RESUMO
Herein, the direct visualization of the dynamic interaction between a photoresponsive transcription factor fusion, GAL4-VVD, and DNA using high-speed atomic force microscopy (HS-AFM) is reported. A series of different GAL4-VVD movements, such as binding, sliding, stalling, and dissociation, was observed. Inter-strand jumping on two double-stranded (ds) DNAs was also observed. Detailed analysis using a long substrate DNA strand containing five GAL4-binding sites revealed that GAL4-VVD randomly moved on the dsDNA using sliding and hopping to rapidly find specific binding sites, and then stalled to the specific sites to form a stable complex formation. These results suggest the existence of different conformations of the protein to enable sliding and stalling. This single-molecule imaging system with nanoscale resolution provides an insight into the searching mechanism used by DNA-binding proteins.
Assuntos
Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Proteínas Fúngicas/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo , Sítios de Ligação , DNA/química , DNA/efeitos da radiação , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/efeitos da radiação , Proteínas Fúngicas/genética , Proteínas Fúngicas/efeitos da radiação , Luz , Microscopia de Força Atômica , Ligação Proteica , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/efeitos da radiação , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/efeitos da radiação , Fatores de Transcrição/genética , Fatores de Transcrição/efeitos da radiaçãoRESUMO
As a key regulator of DNA translesion synthesis (TLS) pathway, RAD18 is reported to be abnormally expressed in many kinds of cancers. In glioma, RAD18 was overexpressed in the primary and recurrent glioblastoma multiforme specimens, and its overexpression weakened ionizing radiation-induced apoptosis in glioma A172 cells. Moreover, A172 cells with mutational P53 also showed enhanced radiation resistance. And RAD18 activation induced by cyclin-dependent kinase 2 (CDK2) was repressed by P53. However, whether P53 involves in RAD18-induced radiation resistance remains unknown. Therefore, this study was conducted to explore the effects and mechanism of RAD18 in the radiation resistance of glioma and study P53 role in this process. Results showed that, RAD18 expression was obviously elevated in glioma tissues and cell lines such as U251, SHG-44, A172, U-87 MG and U-118 MG as compared with the normal brain tissues and neuroglia cells. Up-regulation of RAD18 in U-118 MG and A172 cells with lentivirus infection significantly increased cell growth and inhibited cell apoptosis, determined by CCK-8 and flow cytometry technologies. Besides, RAD18 overexpression enhanced cell growth and inhibited cell apoptosis after U-118 MG or A172 cells were irradiated at a dose of 4 Gy. On the contrary, silencing of endogenous RAD18 sensitized U-118 MG and A172 cells to radiation. Moreover, RAD18 and P53 proteins were co-located in the nucleus, and up-regulation of RAD18 decreased the expression of P53 protein and facilitated its nuclear export. Furthermore, cell growth promotion and cell apoptosis inhibition induced by RAD18 up-regulation were impaired when P53 expression was up-regulated under radiation condition. In a word, this study clarifies that RAD18 functions as a promoter in glioma progression and reduces glioma cells' sensibility to radiation through down-regulating P53, which provides new strategies to overcome the radiation resistance in glioma.
Assuntos
Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/radioterapia , Proteínas de Ligação a DNA/biossíntese , Glioma/metabolismo , Glioma/radioterapia , Proteína Supressora de Tumor p53/metabolismo , Ubiquitina-Proteína Ligases/biossíntese , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/efeitos da radiação , Regulação para Baixo/fisiologia , Regulação para Baixo/efeitos da radiação , Regulação Neoplásica da Expressão Gênica/efeitos da radiação , Humanos , Proteína Supressora de Tumor p53/antagonistas & inibidores , Proteína Supressora de Tumor p53/efeitos da radiação , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/efeitos da radiaçãoRESUMO
Electron transfer through π-stacked arrays of double-stranded DNA contributes to the redox chemistry of bases, including guanine oxidation and thymine-thymine dimer repair by photolyase. 5-Bromouracil is an attractive photoreactive thymine analogue that can be used to investigate electron transfer in DNA, and is a useful probe for protein-DNA interaction analysis. In the present study using BrU we found that UV irradiation facilitated electron injection from mitochondrial transcription factor A into DNA. We also observed that this electron injection could lead to repair of a thymine-thymine dimer.
Assuntos
Reparo do DNA/efeitos da radiação , Proteínas de Ligação a DNA/química , DNA/química , Elétrons , Proteínas Mitocondriais/química , Dímeros de Pirimidina/química , Fatores de Transcrição/química , Sequência de Bases , Bromouracila/química , Bromouracila/efeitos da radiação , DNA/genética , DNA/metabolismo , DNA/efeitos da radiação , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/efeitos da radiação , Humanos , Proteínas Mitocondriais/metabolismo , Proteínas Mitocondriais/efeitos da radiação , Regiões Promotoras Genéticas/efeitos da radiação , Ligação Proteica , Dímeros de Pirimidina/efeitos da radiação , Fatores de Transcrição/metabolismo , Fatores de Transcrição/efeitos da radiação , Raios UltravioletaRESUMO
Plant endogenous clock consists of self-sustained interlocked transcriptional/translational feedback loops whose oscillation regulates many circadian processes, including gene expression. Its free running rhythm can be entrained by external cues, which can influence all clock parameters. Among external cues, the geomagnetic field (GMF) has been demonstrated to influence plant growth and development. We evaluated the quantitative expression (qRT-PCR) of three clock genes (LHY, GI and PRR7) in time-course experiments under either continuous darkness (CD) or long days (LD) conditions in Arabidopsis thaliana seedlings exposed to GMF (â¼40 µT) and Near Null Magnetic Field (NNMF; â¼40 nT) conditions. Under both LD and CD conditions, reduction of GMF to NNMF prompted a significant increase of the gene expression of LHY and PRR7, whereas an opposite trend was found for GI gene expression. Exposure of Arabidopsis to NNMF altered clock gene amplitude, regardless the presence of light, by reinforcing the morning loop. Our data are consistent with the existence of a plant magnetoreceptor that affects the Arabidopsis endogenous clock.
Assuntos
Arabidopsis/efeitos da radiação , Relógios Biológicos/efeitos da radiação , Genes de Plantas/efeitos da radiação , Campos Magnéticos , Arabidopsis/genética , Arabidopsis/fisiologia , Proteínas de Arabidopsis/fisiologia , Proteínas de Arabidopsis/efeitos da radiação , Relógios Biológicos/genética , Proteínas de Ligação a DNA/fisiologia , Proteínas de Ligação a DNA/efeitos da radiação , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas/efeitos da radiação , Genes de Plantas/fisiologia , Luz , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Repressoras/fisiologia , Proteínas Repressoras/efeitos da radiação , Fatores de Transcrição/fisiologia , Fatores de Transcrição/efeitos da radiaçãoRESUMO
DNA is associated with proteins that are involved in its folding and transaction processes. When cells are exposed to chemical cross-linking agents or free radical-generating ionizing radiation, DNA-associated proteins are covalently trapped within the DNA to produce DNA-protein cross-links (DPCs). DPCs produced by these agents contain cross-linked proteins in an undisrupted DNA strand. Some DNA-metabolizing enzymes that form covalent reaction intermediates can also be irreversibly trapped in the presence of inhibitors or DNA damage to give rise to abortive DPCs. The abortive DPCs often contain cross-linked proteins attached to the 5' or 3' end of a DNA strand break. In vitro studies show that steric hindrance caused by cross-linked proteins impedes the progression of DNA helicases and polymerases and of RNA polymerases. The modes and consequences by which DPCs impede replication and transcription processes are considerably different from those with conventional DNA lesions. Thus, DPCs are formidable challenges to maintaining genome integrity and faithful gene expression. Current models of DPC repair involve direct and indirect removal of DPCs. The direct mechanism works for DPCs that contain topoisomerase 2 attached to the 5' end of DNA. The Mre11-Rad50-Nbs1 complex cleaves the site internal to the DPC and directly releases a DPC-containing oligonucleotide. The indirect mechanism involves degradation of cross-linked proteins by proteasomes or the recently identified DPC proteases Wss1 and Sprtn to relieve steric hindrance of DPCs. The resulting peptide-cross-links might be processed by translesion synthesis or other canonical repair mechanisms: however, the exact mechanism remains to be elucidated.
Assuntos
Adutos de DNA/metabolismo , Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Animais , Reagentes de Ligações Cruzadas/farmacologia , Reagentes de Ligações Cruzadas/toxicidade , DNA/efeitos dos fármacos , DNA/efeitos da radiação , Proteínas de Ligação a DNA/efeitos dos fármacos , Proteínas de Ligação a DNA/efeitos da radiação , Eucariotos/efeitos dos fármacos , Eucariotos/genética , Eucariotos/metabolismo , Eucariotos/efeitos da radiação , Humanos , Proteólise , Radiação IonizanteRESUMO
DNA-protein crosslinks (DPCs) are a specific type of DNA lesion consisting of a protein covalently and irreversibly bound to DNA, which arise after exposure to physical and chemical crosslinking agents. DPCs can be bulky and thereby pose a barrier to DNA replication and transcription. The persistence of DPCs during S phase causes DNA replication stress and genome instability. The toxicity of DPCs is exploited in cancer therapy: many common chemotherapeutics kill cancer cells by inducing DPC formation. Recent work from several laboratories discovered a specialized repair pathway for DPCs, namely DPC proteolysis (DPCP) repair. DPCP repair is carried out by replication-coupled DNA-dependent metalloproteases: Wss1 in yeast and SPRTN in metazoans. Mutations in SPRTN cause premature ageing and liver cancer in humans and mice; thus, defective DPC repair has great clinical ramifications. In the present review, we will revise the current knowledge on the mechanisms of DPCP repair and on the regulation of DPC protease activity, while highlighting the most significant unresolved questions in the field. Finally, we will discuss the impact of faulty DPC repair on disease and cancer therapy.
Assuntos
Adutos de DNA/metabolismo , Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Animais , Reagentes de Ligações Cruzadas/farmacologia , Reagentes de Ligações Cruzadas/toxicidade , DNA/efeitos dos fármacos , DNA/efeitos da radiação , Proteínas de Ligação a DNA/efeitos dos fármacos , Proteínas de Ligação a DNA/efeitos da radiação , Eucariotos/efeitos dos fármacos , Eucariotos/genética , Eucariotos/metabolismo , Eucariotos/efeitos da radiação , Humanos , Proteólise , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/efeitos da radiação , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Formation of the neutral flavin radical in the light-oxygen-voltage-sensing (LOV-sensing) domain of photozipper, based on VfAUREO1, was investigated by electron paramagnetic resonance spectroscopy. The flavin radical was observed in the presence of dithiothreitol by illumination of a LOV-domain mutant (C254S), in which a photoactive cysteine residue in close proximity to flavin was replaced with a serine. The radical did not form under low initial protein-concentration conditions (less than 20 µM). The flavin radicals accumulated with logistic time-dependent kinetics when the protein concentrations were higher than 30 µM. These results indicate that the radical is produced by concerted reactions involving protein interactions and that the radical is formed from the LOV dimer but not the LOV monomer. In contrast, logistic time dependencies were not observed for the sample adapted to the dark following radical formation by illumination, indicating that initialization of the proton pathway is essential for this fast sensing reaction.
Assuntos
Proteínas de Ligação a DNA/química , Mononucleotídeo de Flavina/química , Radicais Livres/química , Cisteína/química , Cisteína/efeitos da radiação , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/efeitos da radiação , Escherichia coli/genética , Mononucleotídeo de Flavina/efeitos da radiação , Luz , Mutação , Domínios Proteicos/efeitos da radiação , Estramenópilas/químicaRESUMO
Absorption of solar ultraviolet (UV) radiation by DNA leads to the formation of the highly mutagenic cyclobutane pyrimidine dimer (CPD). The mutagenicity of CPD is caused, in part, by the fact that their recognition and repair by the nucleotide excision repair (NER) pathway is challenging and slow. It has been previously shown that a pre-stimulation with genotoxic agents improve NER efficiency of CPD, indicating a potential adaptive response of this repair pathway. We have pre-treated human dermal fibroblasts with repeated subletal low doses of UVB (chronic low-dose of UVB; CLUV) to determine whether it could enhance NER capacity to repair CPD. Our results show that CLUV pre-treatment greatly enhances CPD repair but have little effect on the repair of another UV-induced bypirimidine photoproduct, the pyrimidine (6-4) pyrimidone photoproducts (6-4 PP). We have determined that the CLUV treatment activates p53 and we found an increase of DDB2 and XPC gene expression. This is consistent with an increasing level of NER recognition proteins, DDB2 and XPC, we found concentrated at the chromatin. This study represents the first demonstration that chronic UVB exposure can stimulate NER pathway. Altogether, these results shed light on the potential adaptability of the NER by chronic UVB irradiation and the mechanisms involved.
Assuntos
Adaptação Fisiológica , Adutos de DNA/metabolismo , Reparo do DNA/fisiologia , Proteínas de Ligação a DNA/genética , Dímeros de Pirimidina/metabolismo , DNA/efeitos da radiação , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/efeitos da radiação , Fibroblastos/metabolismo , Fibroblastos/fisiologia , Fibroblastos/efeitos da radiação , Regulação da Expressão Gênica , Humanos , Transdução de Sinais/efeitos da radiação , Proteína Supressora de Tumor p53/metabolismo , Proteína Supressora de Tumor p53/fisiologia , Proteína Supressora de Tumor p53/efeitos da radiação , Raios UltravioletaRESUMO
Cervical cancer is a public health problem and the molecular mechanisms underlying radioresistance are still poorly understood. Here, we evaluated the modulation of key molecules involved in cell proliferation, cell cycle and DNA repair in cervical cancer cell lines (CASKI and C33A) and in malignant tissues biopsied from 10 patients before and after radiotherapy. The expression patterns of epidermal growth factor receptor (EGFR), excision repair cross-complementation group 1 (ERCC1) and p53 were evaluated in cancer cell lines by quantitative PCR and western blotting, and in human malignant tissues by immunohistochemistry. The mutation status of TP53 gene was evaluated by direct sequencing. Among cell lines, absent or weak modulations of EGFR, ERCC1 and p53 were observed after exposure to 1.8 Gy. Conversely, increased expressions of p53 (5/10 patients; P=0.0239), ERCC1 (5/10 patients; P=0.0294) and EGFR (4/10 patients; P=0.1773) were observed in malignant tissues after radiotherapy with the same radiation dose. TP53 mutations were found only in one patient. Here we show that a single dose of radiotherapy induced EGFR, ERCC1 and p53 expression in malignant tissues from cervical cancer patients but not in cancer cell lines, highlighting the gap between in vitro and in vivo experimental models. Studies on larger patient cohorts are needed to allow an interpretation that an upregulation of p53, EGFR and ERCC1 may be part of a radioresistance mechanism.
Assuntos
Humanos , Feminino , Adulto , Pessoa de Meia-Idade , Idoso , Carcinoma de Células Escamosas/radioterapia , Neoplasias do Colo do Útero/radioterapia , Genes p53/efeitos da radiação , Genes erbB-1/efeitos da radiação , Proteínas de Ligação a DNA/efeitos da radiação , Endonucleases/efeitos da radiação , Imuno-Histoquímica , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patologia , Ensaio Tumoral de Célula-Tronco , Western Blotting , Estudos Prospectivos , Linhagem Celular Tumoral , MutaçãoRESUMO
Cervical cancer is a public health problem and the molecular mechanisms underlying radioresistance are still poorly understood. Here, we evaluated the modulation of key molecules involved in cell proliferation, cell cycle and DNA repair in cervical cancer cell lines (CASKI and C33A) and in malignant tissues biopsied from 10 patients before and after radiotherapy. The expression patterns of epidermal growth factor receptor (EGFR), excision repair cross-complementation group 1 (ERCC1) and p53 were evaluated in cancer cell lines by quantitative PCR and western blotting, and in human malignant tissues by immunohistochemistry. The mutation status of TP53 gene was evaluated by direct sequencing. Among cell lines, absent or weak modulations of EGFR, ERCC1 and p53 were observed after exposure to 1.8 Gy. Conversely, increased expressions of p53 (5/10 patients; P=0.0239), ERCC1 (5/10 patients; P=0.0294) and EGFR (4/10 patients; P=0.1773) were observed in malignant tissues after radiotherapy with the same radiation dose. TP53 mutations were found only in one patient. Here we show that a single dose of radiotherapy induced EGFR, ERCC1 and p53 expression in malignant tissues from cervical cancer patients but not in cancer cell lines, highlighting the gap between in vitro and in vivo experimental models. Studies on larger patient cohorts are needed to allow an interpretation that an upregulation of p53, EGFR and ERCC1 may be part of a radioresistance mechanism.
Assuntos
Carcinoma de Células Escamosas/radioterapia , Proteínas de Ligação a DNA/efeitos da radiação , Endonucleases/efeitos da radiação , Genes erbB-1/efeitos da radiação , Genes p53/efeitos da radiação , Neoplasias do Colo do Útero/radioterapia , Adulto , Idoso , Western Blotting , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos da radiação , Reparo do DNA/efeitos da radiação , Proteínas de Ligação a DNA/metabolismo , Relação Dose-Resposta à Radiação , Endonucleases/metabolismo , Receptores ErbB/metabolismo , Feminino , Expressão Gênica , Humanos , Imuno-Histoquímica , Pessoa de Meia-Idade , Mutação , Estudos Prospectivos , Reação em Cadeia da Polimerase em Tempo Real , Reprodutibilidade dos Testes , Resultado do Tratamento , Ensaio Tumoral de Célula-Tronco , Proteína Supressora de Tumor p53/metabolismo , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/patologiaRESUMO
Nucleic acids are responsible for the storage, transfer and realization of genetic information in the cell, which provides correct development and functioning of organisms. DNA interaction with ligands ensures the safety of this information. Over the past 10 years, advances in electron microscopy and image processing allowed to obtain the structures of key DNA-protein complexes with resolution below 4Å. However, radiation damage is a limiting factor to the potentially attainable resolution in cryo-EM. The prospect and limitations of studying protein-DNA complex interactions using cryo-electron microscopy are discussed here. We reviewed the ways to minimize radiation damage in biological specimens and the possibilities of using radiation damage (so-called 'bubblegrams') to obtain additional structural information.
Assuntos
Microscopia Crioeletrônica/métodos , Proteínas de Ligação a DNA/efeitos da radiação , Estrutura Terciária de Proteína/efeitos da radiação , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/genética , Processamento de Imagem Assistida por Computador/métodos , Imageamento Tridimensional/métodos , Modelos MolecularesRESUMO
PDRG1, is short for P53 and DNA damage-regulated gene, which have been found over 10 years. Although severe studies have described the roles of PDRG1 separately in many kinds of tumors, how to act as an oncogene are unclear. To better verify the function of PDRG1 in lung cancer, both loss-function and gain-function of PDRG1 studies based on two human lung cancer lines were performed. Following the transfection of PDRG1, both A549 and 95-D cells showed significant changes in cell viability, the expression of some protein and apoptosis, which were all implied the PDRG1 is an oncogene. Another interesting finding is PDRG1 could promote radioresistance involved the ATM-p53 signaling pathway in lung cancer. If we combine radiotherapy with gene-targeted therapy together effectively, predominant effect may be acquired, which is a huge milestone in clinical cure about lung cancer.
Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas de Ligação a DNA/genética , Neoplasias Pulmonares/genética , Oncogenes/genética , Tolerância a Radiação/genética , Proteína Supressora de Tumor p53/genética , Animais , Proteínas Mutadas de Ataxia Telangiectasia/efeitos da radiação , Linhagem Celular Tumoral , Sobrevivência Celular/genética , Sobrevivência Celular/efeitos da radiação , Proteínas de Ligação a DNA/administração & dosagem , Proteínas de Ligação a DNA/efeitos da radiação , Humanos , Neoplasias Pulmonares/radioterapia , Camundongos , Camundongos Nus , Oncogenes/efeitos da radiação , Transdução de Sinais/genética , Transdução de Sinais/efeitos da radiação , Proteína Supressora de Tumor p53/efeitos da radiaçãoRESUMO
The bioluminescence induced by luciferases of marine bacteria promotes repair of UV damaged DNA of Escherichia coli AB1886 uvrA6. It is shown that bacterial photolyase that implements photoreactivation activity is the major contributor to DNA repair. However, the intensity of bioluminescence increasing induced by UV-irradiation (SOS-induction) in bacterial cells is not enough for efficient photoreactivation.
Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , Luciferases Bacterianas/metabolismo , Resposta SOS em Genética , Raios Ultravioleta , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/efeitos da radiação , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/efeitos da radiação , Escherichia coli/efeitos da radiação , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/efeitos da radiação , Luciferases Bacterianas/genética , Luciferases Bacterianas/efeitos da radiação , Photobacterium/enzimologiaRESUMO
Aureochrome-1 (AUREO1) has been identified as a blue light (BL) receptor responsible for the BL-induced blanching of a stramenopile alga, Vaucheria frigida. BL induces the dimerization of monomeric AUREO1, which subsequently increases its affinity for the target sequence. We made a synthetic gene encoding N-terminally truncated monomeric AUREO1 (Photozipper protein) containing a basic region/leucine zipper (bZIP) domain and a light-oxygen-voltage-sensing domain. In the present study, yellow fluorescent protein or mCherry protein was fused with the Photozipper (PZ) protein, and their oligomeric structures and DNA-binding were compared in the dark and light states. Dynamic light scattering and size exclusion chromatography demonstrated that the hydrodynamic radii and molecular masses of the fusion proteins increased upon BL illumination, suggesting that fusion PZs underwent BL-induced dimerization. Moreover, BL-induced dimerization enhanced their affinities for the target sequence. Taken together, PZ likely functions as a BL-regulated bZIP module in fusion proteins, and can possibly provide a new approach for controlling bZIP transcription factors.
Assuntos
Proteínas de Algas/química , Proteínas de Algas/metabolismo , Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Zíper de Leucina/efeitos da radiação , Luz , Proteínas de Algas/efeitos da radiação , Sequência de Bases , Sítios de Ligação , DNA/química , DNA/genética , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/efeitos da radiação , Ligação Proteica/efeitos da radiaçãoRESUMO
There are many complex interactions between transposable elements (TEs) and host genomes. Environmental changes that induce stressful conditions help to contribute for increasing complexity of these interactions. The transposon mariner-Mos1 increases its mobilization under mild heat stress. It has putative heat shock elements (HSEs), which are probably activated by heat shock factors (HSFs). Ultraviolet radiation (UVC) is a stressor that has been suggested as able to activate heat shock protein genes (Hsp). In this study, we test the hypothesis that if UVC induces Hsp expression, as heat does, it could also promote mariner-Mos1 transposition and mobilization. The Drosophila simulans white-peach is a mutant lineage that indicates the mariner-Mos1 transposition phenotypically through the formation of mosaic eyes. This lineage was exposed to UVC or mild heat stress (28 °C) in order to evaluate the induction of mariner-Mos1 expression by RT-qPCR, as well as the mariner-Mos1 mobilization activity based on the count number of red spots in the eyes. The effects of both treatments on the developmental time of flies and cell cycle progression were also investigated. Both the analysis of eyes and mariner-Mos1 gene expression indicate that UVC radiation has no effect in mariner-Mos1 transposition, although heat increases the expression and mobilization of this TE soon after the treatment. However, the expression of Hsp70 gene increased after 24 h of UVC exposure, suggesting different pathway of activation. These results showed that heat promotes mariner-Mos1 mobilization, although UVC does not induce the expression or mobilization of this TE.
Assuntos
Elementos de DNA Transponíveis/genética , Elementos de DNA Transponíveis/efeitos da radiação , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/fisiologia , Proteínas de Ligação a DNA/efeitos da radiação , Drosophila simulans/genética , Temperatura Alta , Fatores de Transcrição/fisiologia , Transposases/genética , Transposases/efeitos da radiação , Raios Ultravioleta , Animais , Fatores de Transcrição de Choque Térmico , MutaçãoRESUMO
To elucidate the mechanisms of DNA repair pathway is critical for developing next-generation radiotherapies and chemotherapeutic drugs for cancer. Ionizing radiation and many chemotherapeutic drugs kill tumor cells mainly by inducing DNA double-strand breaks (DSBs). The classical nonhomologous DNA-end joining (NHEJ) (C-NHEJ) pathway repairs a predominant fraction of DSBs in mammalian cells. The C-NHEJ pathway appears to start with the binding of Ku (heterodimer of Ku70 and Ku80) to DNA break ends. Therefore, recruitment of Ku to DSB sites might play a critical role in regulating NHEJ activity. Indeed, human Ku70 and Ku80 localize in the nuclei and accumulate at microirradiated DSB sites. However, the localization and regulation mechanisms of Ku70 and Ku80 homologues in animal models, such as mice and other species, have not been elucidated in detail, particularly in cells immediately after microirradiation. Here, we show that EYFP-tagged mouse Ku70 localizes in the interphase nuclei of mouse fibroblasts and epithelial cells. Furthermore, our findings indicate that EYFP-mouse Ku70 accumulates with its heterodimeric partner Ku80 immediately at laser-microirradiated DSB sites. We also confirmed that the structure of Ku70 nuclear localization signal (NLS) is highly conserved among various rodent species, such as the mouse, rat, degu and ground squirrel, supporting the idea that NLS is important for the regulation of rodent Ku70 function. Collectively, these results suggest that the mechanisms of regulating the localization and accumulation of Ku70 at DSBs might be well conserved between the mouse and human species.
Assuntos
Antígenos Nucleares/efeitos da radiação , Núcleo Celular/metabolismo , Dano ao DNA/efeitos da radiação , Proteínas de Ligação a DNA/efeitos da radiação , Interfase/efeitos da radiação , Animais , Antígenos Nucleares/fisiologia , Linhagem Celular , Núcleo Celular/efeitos da radiação , Reparo do DNA por Junção de Extremidades/efeitos da radiação , Reparo do DNA/efeitos da radiação , Proteínas de Ligação a DNA/fisiologia , Imunofluorescência , Autoantígeno Ku , CamundongosRESUMO
Deinococcus radiodurans (Dr) has a significantly more robust DNA repair response than Escherichia coli (Ec), which helps it survive extremely high doses of ionizing radiation and prolonged periods of desiccation. DrRecA protein plays an essential part in this DNA repair capability. In this study we directly compare the binding of DrRecA and EcRecA to the same set of short, defined single (ss) and double stranded (ds) DNA oligomers. In the absence of cofactors (ATPγS or ADP), DrRecA binds to dsDNA oligomers more than 20 fold tighter than EcRecA, and binds ssDNA up to 9 fold tighter. Binding to dsDNA oligomers in the absence of cofactor presumably predominantly monitors DNA end binding, and thus suggests a significantly higher affinity of DrRecA for ds breaks. Upon addition of ATPγS, this species-specific affinity difference is nearly abolished, as ATPγS significantly decreases the affinity of DrRecA for DNA. Other findings include that: (1) both proteins exhibit a dependence of binding affinity on the length of the ssDNA oligomer, but not the dsDNA oligomer; (2) the salt dependence of binding is modest for both species of RecA, and (3) in the absence of DNA, DrRecA produces significantly shorter and/or fewer free-filaments in solution than does EcRecA. The results suggest intrinsic biothermodynamic properties of DrRecA contribute directly to the more robust DNA repair capabilities of D. radiodurans.
Assuntos
Proteínas de Bactérias/genética , DNA Bacteriano/isolamento & purificação , Deinococcus/genética , Recombinases Rec A/genética , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/deficiência , Trifosfato de Adenosina/genética , Trifosfato de Adenosina/metabolismo , Proteínas de Bactérias/metabolismo , Reparo do DNA/efeitos da radiação , DNA Bacteriano/genética , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/efeitos da radiação , Deinococcus/enzimologia , Relação Dose-Resposta à Radiação , Escherichia coli/genética , Radiação Ionizante , Recombinases Rec A/metabolismoRESUMO
Immunohistochemistry (IHC) testing for mismatch repair proteins (MMRP) is currently being used primarily in colorectal cancer resection specimens. We aimed to compare the results of IHC staining performed on biopsy specimens obtained at endoscopy with that performed on surgical specimens after neoadjuvant therapy. Thirty-two rectal cancer subjects had paired preneoadjuvant and postneoadjuvant tissue available for IHC staining (MLH1, MSH2, MSH6, and PMS2), whereas 39 rectosigmoid cancer patients who did not receive neoadjuvant treatment served as controls. Each slide received a qualitative (absent, focal, and strong) and quantitative score (immunoreactivity [0-3] × percent positivity [0-4]). The quantitative scores of MMRP from the operative material were significantly lower in the neoadjuvant group than in the control (P < .05 for all).The scores of all MMRP from endoscopic biopsies were not significantly different between the neoadjuvant and the control groups. Disagreement between the endoscopic biopsy and the operative material was evident in 23 of 128 stains (18.5%) in the neoadjuvant group and in 12 of 156 stains (7.7%) in the control group (P = .009). In the neoadjuvant group, a disagreement pattern of "endoscopic strong operative focal" was observed in 28.1% for PMS2, 12.5% for MSH6, 12.5% for MLH1, and 6.3% for MSH2, and in the control group, this same disagreement pattern was found in 12.8% for PMS2, 7.7% for MSH6, 7.7% for MLH1, and 0% for MSH2. Based on our findings, we suggest that for rectal cancer, the endoscopic material rather than the operative material should serve as the primary material for IHC staining.
Assuntos
Neoplasias Colorretais/genética , Reparo de Erro de Pareamento de DNA , Imuno-Histoquímica/normas , Terapia Neoadjuvante , Proteínas de Neoplasias/análise , Proteínas Adaptadoras de Transdução de Sinal/análise , Proteínas Adaptadoras de Transdução de Sinal/efeitos dos fármacos , Proteínas Adaptadoras de Transdução de Sinal/efeitos da radiação , Adenosina Trifosfatases/análise , Adenosina Trifosfatases/efeitos dos fármacos , Adenosina Trifosfatases/efeitos da radiação , Idoso , Quimiorradioterapia , Neoplasias Colorretais/terapia , Enzimas Reparadoras do DNA/análise , Enzimas Reparadoras do DNA/efeitos dos fármacos , Enzimas Reparadoras do DNA/efeitos da radiação , Proteínas de Ligação a DNA/análise , Proteínas de Ligação a DNA/efeitos dos fármacos , Proteínas de Ligação a DNA/efeitos da radiação , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Endonuclease PMS2 de Reparo de Erro de Pareamento , Proteína 1 Homóloga a MutL , Proteína 2 Homóloga a MutS/análise , Proteína 2 Homóloga a MutS/efeitos dos fármacos , Proteína 2 Homóloga a MutS/efeitos da radiação , Proteínas de Neoplasias/efeitos dos fármacos , Proteínas de Neoplasias/efeitos da radiação , Proteínas Nucleares/análise , Proteínas Nucleares/efeitos dos fármacos , Proteínas Nucleares/efeitos da radiação , Reprodutibilidade dos TestesRESUMO
With their utilization of light-driven allostery to control biochemical activities, photosensory proteins are of great interest as model systems and novel reagents for use by the basic science and engineering communities. One such protein, the light-activated EL222 transcription factor, from the marine bacterium Erythrobacter litoralis HTCC2594, is appealing for such studies, as it harnesses blue light to drive the reorientation of light-oxygen-voltage (LOV) sensory and helix-turn-helix (HTH) effector domains to allow photoactivation of gene transcription in natural and artificial systems. The protein conformational changes required for this process are not well understood, in part because of the relatively short lifetime of the EL222 photoexcited state (τ â¼ 29 s), which complicates its characterization via certain biophysical methods. Here we report how we have circumvented this limitation by creating an EL222 variant harboring V41I, L52I, A79Q, and V121I point mutations (AQTrip) that stabilizes the photoactivated state. Using the wild-type and AQTrip EL222 proteins, we have probed EL222 activation using a combination of solution scattering, nuclear magnetic resonance (NMR), and electromobility shift assays. Size-exclusion chromatography and light scattering indicate that AQTrip oligomerizes in the absence of DNA and selects for an EL222 dimer-DNA complex in the presence of DNA substrates. These results are confirmed in wild-type EL222 with a high-affinity DNA-binding site that stabilizes the complex. NMR analyses of the EL222-DNA complex confirm a 2:1 stoichiometry in the presence of a previously characterized DNA substrate. Combined, these novel approaches have validated a key mechanistic step, whereby blue light induces EL222 dimerization through LOV and HTH interfaces.
Assuntos
Proteínas de Bactérias/química , Proteínas de Ligação a DNA/química , Fatores de Transcrição/química , Alphaproteobacteria/química , Alphaproteobacteria/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/efeitos da radiação , Domínio Catalítico , Cromatografia em Gel , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/efeitos da radiação , Luz , Ressonância Magnética Nuclear Biomolecular , Multimerização Proteica , Estrutura Terciária de Proteína , Espalhamento de Radiação , Fatores de Transcrição/genética , Fatores de Transcrição/efeitos da radiaçãoRESUMO
PURPOSE: Ataxia telangiectasia mutated (ATM) is a serine/threonine kinase critical to the cellular DNA-damage response, including from DNA double-strand breaks. ATM activation results in the initiation of a complex cascade of events including DNA damage repair, cell cycle checkpoint control, and survival. We sought to create a bioluminescent reporter that dynamically and noninvasively measures ATM kinase activity in living cells and subjects. METHODS AND MATERIALS: Using the split luciferase technology, we constructed a hybrid cDNA, ATM-reporter (ATMR), coding for a protein that quantitatively reports on changes in ATM kinase activity through changes in bioluminescence. RESULTS: Treatment of ATMR-expressing cells with ATM inhibitors resulted in a dose-dependent increase in bioluminescence activity. In contrast, induction of ATM kinase activity upon irradiation resulted in a decrease in reporter activity that correlated with ATM and Chk2 activation by immunoblotting in a time-dependent fashion. Nuclear targeting improved ATMR sensitivity to both ATM inhibitors and radiation, whereas a mutant ATMR (lacking the target phosphorylation site) displayed a muted response. Treatment with ATM inhibitors and small interfering (si)RNA-targeted knockdown of ATM confirm the specificity of the reporter. Using reporter expressing xenografted tumors demonstrated the ability of ATMR to report in ATM activity in mouse models that correlated in a time-dependent fashion with changes in Chk2 activity. CONCLUSIONS: We describe the development and validation of a novel, specific, noninvasive bioluminescent reporter that enables monitoring of ATM activity in real time, in vitro and in vivo. Potential applications of this reporter include the identification and development of novel ATM inhibitors or ATM-interacting partners through high-throughput screens and in vivo pharmacokinetic/pharmacodynamic studies of ATM inhibitors in preclinical models.