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1.
Brain Sci ; 14(1)2024 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-38248298

RESUMO

This paper proposes the "calm contact" technique: an imaginative scenario where someone is in gentle contact with a loved one where the essence of the experience is to enjoy safety and calmness in peaceful social contact. The theoretical background is outlined by combining the brain mechanisms of stress reactions and hypnosis. In addition to the ancient stress responses (flight or fight or freeze), there are oxytocin-based options at the human level: tend and befriend behavior and the state of calm and connection, which is not a stress reaction but a resting reaction. These social-based reactions could prevent the organism from the primitive freezing response. Some studies proved that "hypnosis" as a setting reduces cortisol levels and could raise oxytocin levels. The beneficial mechanisms of the "calm contact" technique are analyzed in relation to "social support" and the psychoaffective effects of central oxytocin. The subjective effects of the proposed technique are outlined based on reports of healthy volunteers. The "calm contact" technique could be an alternative or adjunct to the "safe place" technique, applying the recent findings of endocrinological brain mechanisms of hypnosis. Clinical implications and limitations are briefly summarized.

2.
Front Psychiatry ; 14: 1168611, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37822796

RESUMO

Background: Borderline personality disorder (BPD) is a severe mental disorder that affects attitudes toward the body. However, whether this condition also affects body schema and perceptual body image remains unclear. Previous questionnaire-based studies found dissatisfaction with one's body in patients with BPD. In addition to attitudinal body image, our study investigates whether body schema and perceptual body image are disturbed in patients with BPD. Method: Our study included 31 patients diagnosed with BPD (25 women) and 30 healthy individuals (19 women) (Mage = 29 for both groups). The SCID-5-PD interview was used to determine personality disorder. Attitudinal body image was measured using the Body Attitude Test (BAT) factors. Body schema and perceptual body image were measured by two conditions of a body representation task, the body portraying method (BPM). Results: BPD patients achieved higher scores in all three BAT factors and were more susceptible to misinformation in both conditions of BPM. Based on the results, BPD patients appear to have more negative attitudes toward their bodies and worse perceptual body image and body schema. Conclusion: The novel finding of our study is that, besides the previously found attitudinal dissatisfaction with the body, individuals with BPD also show disturbances at the levels of body schema and perceptual body image. Our findings concerning disturbances in body schema and perceptual body need further research into their etiological factors and provide new therapeutic targets for the treatment of BPD.

3.
PLoS One ; 17(7): e0266201, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35834562

RESUMO

OBJECTIVES AND METHODS: In order to assess the internal consistency, fit indexes, test-retest reliability, and validity of the Personality Inventory for the DSM-5 (PID-5) and its associations with age, gender, and education, 471 non-clinical (69,6% female; mean age: 37,63) and 314 clinical participants (69,7% female, mean age: 37,41) were administered the Hungarian translation of the PID-5, as well as the SCL-90-R and the SCID-II Personality Questionnaire. RESULTS: We found that; (a) temporal consistency of the Hungarian PID-5 was confirmed by one-month test-retest reliability analysis, (b) validity of the PID-5 instrument is acceptable in the clinical and the non-clinical sample as well, based on significant correlations with SCID-II and SCL-90-R, (c) PID-5 facets' and domains' associations with gender, age, and level of education are in accordance with previous findings. CONCLUSION: These findings support that the Hungarian PID-5 is a reliable and valid instrument for both clinical and non-clinical populations.


Assuntos
Transtornos da Personalidade , Personalidade , Manual Diagnóstico e Estatístico de Transtornos Mentais , Feminino , Humanos , Hungria , Masculino , Transtornos da Personalidade/diagnóstico , Inventário de Personalidade , Psicometria , Reprodutibilidade dos Testes
4.
Orv Hetil ; 160(38): 1495-1502, 2019 Sep.
Artigo em Húngaro | MEDLINE | ID: mdl-31537098

RESUMO

A significant proportion of cancer patients reports a decline in their usual cognitive functions. This impairment may be the consequence of either the malignant disorder itself or the side-effect of the treatment. This impairment in cognitive skills was referred to with a sticky witty name as chemobrain or chemofog, reflecting on the old conception based on the assumption that solely the neurotoxic effect of chemotherapy was responsible for this kind of disturbances. However, today there is increasing evidence proving that such hypotheses can only partially explain the cognitive decline of cancer patients and cancer survivors. As a matter of fact, the cognitive impairment of cancer patients is a much more complex consequence of the malignant disorder than chemobrain or chemofog. Nowadays, this process is described as "cancer-related cognitive impairment" (CRCI). Orv Hetil. 2019; 160(38): 1495-1502.


Assuntos
Antineoplásicos/efeitos adversos , Transtornos Cognitivos/induzido quimicamente , Disfunção Cognitiva/induzido quimicamente , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Neoplasias/tratamento farmacológico , Antineoplásicos/uso terapêutico , Cognição , Transtornos Cognitivos/diagnóstico , Disfunção Cognitiva/diagnóstico , Humanos , Neoplasias/complicações
5.
J Pain Res ; 12: 1155-1178, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31114299

RESUMO

Objectives: Pain catastrophizing is reliably associated with pain reports during experimental pain in healthy, pain-free subjects and in people with chronic pain. It also correlates with self-reports of clinical pain intensity/severity in a variety of disorders characterized by chronic pain in adults, adolescents and children. However, processes, through which it exerts its effects are yet unclear. In this paper, our primary aim was to synthesize neuroimaging research to open a window to possible mechanisms underlying pain catastrophizing in both chronic pain patients and healthy controls. We also aimed to compare whether the neural correlates of pain catastrophizing are similar in these two groups. Methods: PubMed and the Web of Science were searched for magnetic resonance imaging (MRI) studies that explored neural correlates of pain catastrophizing. Results: Twenty articles met the inclusion criteria. The results of our review show a connection between pain catastrophizing and brain areas tightly connected to pain perception (including the somatosensory cortices, anterior insula, anterior cingulate cortex and thalamus) and/or modulation (eg, the dorsolateral prefrontal cortex). Our results also highlight that these processes - in relation to pain catastrophizing - are more pronounced in chronic pain patients, suggesting that structural and functional brain alterations (and perhaps mechanisms) related to pain catastrophizing may depend on prior and/or relatively stable/constant pain experience. However, we also found methodological issues and differences that could lead to divergent results. Discussion: Based on our results, pain catastrophizing might be related to salience detection, pain processing, and top-down attentional processes. More research is recommended to explore neural changes to specific types of catastrophizing thoughts (eg, experimentally induced and/or state). Furthermore, we provide ideas regarding pain catastrophizing studies in the future for a more standardized approach.

6.
Nat Commun ; 10(1): 1288, 2019 03 20.
Artigo em Inglês | MEDLINE | ID: mdl-30894545

RESUMO

The TFIIH subunit XPB is involved in combined Xeroderma Pigmentosum and Cockayne syndrome (XP-B/CS). Our analyses reveal that XPB interacts functionally with KAT2A, a histone acetyltransferase (HAT) that belongs to the hSAGA and hATAC complexes. XPB interacts with KAT2A-containing complexes on chromatin and an XP-B/CS mutation specifically elicits KAT2A-mediated large-scale chromatin decondensation. In XP-B/CS cells, the abnormal recruitment of TFIIH and KAT2A to chromatin causes inappropriate acetylation of histone H3K9, leading to aberrant formation of transcription initiation complexes on the promoters of several hundred genes and their subsequent overexpression. Significantly, this cascade of events is similarly sensitive to KAT2A HAT inhibition or to the rescue with wild-type XPB. In agreement, the XP-B/CS mutation increases KAT2A HAT activity in vitro. Our results unveil a tight connection between TFIIH and KAT2A that controls higher-order chromatin structure and gene expression and provide new insights into transcriptional misregulation in a cancer-prone DNA repair-deficient disorder.


Assuntos
Cromatina/química , Síndrome de Cockayne/genética , Histona Acetiltransferases/genética , Histonas/metabolismo , Subunidades Proteicas/genética , Fator de Transcrição TFIIH/genética , Xeroderma Pigmentoso/genética , Acetilação , Proteína 9 Associada à CRISPR/genética , Proteína 9 Associada à CRISPR/metabolismo , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Cromatina/metabolismo , Síndrome de Cockayne/metabolismo , Síndrome de Cockayne/patologia , Fibroblastos/citologia , Fibroblastos/metabolismo , Edição de Genes , Regulação da Expressão Gênica , Histona Acetiltransferases/antagonistas & inibidores , Histona Acetiltransferases/metabolismo , Histonas/genética , Humanos , Modelos Biológicos , Osteoblastos/citologia , Osteoblastos/metabolismo , Cultura Primária de Células , Subunidades Proteicas/metabolismo , RNA Guia de Cinetoplastídeos/genética , RNA Guia de Cinetoplastídeos/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Fator de Transcrição TFIIH/metabolismo , Iniciação da Transcrição Genética , Xeroderma Pigmentoso/metabolismo , Xeroderma Pigmentoso/patologia
7.
Sci Rep ; 9(1): 2753, 2019 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-30808882

RESUMO

Ultraviolet light induced pyrimidine dimer is a helix distortion DNA damage type, which recruits repair complexes. However, proteins of these complexes that take part in both DNA damage recognition and repair have been well-described, the regulation of the downstream steps of nucleotide excision repair (NER) have not been clearly clarified yet. In a high-throughput screen, we identified SerpinB2 (SPB2) as one of the most dramatically upregulated gene in keratinocytes following UV irradiation. We found that both the mRNA and the protein levels of SPB2 were increased upon UV irradiation in various cell lines. Additionally, UV damage induced translocation of SPB2 from the cytoplasm to the nucleus as well as the damage induced foci formation of it. Here we show that SPB2 co-localizes with XPB involved in the NER pathway at UV-induced repair foci. Finally, we demonstrated that UV irradiation promoted the association of SPB2 with ubiquitylated proteins. In basal cell carcinoma tumour cells, we identified changes in the subcellular localization of SPB2. Based on our results, we conclude that SPB2 protein has a novel role in UV-induced NER pathway, since it regulates the removal of the repair complex from the damaged site leading to cancerous malformation.


Assuntos
Dano ao DNA , Reparo do DNA , Melanoma/patologia , Osteossarcoma/patologia , Inibidor 2 de Ativador de Plasminogênio/metabolismo , Raios Ultravioleta/efeitos adversos , Neoplasias Ósseas/etiologia , Neoplasias Ósseas/patologia , Carcinoma Basocelular/etiologia , Carcinoma Basocelular/patologia , DNA Helicases/genética , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Humanos , Melanoma/etiologia , Osteossarcoma/etiologia , Inibidor 2 de Ativador de Plasminogênio/genética , Dímeros de Pirimidina , Células Tumorais Cultivadas
8.
Mol Cell ; 65(3): 504-514.e4, 2017 Feb 02.
Artigo em Inglês | MEDLINE | ID: mdl-28157507

RESUMO

Transcription starts with the assembly of pre-initiation complexes on promoters followed by their opening. Current models suggest that class II gene transcription requires ATP and the TFIIH XPB subunit to open a promoter. Here, we observe that XPB depletion surprisingly leaves transcription virtually intact. In contrast, inhibition of XPB ATPase activity affects transcription, revealing that mRNA expression paradoxically accommodates the absence of XPB while being sensitive to the inhibition of its ATPase activity. The XPB-depleted TFIIH complex is recruited to active promoters and contributes to transcription. We finally demonstrate that the XPB ATPase activity is only used to relieve a transcription initiation block imposed by XPB itself. In the absence of this block, transcription initiation can take place without XPB ATPase activity. These results suggest that a helicase is dispensable for mRNA transcription, thereby unifying the mechanism of promoter DNA opening for the three eukaryotic RNA polymerases.


Assuntos
DNA Helicases/genética , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Transcrição Gênica , Adenosina Trifosfatases/metabolismo , Linhagem Celular , Perfilação da Expressão Gênica/métodos , Humanos , Mutação , Regiões Promotoras Genéticas , Análise de Sequência de RNA/métodos , Fator de Transcrição TFIIH/química , Fator de Transcrição TFIIH/metabolismo
9.
PLoS Genet ; 12(2): e1005791, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26845027

RESUMO

DNA lesions are sensed by a network of proteins that trigger the DNA damage response (DDR), a signaling cascade that acts to delay cell cycle progression and initiate DNA repair. The Mediator of DNA damage Checkpoint protein 1 (MDC1) is essential for spreading of the DDR signaling on chromatin surrounding Double Strand Breaks (DSBs) by acting as a scaffold for PI3K kinases and for ubiquitin ligases. MDC1 also plays a role both in Non-Homologous End Joining (NHEJ) and Homologous Recombination (HR) repair pathways. Here we identify two novel binding partners of MDC1, the poly (ADP-ribose) Polymerases (PARPs) TNKS1 and 2. We find that TNKSs are recruited to DNA lesions by MDC1 and regulate DNA end resection and BRCA1A complex stabilization at lesions leading to efficient DSB repair by HR and proper checkpoint activation.


Assuntos
Quebras de DNA de Cadeia Dupla , Recombinação Homóloga , Tanquirases/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Sítios de Ligação , Proteínas de Ciclo Celular , Linhagem Celular , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Humanos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo , Tanquirases/genética , Transativadores/genética , Transativadores/metabolismo , Ubiquitina-Proteína Ligases
10.
J Cell Biol ; 206(5): 589-98, 2014 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-25154395

RESUMO

In nucleotide excision repair (NER), damage recognition by XPC-hHR23b is described as a critical step in the formation of the preincision complex (PInC) further composed of TFIIH, XPA, RPA, XPG, and ERCC1-XPF. To obtain new molecular insights into the assembly of the PInC, we analyzed its formation independently of DNA damage by using the lactose operator/repressor reporter system. We observed a sequential and ordered self-assembly of the PInC operating upon immobilization of individual NER factors on undamaged chromatin and mimicking that functioning on a bona fide NER substrate. We also revealed that the recruitment of the TFIIH subunit TTDA, involved in trichothiodystrophy group A disorder (TTD-A), was key in the completion of the PInC. TTDA recruits XPA through its first 15 amino acids, depleted in some TTD-A patients. More generally, these results show that proteins forming large nuclear complexes can be recruited sequentially on chromatin in the absence of their natural DNA target and with no reciprocity in their recruitment.


Assuntos
Cromatina/metabolismo , Linhagem Celular Tumoral , Dano ao DNA , Reparo do DNA , Enzimas Reparadoras do DNA/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Endonucleases/metabolismo , Humanos , Proteínas Imobilizadas/química , Proteínas Imobilizadas/metabolismo , Proteínas Nucleares/metabolismo , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Transporte Proteico , Fator de Transcrição TFIIH/metabolismo , Fatores de Transcrição/metabolismo , Proteína de Xeroderma Pigmentoso Grupo A/química , Proteína de Xeroderma Pigmentoso Grupo A/metabolismo
12.
EMBO J ; 29(14): 2381-94, 2010 Jul 21.
Artigo em Inglês | MEDLINE | ID: mdl-20562830

RESUMO

All DNA-related processes rely on the degree of chromatin compaction. The highest level of chromatin condensation accompanies transition to mitosis, central for cell cycle progression. Covalent modifications of histones, mainly deacetylation, have been implicated in this transition, which also involves transcriptional repression. Here, we show that the Gcn5-containing histone acetyl transferase complex, Ada Two A containing (ATAC), controls mitotic progression through the regulation of the activity of non-histone targets. RNAi for the ATAC subunits Ada2a/Ada3 results in delayed M/G1 transition and pronounced cell division defects such as centrosome multiplication, defective spindle and midbody formation, generation of binucleated cells and hyperacetylation of histone H4K16 and alpha-tubulin. We show that ATAC localizes to the mitotic spindle and controls cell cycle progression through direct acetylation of Cyclin A/Cdk2. Our data describes a new pathway in which the ATAC complex controls Cyclin A/Cdk2 mitotic function: ATAC/Gcn5-mediated acetylation targets Cyclin A for degradation, which in turn regulates the SIRT2 deacetylase activity. Thus, we have uncovered an essential function for ATAC in regulating Cyclin A activity and consequent mitotic progression.


Assuntos
Acetiltransferases/metabolismo , Mitose/fisiologia , Acetiltransferases/genética , Sequência de Aminoácidos , Animais , Ciclina A/metabolismo , Quinase 2 Dependente de Ciclina/metabolismo , Células HeLa , Humanos , Camundongos , Dados de Sequência Molecular , Células NIH 3T3 , Interferência de RNA , Fuso Acromático/metabolismo , Tubulina (Proteína)/metabolismo , Fatores de Transcrição de p300-CBP/genética , Fatores de Transcrição de p300-CBP/metabolismo
13.
Cell Mol Life Sci ; 67(4): 611-28, 2010 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19936620

RESUMO

Histone acetyl transferases (HATs) play a crucial role in eukaryotes by regulating chromatin architecture and locus-specific transcription. The GCN5 HAT was identified as a subunit of the SAGA (Spt-Ada-Gcn5-Acetyltransferase) multiprotein complex. Vertebrate cells express a second HAT, PCAF, that is 73% identical to GCN5. Here, we report the characterization of the mammalian ATAC (Ada-Two-A-Containing) complexes containing either GCN5 or PCAF in a mutually exclusive manner. In vitro ATAC complexes acetylate lysine 14 of histone H3. Moreover, ATAC- or SAGA-specific knock-down experiments suggest that both ATAC and SAGA are involved in the acetylation of histone H3K9 and K14 residues. Despite their catalytic similarities, SAGA and ATAC execute their coactivator functions on distinct sets of inducible target genes. Interestingly, ATAC strongly influences the global phosphorylation level of histone H3S10, suggesting that in mammalian cells a cross-talk exists linking ATAC function to H3S10 phosphorylation.


Assuntos
Regulação da Expressão Gênica , Histona Acetiltransferases/metabolismo , Complexos Multienzimáticos/metabolismo , Fatores de Transcrição de p300-CBP/metabolismo , Acetilação , Animais , Catálise , Drosophila , Técnicas de Silenciamento de Genes , Células HeLa , Histona Acetiltransferases/genética , Histonas/metabolismo , Humanos , Complexos Multienzimáticos/genética , Fosforilação , Fatores de Transcrição de p300-CBP/genética
14.
Trends Cell Biol ; 19(11): 617-29, 2009 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-19819145

RESUMO

Faithful repair of DNA damage is essential for the maintenance of genome integrity. Recent advances in the local induction of DNA damage and in cell biological imaging techniques have extended our understanding of DNA repair gained from biochemical and genetic approaches; these advances now reveal that the assembly of DNA repair complexes at sites of DNA damage is spatially and temporally regulated. Visualization of the dynamics of double strand breaks in living cells has also provided valuable insights into how chromosomal translocations form. Here we outline the most commonly used tools to induce and visualize the DNA repair process and we discuss the advantages and disadvantages inherent to each method.


Assuntos
Técnicas Citológicas , Reparo do DNA , DNA/genética , Técnicas Genéticas , Animais , DNA/metabolismo , Dano ao DNA , Desoxirribonucleases/metabolismo , Humanos , Translocação Genética
15.
Mol Cell Biol ; 29(6): 1649-60, 2009 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-19114550

RESUMO

One of the central questions in eukaryotic transcription is how activators can transmit their signal to stimulate gene expression in the context of chromatin. The multisubunit SAGA coactivator complex has both histone acetyltransferase and deubiquitination activities and remodels chromatin to allow transcription. Whether and how SAGA is able to regulate transcription at specific loci is poorly understood. Using mass spectrometry, immunoprecipitation, and Western blot analysis, we have identified human SPT20 (hSPT20) as the human homologue of the yeast Spt20 and show that hSPT20 is a bona fide subunit of the human SAGA (hSAGA; previously called TFTC/STAGA/PCAF) complex and that hSPT20 is required for the integrity of the hSAGA complex. We demonstrate that hSPT20 and other hSAGA subunits, together with RNA polymerase II, are specifically recruited to genes induced by endoplasmic reticulum (ER) stress. In good agreement with the recruitment of hSAGA to the ER stress-regulated genes, knockdown of hSTP20 hampers ER stress response. Surprisingly, hSPT20 recruitment was not observed for genes induced by another type of stress. These results provide evidence for a direct and specific role of the hSPT20-containing SAGA complex in transcriptional induction of ER stress-responsive genes. Thus, hSAGA regulates the transcription of stress-responsive genes in a stress type-dependent manner.


Assuntos
Retículo Endoplasmático/metabolismo , Fatores de Transcrição/metabolismo , Fatores de Transcrição de p300-CBP/metabolismo , Acetilação , Sequência de Aminoácidos , Regulação da Expressão Gênica , Células HeLa , Humanos , Dados de Sequência Molecular , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , RNA Polimerase II/metabolismo , Fatores de Transcrição/genética , Transcrição Gênica , Fatores de Transcrição de p300-CBP/genética
16.
BMC Mol Biol ; 9: 57, 2008 Jun 12.
Artigo em Inglês | MEDLINE | ID: mdl-18549481

RESUMO

BACKGROUND: The tumour suppressor protein p53 is a sequence specific DNA-binding transcription regulator, which exerts its versatile roles in genome protection and apoptosis by affecting the expression of a large number of genes. In an attempt to obtain a better understanding of the mechanisms by which p53 transcription function is regulated, we studied p53 interactions. RESULTS: We identified BIP2 (Bric-à-brac interacting protein 2), the fly homolog of TAF3, a histone fold and a plant homeodomain containing subunit of TFIID, as an interacting partner of Drosophila melanogaster p53 (Dmp53). We detected physical interaction between the C terminus of Dmp53 and the central region of TAF3 both in yeast two hybrid assays and in vitro. Interestingly, DmTAF3 can also interact with human p53, and mammalian TAF3 can bind to both Dmp53 and human p53. This evolutionarily conserved interaction is functionally significant, since elevated TAF3 expression severely and selectively inhibits transcription activation by p53 in human cell lines, and it decreases the level of the p53 protein as well. CONCLUSION: We identified TAF3 as an evolutionarily conserved negative regulator of p53 transcription activation function.


Assuntos
Proteínas de Drosophila/metabolismo , Fator de Transcrição TFIID/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Animais , Western Blotting , Linhagem Celular , Linhagem Celular Tumoral , Proteínas de Drosophila/genética , Drosophila melanogaster , Células HeLa , Humanos , Imunoprecipitação , Modelos Biológicos , Ligação Proteica , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Spodoptera , Fatores Associados à Proteína de Ligação a TATA , Fator de Transcrição TFIID/genética , Transcrição Gênica , Transfecção , Proteína Supressora de Tumor p53/genética , Técnicas do Sistema de Duplo-Híbrido
17.
J Mol Biol ; 375(2): 337-52, 2008 Jan 11.
Artigo em Inglês | MEDLINE | ID: mdl-18022196

RESUMO

Inverted repeats of insertion sequences (ISs) are indispensable for transposition. We demonstrate that sub-terminal sequences adjacent to the inverted repeats of IS30 are also required for optimal transposition activity. We have developed a cell-free recombination system and showed that the transposase catalyses formation of a figure-of-eight transposition intermediate, where a 2 bp long single strand bridge holds the inverted repeat sequences (IRs) together. This is the first demonstration of the figure-of-eight structure in a non-IS3 family element, suggesting that this mechanism is likely more widely adopted among IS families. We show that the absence of sub-terminal IS30 sequences negatively influences figure-of-eight production both in vivo and in vitro. These regions enhance IR-IR junction formation and IR-targeting events in vivo. Enhancer elements have been identified within 51 bp internal to IRL and 17 bp internal to IRR. In the right end, a decanucleotide, 5'-GAGATAATTG-3', is responsible for wild-type activity, while in the left end, a complex assembly of repetitive elements is required. Functioning of the 10 bp element in the right end is position-dependent and the repetitive elements in the left end act cooperatively and may influence bendability of the end. In vitro kinetic experiments suggest that the sub-terminal enhancers may, at least partly, be transposase-dependent. Such enhancers may reflect a subtle regulatory mechanism for IS30 transposition.


Assuntos
Elementos de DNA Transponíveis , Sequências Repetidas Terminais , Transposases/metabolismo , Sequência de Bases , Sítios de Ligação , Sistema Livre de Células , Análise Mutacional de DNA , Primers do DNA , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Bacteriano/isolamento & purificação , DNA Circular/genética , Dimerização , Eletroforese em Gel de Poliacrilamida , Elementos Facilitadores Genéticos , Escherichia coli/genética , Genes Bacterianos , Técnicas In Vitro , Cinética , Modelos Genéticos , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Insercional , Conformação de Ácido Nucleico , Fases de Leitura Aberta , Plasmídeos , Mutação Puntual , Reação em Cadeia da Polimerase , Recombinação Genética , Salmonella typhimurium/genética , Transposases/química , Transposases/genética , Transposases/isolamento & purificação
18.
Mol Microbiol ; 63(6): 1731-47, 2007 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-17367392

RESUMO

We have analysed the transposition and target selection strategy of IS1655, a typical IS30 family member resident in Neisseria meningitidis. We have redefined IS1655 as a 1080 bp long element with 25 bp imperfect inverted repeats (IRs), which generates a 3 bp target duplication and have shown that it transposes using an intermediate with abutted IRs separated by 2 bp. IS1655 exhibits bipartite target specificity inserting preferentially either next to sequences similar to its IRs or into an unrelated but well defined sequence. IR-targeting leads to the formation of a new junction in which the targeted IR and one of the donor IRs are separated by 2 bp. The non-IR targets were characterized as an imperfect 19 bp palindrome in which the central five positions show slight GC excess and the distal region is AT-rich. Artificial targets designed according to the consensus were recognized by the element as hot spots for insertion. The organization of IS1655 is similar to that of other IS30 family members. Moreover, it shows striking similarity to IS30 in transposition strategy even though their transposases differ in their N-terminal regions, which, for IS30, appears to determine target specificity. Comparative analysis of the transposases and the evolutionary aspects of sequence variants are also briefly discussed.


Assuntos
Elementos de DNA Transponíveis/fisiologia , Neisseria meningitidis/genética , Transposases/metabolismo , DNA Bacteriano/química , Genoma Bacteriano , Dados de Sequência Molecular , Mutagênese Insercional
19.
PLoS One ; 2(3): e316, 2007 Mar 21.
Artigo em Inglês | MEDLINE | ID: mdl-17375202

RESUMO

TFIID plays a role in nucleating RNA polymerase II preinitiation complex assembly on protein-coding genes. TFIID is a multisubunit complex comprised of the TATA box binding protein (TBP) and 14 TBP-associated factors (TAFs). Another class of multiprotein transcriptional regulatory complexes having histone acetyl transferase (HAT) activity, and containing TAFs, includes TFTC, STAGA and the PCAF/GCN5 complex. Looking for as yet undiscovered subunits by a proteomic approach, we had identified TAF8 and SPT7L in human TFTC preparations. Subsequently, however, we demonstrated that TAF8 was not a stable component of TFTC, but that it is present in a small TAF complex (SMAT), containing TAF8, TAF10 and SPT7L, that co-purified with TFTC. Thus, TAF8 is a subunit of both TFIID and SMAT. The latter has to be involved in a pathway of complex formation distinct from the other known TAF complexes, since these three histone fold (HF)-containing proteins (TAF8, TAF10 and SPT7L) can never be found together either in TFIID or in STAGA/TFTC HAT complexes. Here we show that TAF8 is absolutely necessary for the integration of TAF10 in a higher order TFIID core complex containing seven TAFs. TAF8 forms a heterodimer with TAF10 through its HF and proline rich domains, and also interacts with SPT7L through its C-terminal region, and the three proteins form a complex in vitro and in vivo. Thus, the TAF8-TAF10 and TAF10-SPT7L HF pairs, and also the SMAT complex, seem to be important regulators of the composition of different TFIID and/or STAGA/TFTC complexes in the nucleus and consequently may play a role in gene regulation.


Assuntos
Proteínas Semelhantes à Proteína de Ligação a TATA-Box/genética , Fatores Associados à Proteína de Ligação a TATA/genética , Sequência de Aminoácidos , Animais , Western Blotting , DNA Complementar/genética , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Amplificação de Genes , Humanos , Camundongos , Reação em Cadeia da Polimerase/métodos , Subunidades Proteicas/química , Proteínas Semelhantes à Proteína de Ligação a TATA-Box/química , Proteínas Semelhantes à Proteína de Ligação a TATA-Box/metabolismo , Fatores Associados à Proteína de Ligação a TATA/química , Fatores de Transcrição/química , Fatores de Transcrição/genética , Transfecção , Leveduras/genética
20.
Mol Cell Biol ; 26(2): 402-12, 2006 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-16382133

RESUMO

Transactivation-transformation domain-associated protein (TRRAP) is a component of several multiprotein histone acetyltransferase (HAT) complexes implicated in transcriptional regulation. TRRAP was shown to be required for the mitotic checkpoint and normal cell cycle progression. MRE11, RAD50, and NBS1 (product of the Nijmegan breakage syndrome gene) form the MRN complex that is involved in the detection, signaling, and repair of DNA double-strand breaks (DSBs). By using double immunopurification, mass spectrometry, and gel filtration, we describe the stable association of TRRAP with the MRN complex. The TRRAP-MRN complex is not associated with any detectable HAT activity, while the isolated other TRRAP complexes, containing either GCN5 or TIP60, are. TRRAP-depleted extracts show a reduced nonhomologous DNA end-joining activity in vitro. Importantly, small interfering RNA knockdown of TRRAP in HeLa cells or TRRAP knockout in mouse embryonic stem cells inhibit the DSB end-joining efficiency and the precise nonhomologous end-joining process, further suggesting a functional involvement of TRRAP in the DSB repair processes. Thus, TRRAP may function as a molecular link between DSB signaling, repair, and chromatin remodeling.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Dano ao DNA , Enzimas Reparadoras do DNA/metabolismo , Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Proteínas Nucleares/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Hidrolases Anidrido Ácido , Proteínas Adaptadoras de Transdução de Sinal , Animais , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Montagem e Desmontagem da Cromatina , Cromatografia em Gel , Enzimas Reparadoras do DNA/genética , Proteínas de Ligação a DNA/genética , Histona Acetiltransferases/genética , Histona Acetiltransferases/metabolismo , Humanos , Lisina Acetiltransferase 5 , Proteína Homóloga a MRE11 , Camundongos , Proteínas Nucleares/genética , Ligação Proteica , RNA Interferente Pequeno/genética , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição Gênica , Fatores de Transcrição de p300-CBP
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