Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 2.601
Filtrar
1.
Nat Commun ; 10(1): 4887, 2019 10 25.
Artigo em Inglês | MEDLINE | ID: mdl-31653834

RESUMO

Accumulation of DNA lesions causing transcription stress is associated with natural and accelerated aging and culminates with profound metabolic alterations. Our understanding of the mechanisms governing metabolic redesign upon genomic instability, however, is highly rudimentary. Using Ercc1-defective mice and Xpg knock-out mice, we demonstrate that combined defects in transcription-coupled DNA repair (TCR) and in nucleotide excision repair (NER) directly affect bioenergetics due to declined transcription, leading to increased ATP levels. This in turn inhibits glycolysis allosterically and favors glucose rerouting through the pentose phosphate shunt, eventually enhancing production of NADPH-reducing equivalents. In NER/TCR-defective mutants, augmented NADPH is not counterbalanced by increased production of pro-oxidants and thus pentose phosphate potentiation culminates in an over-reduced redox state. Skin fibroblasts from the TCR disease Cockayne syndrome confirm results in animal models. Overall, these findings unravel a mechanism connecting DNA damage and transcriptional stress to metabolic redesign and protective antioxidant defenses.


Assuntos
Trifosfato de Adenosina/metabolismo , Antioxidantes/metabolismo , Dano ao DNA/genética , Reparo do DNA/genética , Glicólise/fisiologia , NADP/metabolismo , Via de Pentose Fosfato/fisiologia , Transcrição Genética/genética , Regulação Alostérica , Animais , Síndrome de Cockayne/metabolismo , Proteínas de Ligação a DNA/genética , Endonucleases/genética , Fibroblastos/metabolismo , Instabilidade Genômica , Metabolômica , Camundongos , Camundongos Knockout , Proteínas Nucleares/genética , Oxirredução , Pele/citologia , Fatores de Transcrição/genética
2.
Cytogenet Genome Res ; 159(1): 48-53, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31610539

RESUMO

Visualizing the spatiotemporal organization of the genome will improve our understanding of how chromatin structure and function are intertwined. Here, we describe a further development of the CRISPR/Cas9-based RNA-guided endonuclease-in situ labeling (RGEN-ISL) method. RGEN-ISL allowed the differentiation between vertebrate-type (TTAGGG)n and Arabidopsis-type (TTTAGGG)n telomere repeats. Using maize as an example, we established a combination of RGEN-ISL, immunostaining, and EdU labeling to visualize in situ specific repeats, histone marks, and DNA replication sites, respectively. The effects of the non-denaturing RGEN-ISL and standard denaturing FISH on the chromatin structure were compared using super-resolution microscopy. 3D structured illumination microscopy revealed that denaturation and acetic acid fixation impaired and flattened the chromatin. The broad range of adaptability of RGEN-ISL to different combinations of methods has the potential to advance the field of chromosome biology.


Assuntos
Amaryllidaceae/genética , Arabidopsis/genética , Sistemas CRISPR-Cas/genética , Replicação do DNA/genética , Zea mays/genética , Cromatina/metabolismo , Cromossomos/genética , DNA de Plantas/genética , Endonucleases/genética , Hibridização in Situ Fluorescente/métodos , RNA Guia/genética , Telômero/genética
3.
Nat Commun ; 10(1): 4906, 2019 10 28.
Artigo em Inglês | MEDLINE | ID: mdl-31659165

RESUMO

The delivery of biologic cargoes to airway epithelial cells is challenging due to the formidable barriers imposed by its specialized and differentiated cells. Among cargoes, recombinant proteins offer therapeutic promise but the lack of effective delivery methods limits their development. Here, we achieve protein and SpCas9 or AsCas12a ribonucleoprotein (RNP) delivery to cultured human well-differentiated airway epithelial cells and mouse lungs with engineered amphiphilic peptides. These shuttle peptides, non-covalently combined with GFP protein or CRISPR-associated nuclease (Cas) RNP, allow rapid entry into cultured human ciliated and non-ciliated epithelial cells and mouse airway epithelia. Instillation of shuttle peptides combined with SpCas9 or AsCas12a RNP achieves editing of loxP sites in airway epithelia of ROSAmT/mG mice. We observe no evidence of short-term toxicity with a widespread distribution restricted to the respiratory tract. This peptide-based technology advances potential therapeutic avenues for protein and Cas RNP delivery to refractory airway epithelial cells.


Assuntos
Proteínas de Bactérias/metabolismo , Sistemas de Liberação de Medicamentos/métodos , Endonucleases/metabolismo , Células Epiteliais/metabolismo , Pneumopatias/terapia , Pulmão/metabolismo , Peptídeos/genética , Animais , Proteínas de Bactérias/genética , Brônquios/citologia , Brônquios/metabolismo , Endonucleases/genética , Terapia Genética , Humanos , Pneumopatias/genética , Pneumopatias/metabolismo , Camundongos , Peptídeos/administração & dosagem , Peptídeos/metabolismo , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Suínos
4.
Nat Protoc ; 14(10): 2986-3012, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31548639

RESUMO

Rapid detection of nucleic acids is integral to applications in clinical diagnostics and biotechnology. We have recently established a CRISPR-based diagnostic platform that combines nucleic acid pre-amplification with CRISPR-Cas enzymology for specific recognition of desired DNA or RNA sequences. This platform, termed specific high-sensitivity enzymatic reporter unlocking (SHERLOCK), allows multiplexed, portable, and ultra-sensitive detection of RNA or DNA from clinically relevant samples. Here, we provide step-by-step instructions for setting up SHERLOCK assays with recombinase-mediated polymerase pre-amplification of DNA or RNA and subsequent Cas13- or Cas12-mediated detection via fluorescence and colorimetric readouts that provide results in <1 h with a setup time of less than 15 min. We also include guidelines for designing efficient CRISPR RNA (crRNA) and isothermal amplification primers, as well as discuss important considerations for multiplex and quantitative SHERLOCK detection assays.


Assuntos
Sistemas CRISPR-Cas , Endonucleases/genética , Ácidos Nucleicos/análise , Primers do DNA , Endonucleases/isolamento & purificação , Endonucleases/metabolismo , Humanos , Leptotrichia/genética , Técnicas de Amplificação de Ácido Nucleico/métodos , Ácidos Nucleicos/genética , Engenharia de Proteínas/métodos , RNA Guia , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Ribonucleases/genética , Ribonucleases/isolamento & purificação , Ribonucleases/metabolismo , Fluxo de Trabalho , Zika virus/genética , Infecção por Zika virus/sangue , Infecção por Zika virus/urina
5.
World J Gastroenterol ; 25(34): 5152-5161, 2019 Sep 14.
Artigo em Inglês | MEDLINE | ID: mdl-31558863

RESUMO

BACKGROUND: The xeroderma pigmentosum group G (XPG) gene at chromosome 13q33 consists of 15 exons, which may be related to the occurrence and development of gastric cancer (GC). AIM: To examine the association of several common single nucleotide polymorphisms (SNPs) of the XPG gene with GC risk and survival. METHODS: Five SNPs of XPG (rs2094258, rs751402, rs873601, rs2296147, and rs1047768) were genotyped by PCR restriction fragment length polymorphism in 956 histologically confirmed GC cases and 1012 controls in North China. GC patients were followed for survival status and, if deceased, cause of death. Logistic regression and Cox regression were used for analysing associations of XPG SNPs with risk of GC and prognosis, respectively. For rs2094258, heterozygous model (CT vs CC), homozygous model (TT vs CC), recessive model (TT vs CT + CC), and dominant model (TT + CT vs CC) were analyzed. RESULTS: None of the examined loci were statistically associated with GC risk, although rs2296147 was marginally associated with GC risk (P = 0.050). GC patients with the rs2094258 CT + CC genotype showed worse survival than those with the TT genotype (log-rank test, P = 0.028), and patients with the CC genotype had a tendency of unfavourable prognosis compared with those with the TT + CT genotype (log-rank test, P = 0.039). The increase in C alleles of rs2094258 [hazard ratio (HR) = 1.19, 95% confidence interval (CI): 1.02-1.45, P = 0.037] were associated with the long-term survival of GC cases. Other risk factors for survival included tumor differentiation (HR = 4.51, 95%CI: 1.99-8.23, P < 0.001), lymphovascular invasion (HR = 1.97, 95%CI: 1.44-3.01, P < 0.001), and use of chemotherapy (HR = 0.81, 95%CI: 0.63-0.98, P = 0.041). CONCLUSION: The XPG rs2094258 polymorphism may be associated with overall survival in GC patients.


Assuntos
Proteínas de Ligação a DNA/genética , Endonucleases/genética , Predisposição Genética para Doença , Proteínas Nucleares/genética , Neoplasias Gástricas/genética , Fatores de Transcrição/genética , Estudos de Casos e Controles , Intervalo Livre de Doença , Feminino , Seguimentos , Humanos , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único , Prognóstico , Medição de Risco , Estômago/patologia , Neoplasias Gástricas/mortalidade , Neoplasias Gástricas/patologia
6.
Chem Commun (Camb) ; 55(78): 11671-11674, 2019 Sep 26.
Artigo em Inglês | MEDLINE | ID: mdl-31497827

RESUMO

We report the design and optimisation of novel oligonucleotide substrates for a sensitive fluorescence assay for high-throughput screening and functional studies of the DNA repair enzyme, XPF-ERCC1, with a view to accelerating inhibitor and drug discovery.


Assuntos
Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Endonucleases/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Dimerização , Endonucleases/química , Endonucleases/genética , Humanos , Oligonucleotídeos/química , Oligonucleotídeos/metabolismo , Especificidade por Substrato , Temperatura Ambiente
7.
Nucleic Acids Res ; 47(19): 10181-10201, 2019 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-31495888

RESUMO

Interstrand crosslinks (ICLs) are highly toxic DNA lesions that are repaired via a complex process requiring the coordination of several DNA repair pathways. Defects in ICL repair result in Fanconi anemia, which is characterized by bone marrow failure, developmental abnormalities, and a high incidence of malignancies. SLX4, also known as FANCP, acts as a scaffold protein and coordinates multiple endonucleases that unhook ICLs, resolve homologous recombination intermediates, and perhaps remove unhooked ICLs. In this study, we explored the role of SLX4IP, a constitutive factor in the SLX4 complex, in ICL repair. We found that SLX4IP is a novel regulatory factor; its depletion sensitized cells to treatment with ICL-inducing agents and led to accumulation of cells in the G2/M phase. We further discovered that SLX4IP binds to SLX4 and XPF-ERCC1 simultaneously and that disruption of one interaction also disrupts the other. The binding of SLX4IP to both SLX4 and XPF-ERCC1 not only is vital for maintaining the stability of SLX4IP protein, but also promotes the interaction between SLX4 and XPF-ERCC1, especially after DNA damage. Collectively, these results demonstrate a new regulatory role for SLX4IP in maintaining an efficient SLX4-XPF-ERCC1 complex in ICL repair.


Assuntos
Proteínas de Transporte/genética , Reparo do DNA/genética , Proteínas de Ligação a DNA/genética , Endonucleases/genética , Recombinação Homóloga/genética , Recombinases/genética , DNA/química , DNA/genética , Proteínas de Ligação a DNA/química , Células HEK293 , Humanos , Ligação Proteica/genética
8.
Nat Commun ; 10(1): 3556, 2019 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-31391465

RESUMO

Cystic fibrosis (CF) is an autosomal recessive disease caused by mutations in the CFTR gene. The 3272-26A>G and 3849+10kbC>T CFTR mutations alter the correct splicing of the CFTR gene, generating new acceptor and donor splice sites respectively. Here we develop a genome editing approach to permanently correct these genetic defects, using a single crRNA and the Acidaminococcus sp. BV3L6, AsCas12a. This genetic repair strategy is highly precise, showing very strong discrimination between the wild-type and mutant sequence and a complete absence of detectable off-targets. The efficacy of this gene correction strategy is verified in intestinal organoids and airway epithelial cells derived from CF patients carrying the 3272-26A>G or 3849+10kbC>T mutations, showing efficient repair and complete functional recovery of the CFTR channel. These results demonstrate that allele-specific genome editing with AsCas12a can correct aberrant CFTR splicing mutations, paving the way for a permanent splicing correction in genetic diseases.


Assuntos
Acidaminococcus/genética , Proteínas Associadas a CRISPR/genética , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Fibrose Cística/terapia , Edição de Genes/métodos , Alelos , Proteínas de Bactérias/genética , Biópsia , Técnicas de Cultura de Células , Linhagem Celular , Fibrose Cística/genética , Fibrose Cística/patologia , Endonucleases/genética , Humanos , Intestinos/patologia , Organoides , Mutação Puntual , Sítios de Splice de RNA/genética , Processamento de RNA/genética
9.
Nucleic Acids Res ; 47(16): 8632-8648, 2019 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-31392984

RESUMO

CRISPR-Cas systems provide heritable immunity against viruses by capturing short invader DNA sequences, termed spacers, and incorporating them into the CRISPR loci of the prokaryotic host genome. Here, we investigate DNA elements that control accurate spacer uptake in the type II-A CRISPR locus of Streptococcus thermophilus. We determined that purified Cas1 and Cas2 proteins catalyze spacer integration with high specificity for CRISPR repeat junctions. We show that 10 bp of the CRISPR leader sequence is critical for stimulating polarized integration preferentially at the repeat proximal to the leader. Spacer integration proceeds through a two-step transesterification reaction where the 3' hydroxyl groups of the spacer target both repeat borders on opposite strands. The leader-proximal end of the repeat is preferentially targeted for the first site of integration through recognition of sequences spanning the leader-repeat junction. Subsequently, second-site integration at the leader-distal end of the repeat is specified by multiple determinants including a length-defining mechanism relying on a repeat element proximal to the second site of integration. Our results highlight the intrinsic ability of type II Cas1/Cas2 proteins to coordinate directional and site-specific spacer integration into the CRISPR locus to ensure precise duplication of the repeat required for CRISPR immunity.


Assuntos
Sistemas CRISPR-Cas , Endonucleases/genética , Edição de Genes , Genoma Bacteriano , Streptococcus thermophilus/genética , Sequência de Bases , Endonucleases/imunologia , Endonucleases/metabolismo , Esterificação , Loci Gênicos , Isoenzimas/genética , Isoenzimas/imunologia , Isoenzimas/metabolismo , Mutagênese Insercional , Plasmídeos/química , Plasmídeos/metabolismo , Streptococcus thermophilus/imunologia , Streptococcus thermophilus/metabolismo , Streptococcus thermophilus/virologia , Vírus/genética , Vírus/metabolismo
10.
Genes (Basel) ; 10(8)2019 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-31374908

RESUMO

The incidence of thyroid cancer (TC), particularly well-differentiated forms (DTC), has been rising and remains the highest among endocrine malignancies. Although ionizing radiation (IR) is well established on DTC aetiology, other environmental and genetic factors may also be involved. DNA repair single nucleotide polymorphisms (SNPs) could be among the former, helping in explaining the high incidence. To further clarify the role of DNA repair SNPs in DTC susceptibility, we analyzed 36 SNPs in 27 DNA repair genes in a population of 106 DTCs and corresponding controls with the aim of interpreting joint data from previously studied isolated SNPs in DNA repair genes. Significant associations with DTC susceptibility were observed for XRCC3 rs861539, XPC rs2228001, CCNH rs2230641, MSH6 rs1042821 and ERCC5 rs2227869 and for a haplotype block on chromosome 5q. From 595 SNP-SNP combinations tested and 114 showing relevance, 15 significant SNP combinations (p < 0.01) were detected on paired SNP analysis, most of which involving CCNH rs2230641 and mismatch repair variants. Overall, a gene-dosage effect between the number of risk genotypes and DTC predisposition was observed. In spite of the volume of data presented, new studies are sought to provide an interpretability of the role of SNPs in DNA repair genes and their combinations in DTC susceptibility.


Assuntos
Reparo do DNA , Predisposição Genética para Doença , Polimorfismo de Nucleotídeo Único , Neoplasias da Glândula Tireoide/genética , Adulto , Idoso , Cromossomos Humanos Par 5/genética , Ciclina H/genética , Proteínas de Ligação a DNA/genética , Endonucleases/genética , Feminino , Haplótipos , Humanos , Masculino , Pessoa de Meia-Idade , Proteínas Nucleares/genética , Fatores de Transcrição/genética
11.
Mol Cell ; 75(4): 875-887.e5, 2019 08 22.
Artigo em Inglês | MEDLINE | ID: mdl-31442426

RESUMO

Diverse ribonucleoprotein complexes control mRNA processing, translation, and decay. Transcripts in these complexes localize to specific regions of the cell and can condense into non-membrane-bound structures such as stress granules. It has proven challenging to map the RNA composition of these large and dynamic structures, however. We therefore developed an RNA proximity labeling technique, APEX-seq, which uses the ascorbate peroxidase APEX2 to probe the spatial organization of the transcriptome. We show that APEX-seq can resolve the localization of RNAs within the cell and determine their enrichment or depletion near key RNA-binding proteins. Matching the spatial transcriptome, as revealed by APEX-seq, with the spatial proteome determined by APEX-mass spectrometry (APEX-MS), obtained precisely in parallel, provides new insights into the organization of translation initiation complexes on active mRNAs and unanticipated complexity in stress granule composition. Our novel technique allows a powerful and general approach to explore the spatial environment of macromolecules.


Assuntos
Grânulos Citoplasmáticos/metabolismo , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , Endonucleases/metabolismo , Enzimas Multifuncionais/metabolismo , Iniciação Traducional da Cadeia Peptídica , RNA/metabolismo , Coloração e Rotulagem , Transcriptoma , Grânulos Citoplasmáticos/genética , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/genética , Endonucleases/genética , Células HEK293 , Humanos , Enzimas Multifuncionais/genética , RNA/genética
12.
Nat Methods ; 16(9): 887-893, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31406383

RESUMO

The ability to modify multiple genetic elements simultaneously would help to elucidate and control the gene interactions and networks underlying complex cellular functions. However, current genome engineering technologies are limited in both the number and the type of perturbations that can be performed simultaneously. Here, we demonstrate that both Cas12a and a clustered regularly interspaced short palindromic repeat (CRISPR) array can be encoded in a single transcript by adding a stabilizer tertiary RNA structure. By leveraging this system, we illustrate constitutive, conditional, inducible, orthogonal and multiplexed genome engineering of endogenous targets using up to 25 individual CRISPR RNAs delivered on a single plasmid. Our method provides a powerful platform to investigate and orchestrate the sophisticated genetic programs underlying complex cell behaviors.


Assuntos
Sistemas CRISPR-Cas , Endonucleases/metabolismo , Edição de Genes , Redes Reguladoras de Genes , Engenharia Genética , Genoma Humano , RNA Guia/genética , Acidaminococcus/enzimologia , Endonucleases/genética , Células HEK293 , Humanos , Plasmídeos/genética , Ativação Transcricional
13.
Extremophiles ; 23(6): 669-679, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31363851

RESUMO

Self-splicing inteins are mobile genetic elements invading host genes via nested homing endonuclease (HEN) domains. All HEN domains residing within inteins are inserted at a highly conserved insertion site. A purifying selection mechanism directing the location of the HEN insertion site has not yet been identified. In this work, we solved the three-dimensional crystal structures of two inteins inserted in the cell division control protein 21 of the hyperthermophilic archaea Pyrococcus abyssi and Pyrococcus horikoshii. A comparison between the structures provides the structural basis for the thermo-stabilization mechanism of inteins that have lost the HEN domain during evolution. The presence of an entire extein domain in the intein structure from Pyrococcus horikoshii suggests the selection mechanism for the highly conserved HEN insertion point.


Assuntos
Proteínas Arqueais/química , Endonucleases/química , Inteínas , Pyrococcus abyssi/enzimologia , Pyrococcus horikoshii/enzimologia , Proteínas Arqueais/genética , Endonucleases/genética , Estabilidade Enzimática , Temperatura Alta , Domínios Proteicos , Pyrococcus abyssi/genética , Pyrococcus horikoshii/genética
14.
Asian Pac J Cancer Prev ; 20(8): 2397-2403, 2019 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-31450912

RESUMO

Background: Environmental carcinogens cause DNA damages which if not repaired properly, may increase the risk of cancer. The Xerodermapigmentosum group D (XPD) and group G (XPG) genes are essential genes for DNA repair and alteration in DNA repair causes cancer. The present study aimed to evaluate the relationship between XPD and XPG polymorphisms and risk of oral pre cancer and cancer. Methods: Present study genotyped 302 samples of oral diseases and 300 controls for XPD (A/C) and XPG (G/C) polymorphisms with PCR-RFLP method. Results: Our result showed that compared to AA genotype frequency of AC and CC genotype for XPD(A/C) polymorphism were significantly lower among cases than in control and are associated with decreased risk of oral diseases (OR= 0.621 and 0.603 respectively). In contrast with reference to GG genotype the frequency of CC genotype of XPG (G/C) was significantly higher in case than in control population (p value=0.004) and found to increase the risk of oral diseases (OR= 2.077). Particularly C allele for XPD A/C polymorphism was found to be associated with decreased risk of Lichen planus and increased risk of ( OR = 0.470 and 1.541 respectively) oral cancer. While C allele of XPG G/C polymorphism significantly increased the risk of Oral Submucous Fibrosis and Leukoplakia (OR= 1.879 and 1.837 respectively) but not of Lichen planus and oral cancer. In combined genotype analysis from the aforesaid polymorphisms presence of C allele for XPD (A/C) polymorphisms were found to decrease the risk of oral diseases. However, the same C allele was observed to increase the chance of having high stage disease (OR= 5.71) with nodal involvement (OR= 6.78) once the cancer been initiated. Conclusion: This work shows association of XPD (A/C), XPG (G/C) polymorphisms with the development of pre oral cancer as well as oral cancer and its clinical courses.


Assuntos
Proteínas de Ligação a DNA/genética , Endonucleases/genética , Neoplasias Bucais/genética , Proteínas Nucleares/genética , Polimorfismo de Nucleotídeo Único , Lesões Pré-Cancerosas/genética , Fatores de Transcrição/genética , Proteína Grupo D do Xeroderma Pigmentoso/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos de Casos e Controles , Criança , Feminino , Predisposição Genética para Doença , Humanos , Índia/epidemiologia , Masculino , Pessoa de Meia-Idade , Neoplasias Bucais/epidemiologia , Neoplasias Bucais/patologia , Lesões Pré-Cancerosas/epidemiologia , Lesões Pré-Cancerosas/patologia , Fatores de Risco , Adulto Jovem
15.
Medicine (Baltimore) ; 98(32): e16541, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31393355

RESUMO

BACKGROUNDS: Previous investigations yielded inconsistent results for the associations between pancreatic cancer (PC) risk and genetic polymorphisms. The study aimed to perform a systematic review and meta-analysis of studies exploring association of some genetic polymorphisms and PC risk. METHODS: We systematically searched on PubMed and Web of Science for association of genetic polymorphisms and PC risk published from 1969 to January 2019. We computed the multivariate odd ratio (OR) and 95% confidence intervals (CI), comparing different genetic types. RESULTS: The present meta-analysis showed significant associations between deoxyribonucleic acid (DNA) repair gene (X-ray repair cross-complementing group 1 (XRCC1) Arg399GIn and Arg194Trp, excision repair cross complementation 1 (ERCC1) rs11615 and rs3212986, ERCC2 rs13181) polymorphisms and PC risk. CONCLUSIONS: Because of the limited sample size and ethnicity enrolled in the present meta-analysis, further larger scaled studies should be performed to demonstrate the association.


Assuntos
Neoplasias Pancreáticas/genética , DNA Glicosilases/genética , Reparo do DNA/genética , Proteínas de Ligação a DNA/genética , Endonucleases/genética , Predisposição Genética para Doença , Humanos , Polimorfismo de Nucleotídeo Único , Proteína 1 Complementadora Cruzada de Reparo de Raio-X/genética , Proteína Grupo D do Xeroderma Pigmentoso/genética
16.
Cuad. bioét ; 30(99): 171-185, mayo-ago. 2019. graf
Artigo em Espanhol | IBECS | ID: ibc-185233

RESUMO

La adaptación del sistema CRISPR como herramienta de edición genética ha supuesto una revolución en numerosos campos de aplicación, pues esta técnica resulta considerablemente más rápida, fácil de realizar y eficaz que las técnicas predecesoras. Sin embargo, algunas de estas aplicaciones suscitan objetivas cuestiones éticas que deben ser abordadas. En este trabajo discutimos, en base a los datos más recientes, las distintas cuestiones relacionadas con las aplicaciones de CRISPR sobre la línea germinal, su introducción en ensayos clínicos, la edición genética de animales y plantas para consumo humano y el novedoso gene drive


The adaptation of the CRISPR system as a genetic editing tool has led to a revolution in many fields of application, as this technique is considerably faster, easier to perform and more efficient than predecessor techniques. However, some of these applications raise objective ethical issues that must be addressed. In this paper we discuss, based on the most recent data, the different issues related to CRISPR applications on the germ line, its introduction in clinical trials, the genetic edition of animals and plants for human consumption and the novel gene drive


Assuntos
Humanos , Edição de Genes/ética , Proteína 9 Associada à CRISPR , Endonucleases/genética , Engenharia Genética/ética , Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Engenharia Genética/métodos
17.
Indian J Pathol Microbiol ; 62(3): 405-412, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31361228

RESUMO

Background: There are several DNA repair pathways that protect cellular DNA from injury, such as nucleotide excision repair (NER) and mismatch repair (MMR). The protein product of the excision repair cross-complementation group 1 (ERCC1) gene plays a pivotal role in NER. The exact relationship between MMR proteins and ERCC1 is not well known in colorectal carcinoma (CRC). Aim of the Study: To investigate expression of ERCC1 and MMR proteins in colorectal mucinous carcinoma (MA) and non-mucinous carcinoma (NMA) using tissue microarray technique. Material and Methods: We studied tumor tissue specimens from 150 patients with colorectal mucinous (MA) and non-mucinous adenocarcinoma (NMA). Tissue microarrays were constructed using modified mechanical pencil tips technique and immunohistochemistry for ERCC1, MLH1, MSH2, MSH6, and PMS2. Results: NMA showed a significantly more frequent aberrant cytoplasmic expression than MA while MA showed a more frequent intact nuclear expression than NMA. There were no significant differences between the NMA and MA groups in the expression of MMR proteins. In NMA cases, ERCC1 expression was significantly related to MMR status while was not significantly related in MA cases. ERCC1 expression was not significantly related to overall and disease-free survival in both NMA and MA groups. Conclusion: this study is the first to investigate the relation between MMR status and ERCC1 expression in colorectal MA and NMA. ERCC1 expression was significantly related to MMR status only in NMA cases. Hence, the current study emphasizes that further research about the relation between various DNA repair pathways is needed.


Assuntos
Adenocarcinoma Mucinoso/genética , Adenocarcinoma/genética , Neoplasias Colorretais/genética , Reparo de Erro de Pareamento de DNA/genética , Proteínas de Ligação a DNA/genética , Endonucleases/genética , Adenocarcinoma/patologia , Adenocarcinoma Mucinoso/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais , Neoplasias Colorretais/diagnóstico , Neoplasias Colorretais/patologia , Feminino , Expressão Gênica , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Prognóstico , Análise Serial de Tecidos , Adulto Jovem
18.
J Exp Clin Cancer Res ; 38(1): 292, 2019 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-31287003

RESUMO

BACKGROUND: Bladder cancer progression has been associated with dysfunctional repair of double-strand breaks (DSB), a deleterious type of DNA lesions that fuel genomic instability. Accurate DSB repair relies on two distinct pathways, homologous recombination (HR) and classical non-homologous end-joining (c-NHEJ). The transcription factor E2F1 supports HR-mediated DSB repair and protects genomic stability. However, invasive bladder cancers (BC) display, in contrast to non-invasive stages, genomic instability despite their high E2F1 levels. Hence, E2F1 is either inefficient in controlling DSB repair in this setting, or rewires the repair apparatus towards alternative, error-prone DSB processing pathways. METHODS: RT-PCR and immunoblotting, in combination with bioinformatics tools were applied to monitor c-NHEJ factors status in high-E2F1-expressing, invasive BC versus low-E2F1-expressing, non-invasive BC. In vivo binding of E2F1 on target gene promoters was demonstrated by ChIP assays and E2F1 CRISPR-Cas9 knockdown. MIR888-dependent inhibition of APLF by E2F1 was demonstrated using overexpression and knockdown experiments, in combination with luciferase assays. Methylation status of MIR888 promoter was monitored by methylation-specific PCR. The changes in invasion potential and the DSB repair efficiency were estimated by Boyden chamber assays and pulse field electrophoresis, correspondingly. RESULTS: Herein, we show that E2F1 directly transactivates the c-NHEJ core factors Artemis, DNA-PKcs, ligase IV, NHEJ1, Ku70/Ku80 and XRCC4, but indirectly inhibits APLF, a chromatin modifier regulating c-NHEJ. Inhibition is achieved by miR-888-5p, a testis-specific, X-linked miRNA which, in normal tissues, is often silenced via promoter methylation. Upon hypomethylation in invasive BC cells, MIR888 is transactivated by E2F1 and represses APLF. Consequently, E2F1/miR-888/APLF rewiring is established, generating conditions of APLF scarcity that compromise proper c-NHEJ function. Perturbation of the E2F1/miR-888/APLF axis restores c-NHEJ and ameliorates cell invasiveness. Depletion of miR-888 can establish a 'high E2F1/APLF/DCLRE1C' signature, which was found to be particularly favorable for BC patient survival. CONCLUSION: Suppression of the 'out-of-context' activity of miR-888 improves DSB repair and impedes invasiveness by restoring APLF.


Assuntos
Reparo do DNA por Junção de Extremidades , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , Fator de Transcrição E2F1/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Neoplasias da Bexiga Urinária/genética , Neoplasias da Bexiga Urinária/metabolismo , Linhagem Celular Tumoral , Quebras de DNA de Cadeia Dupla , Metilação de DNA , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/metabolismo , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/antagonistas & inibidores , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Fator de Transcrição E2F1/genética , Endonucleases/genética , Endonucleases/metabolismo , Técnicas de Silenciamento de Genes , Recombinação Homóloga , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , Invasividade Neoplásica , Proteínas de Ligação a Poli-ADP-Ribose/antagonistas & inibidores , Proteínas de Ligação a Poli-ADP-Ribose/genética , Regiões Promotoras Genéticas , Ativação Transcricional , Neoplasias da Bexiga Urinária/patologia
19.
Genome Res ; 29(7): 1067-1077, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31221724

RESUMO

Nucleotide excision repair (NER) is one of the main DNA repair pathways that protect cells against genomic damage. Disruption of this pathway can contribute to the development of cancer and accelerate aging. Mutational characteristics of NER-deficiency may reveal important diagnostic opportunities, as tumors deficient in NER are more sensitive to certain treatments. Here, we analyzed the genome-wide somatic mutational profiles of adult stem cells (ASCs) from NER-deficient Ercc1 -/Δ mice. Our results indicate that NER-deficiency increases the base substitution load twofold in liver but not in small intestinal ASCs, which coincides with the tissue-specific aging pathology observed in these mice. Moreover, NER-deficient ASCs of both tissues show an increased contribution of Signature 8 mutations, which is a mutational pattern with unknown etiology that is recurrently observed in various cancer types. The scattered genomic distribution of the base substitutions indicates that deficiency of global-genome NER (GG-NER) underlies the observed mutational consequences. In line with this, we observe increased Signature 8 mutations in a GG-NER-deficient human organoid culture, in which XPC was deleted using CRISPR-Cas9 gene-editing. Furthermore, genomes of NER-deficient breast tumors show an increased contribution of Signature 8 mutations compared with NER-proficient tumors. Elevated levels of Signature 8 mutations could therefore contribute to a predictor of NER-deficiency based on a patient's mutational profile.


Assuntos
Reparo do DNA/genética , Mutação , Neoplasias/genética , Células-Tronco Adultas , Animais , Neoplasias da Mama/genética , Estudos de Coortes , Análise Mutacional de DNA , DNA de Neoplasias , Proteínas de Ligação a DNA/genética , Endonucleases/genética , Feminino , Humanos , Camundongos , Organoides , Técnicas de Cultura de Tecidos , Sequenciamento Completo do Genoma
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA