/metabolismo , Instabilidade Genômica , Humanos , Proteínas de Membrana Transportadoras/fisiologia , Mutagênese , Mutação , Fatores de Transcrição/metabolismo
18.
Proc Natl Acad Sci U S A ; 116(7): 2561-2570, 2019 02 12.
Artigo em Inglês | MEDLINE | ID: mdl-30692263

RESUMO

Fanconi anemia (FA) is a disease of DNA repair characterized by bone marrow failure and a reduced ability to remove DNA interstrand cross-links. Here, we provide evidence that the FA protein FANCI also functions in ribosome biogenesis, the process of making ribosomes that initiates in the nucleolus. We show that FANCI localizes to the nucleolus and is functionally and physically tied to the transcription of pre-ribosomal RNA (pre-rRNA) and to large ribosomal subunit (LSU) pre-rRNA processing independent of FANCD2. While FANCI is known to be monoubiquitinated when activated for DNA repair, we find that it is predominantly in the deubiquitinated state in the nucleolus, requiring the nucleoplasmic deubiquitinase (DUB) USP1 and the nucleolar DUB USP36. Our model suggests a possible dual pathophysiology for FA that includes defects in DNA repair and in ribosome biogenesis.


Assuntos
Proteínas de Grupos de Complementação da Anemia de Fanconi/fisiologia , Ribossomos/metabolismo , Western Blotting , Nucléolo Celular/metabolismo , Reparo do DNA/fisiologia , Eletroforese em Gel de Poliacrilamida , Anemia de Fanconi/fisiopatologia , Proteínas de Grupos de Complementação da Anemia de Fanconi/genética , Proteínas de Grupos de Complementação da Anemia de Fanconi/metabolismo , Células HEK293 , Células HeLa , Humanos , Mutação , Biossíntese de Proteínas , Precursores de RNA/genética , RNA Ribossômico/genética , Transcrição Genética , Ubiquitinação
19.
Oncogene ; 38(19): 3616-3635, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30651597

RESUMO

The melanocytic lineage, which is prominently exposed to ultraviolet radiation (UVR) and radiation-independent oxidative damage, requires specific DNA-damage response mechanisms to maintain genomic and transcriptional homeostasis. The coordinate lineage-specific regulation of intricately intertwined DNA repair and transcription is incompletely understood. Here we demonstrate that the Microphthalmia-associated transcription factor (MITF) directly controls general transcription and UVR-induced nucleotide excision repair by transactivation of GTF2H1 as a core element of TFIIH. Thus, MITF ensures the rapid resumption of transcription after completion of strand repair and maintains transcriptional output, which is indispensable for survival of the melanocytic lineage including melanoma in vitro and in vivo. Moreover, MITF controls c-MYC implicated in general transcription by transactivation of far upstream binding protein 2 (FUBP2/KSHRP), which induces c-MYC pulse regulation through TFIIH, and experimental depletion of MITF results in consecutive loss of CDK7 in the TFIIH-CAK subcomplex. Targeted for proteasomal degradation, CDK7 is dependent on transactivation by MITF or c-MYC to maintain a steady state. The dependence of TFIIH-CAK on sequence-specific MITF and c-MYC constitutes a previously unrecognized mechanism feeding into super-enhancer-driven or other oncogenic transcriptional circuitries, which supports the concept of a transcription-directed therapeutic intervention in melanoma.


Assuntos
Reparo do DNA/fisiologia , Fator de Transcrição Associado à Microftalmia/metabolismo , Fosfoproteínas/metabolismo , Fator de Transcrição TFIIH/metabolismo , Fatores de Transcrição TFII/metabolismo , Animais , Células Cultivadas , Reparo do DNA/efeitos da radiação , Receptor com Domínio Discoidina 1/genética , Receptor com Domínio Discoidina 1/metabolismo , Feminino , Genes myc , Humanos , Melanócitos/fisiologia , Melanócitos/efeitos da radiação , Melanoma/metabolismo , Melanoma/patologia , Camundongos SCID , Fator de Transcrição Associado à Microftalmia/genética , Fosfoproteínas/genética , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/patologia , Fator de Transcrição TFIIH/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição TFII/genética , Transcrição Genética , Raios Ultravioleta
20.
Oncol Rep ; 41(1): 552-558, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30365131

RESUMO

Breast cancer metastasis suppressor 1 (BRMS1) is a tumor metastasis suppressor implicated in multiple steps during the metastatic cascade. Many proteins interacting with BRMS1 have been identified to unravel the intracellular signaling mechanisms. In the present study, we report that FANCI is a novel interacting protein of BRMS1 as determined by co­immunoprecipitation assay. The linker region between two coiled­coil motifs of BRMS1 is required for BRMS1­FANCI interaction. FANCI is an essential protein in the Fanconi anemia (FA) pathway responsible for the repair of DNA interstrand crosslinks (ICLs). We demonstrated that knockdown or knockout of BRMS1 significantly diminished the monoubiquitination of FANCI and FANCD2 in response to DNA ICL damage. BRMS1­deficient cells exhibited suppressed FANCD2 foci formation and hypersensitivity to ICLs. Moreover, rescue assays by utilizing different BRMS1 constructs suggested that BRMS1­FANCI interaction is necessary for the regulatory role of BRMS1 in the FA pathway. Overall, our findings characterize BRMS1 as a novel regulatory protein functioning in the DNA repair pathway via protein interaction.


Assuntos
Dano ao DNA/fisiologia , DNA/metabolismo , Proteínas de Grupos de Complementação da Anemia de Fanconi/metabolismo , Proteínas Repressoras/metabolismo , Linhagem Celular , Reparo do DNA/fisiologia , Anemia de Fanconi/metabolismo , Células HEK293 , Humanos , Transdução de Sinais/fisiologia , Ubiquitinação/fisiologia
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