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  1 / 1726 MEDLINE  
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[PMID]:28863186
[Au] Autor:Olszewski M; Spibida M; Bilek M; Krawczyk B
[Ad] Endereço:Gdansk University of Technology, Department of Molecular Biotechnology and Microbiology, ul. G. Narutowicza 11/12, Gdansk, Poland.
[Ti] Título:Fusion of Taq DNA polymerase with single-stranded DNA binding-like protein of Nanoarchaeum equitans-Expression and characterization.
[So] Source:PLoS One;12(9):e0184162, 2017.
[Is] ISSN:1932-6203
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:DNA polymerases are present in all organisms and are important enzymes that synthesise DNA molecules. They are used in various fields of science, predominantly as essential components for in vitro DNA syntheses, known as PCR. Modern diagnostics, molecular biology and genetic engineering need DNA polymerases which demonstrate improved performance. This study was aimed at obtaining a new NeqSSB-TaqS fusion DNA polymerase from the Taq DNA Stoffel domain and a single-stranded DNA binding-like protein of Nanoarchaeum equitans in order to significantly improve the properties of DNA polymerase. The DNA coding sequence of Taq Stoffel DNA polymerase and the nonspecific DNA-binding protein of Nanoarchaeum equitans (NeqSSB-like protein) were fused. A novel recombinant gene was obtained which was cloned into the pET-30 Ek/LIC vector and introduced into E. coli for expression. The recombinant enzyme was purified and its enzymatic properties including DNA polymerase activity, PCR amplification rate, thermostability, processivity and resistance to inhibitors, were tested. The yield of the target protein reached approximately 18 mg/l after 24 h of the IPTG induction. The specific activity of the polymerase was 2200 U/mg. The recombinant NeqSSB-TaqS exhibited a much higher extension rate (1000 bp template in 20 s), processivity (19 nt), thermostability (half-life 35 min at 95°C) and higher tolerance to PCR inhibitors (0.3-1.25% of whole blood, 0.84-13.5 µg of lactoferrin and 4.7-150 ng of heparin) than Taq Stoffel DNA polymerase. Furthermore, our studies show that NeqSSB-TaqS DNA polymerase has a high level of flexibility in relation to Mg2+ ions (from 1 to 5 mM) and KCl or (NH4)2SO4 salts (more than 60 mM and 40 mM, respectively). Using NeqSSB-TaqS DNA polymerase instead of the Taq DNA polymerase could be a better choice in many PCR applications.
[Mh] Termos MeSH primário: DNA de Cadeia Simples
Proteínas de Ligação a DNA/metabolismo
Nanoarchaeota/enzimologia
Taq Polimerase/metabolismo
[Mh] Termos MeSH secundário: Proteínas de Ligação a DNA/genética
Escherichia coli/genética
Escherichia coli/metabolismo
Engenharia Genética
Nanoarchaeota/genética
Plasmídeos/metabolismo
Reação em Cadeia da Polimerase
Proteínas Recombinantes/genética
Proteínas Recombinantes/metabolismo
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (DNA, Single-Stranded); 0 (DNA-Binding Proteins); 0 (Recombinant Proteins); EC 2.7.7.- (Taq Polymerase)
[Em] Mês de entrada:1710
[Cu] Atualização por classe:171023
[Lr] Data última revisão:
171023
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:170902
[St] Status:MEDLINE
[do] DOI:10.1371/journal.pone.0184162


  2 / 1726 MEDLINE  
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[PMID]:28475413
[Au] Autor:Ryabinin VA; Kostina EV; Sinyakov AN
[Ad] Endereço:a Institute of Chemical Biology and Fundamental Medicine, Siberian Branch, Russian Academy of Sciences , Novosibirsk , Russia.
[Ti] Título:Unexpected transformation of black hole quenchers in electrophoretic purification of the fluorescein-containing TaqMan probes.
[So] Source:Nucleosides Nucleotides Nucleic Acids;36(6):418-427, 2017 Jun 03.
[Is] ISSN:1532-2335
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:The fluorescence quenchers BHQ1 and BHQ2 can be modified by trace amounts of ammonium persulfate, used for initiating gel polymerization, in electrophoretic purification of TaqMan probes using a denaturing polyacrylamide gel. The case study of BHQ1 quencher has demonstrated that a Boyland-Sims reaction proceeds in the presence of ammonium persulfate to give the corresponding sulfate. The absorption maximum of the resulting quencher shifts to the short-wavelength region relative to the absorption maximum of the initial BHQ1. The TaqMan probe containing such a quencher is less efficient as compared with the probe carrying an unmodified BHQ1. The presence of fluorescein in TaqMan probe plays decisive role in this transformation: the quencher modification proceeds at a considerably lower rate when the fluorescein is absent or replaced with a rhodamine dye (for example, R6G). It is assumed that the observed reaction can take place in two ways-both in darkness and in the reaction of the quencher in an excited state due to energy transfer from the fluorophore irradiated by light.
[Mh] Termos MeSH primário: Eletroforese
Fluoresceína/química
Fluoresceína/isolamento & purificação
Corantes Fluorescentes/química
Corantes Fluorescentes/isolamento & purificação
Taq Polimerase/química
Taq Polimerase/metabolismo
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Fluorescent Dyes); EC 2.7.7.- (Taq Polymerase); TPY09G7XIR (Fluorescein)
[Em] Mês de entrada:1711
[Cu] Atualização por classe:171113
[Lr] Data última revisão:
171113
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:170506
[St] Status:MEDLINE
[do] DOI:10.1080/15257770.2017.1310384


  3 / 1726 MEDLINE  
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[PMID]:28421451
[Au] Autor:Sochivko DG; Fedorov AA; Alekseev YI; Kurochkin VE; Dubina MV
[Ad] Endereço:JSC Syntol, Moscow, 127550, Russia.
[Ti] Título:Mathematical model of polymerase chain reaction with temperature-dependent parameters.
[So] Source:Dokl Biochem Biophys;472(1):77-80, 2017 Jan.
[Is] ISSN:1608-3091
[Cp] País de publicação:Russia (Federation)
[La] Idioma:eng
[Ab] Resumo:The course of the real-time polymerase chain reaction (PCR) is determined by the temperature dependence of the kinetics of the component reactions, particularly the DNA strand hybridization. To investigate the effect of thermal processes on the reaction behavior, a mathematical model in which the variable rate constant of dissociation of "primer-single strand" complexes depends on temperature was proposed. The reaction medium temperature, which depends on time, was also introduced into the model. The proposed model of real-time PCR makes it possible to analyze different aspects of the reaction, which are important for the development of instruments and reagents for PCR.
[Mh] Termos MeSH primário: Modelos Teóricos
Reação em Cadeia da Polimerase/métodos
Taq Polimerase/metabolismo
[Mh] Termos MeSH secundário: Pareamento de Bases
DNA/química
Desnaturação de Ácido Nucleico
Taq Polimerase/química
Temperatura Ambiente
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
9007-49-2 (DNA); EC 2.7.7.- (Taq Polymerase)
[Em] Mês de entrada:1707
[Cu] Atualização por classe:171019
[Lr] Data última revisão:
171019
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:170420
[St] Status:MEDLINE
[do] DOI:10.1134/S1607672917010240


  4 / 1726 MEDLINE  
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[PMID]:28384080
[Au] Autor:Ding W; Weng H; Jin P; Du G; Chen J; Kang Z
[Ad] Endereço:a The Key Laboratory of Industrial Biotechnology, Ministry of Education , School of Biotechnology, Jiangnan University , Wuxi , China.
[Ti] Título:Scarless assembly of unphosphorylated DNA fragments with a simplified DATEL method.
[So] Source:Bioengineered;8(3):296-301, 2017 May 04.
[Is] ISSN:2165-5987
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:Efficient assembly of multiple DNA fragments is a pivotal technology for synthetic biology. A scarless and sequence-independent DNA assembly method (DATEL) using thermal exonucleases has been developed recently. Here, we present a simplified DATEL (sDATEL) for efficient assembly of unphosphorylated DNA fragments with low cost. The sDATEL method is only dependent on Taq DNA polymerase and Taq DNA ligase. After optimizing the committed parameters of the reaction system such as pH and the concentration of Mg and NAD+, the assembly efficiency was increased by 32-fold. To further improve the assembly capacity, the number of thermal cycles was optimized, resulting in successful assembly 4 unphosphorylated DNA fragments with an accuracy of 75%. sDATEL could be a desirable method for routine manual and automated assembly.
[Mh] Termos MeSH primário: Fragmentação do DNA
DNA/química
DNA/genética
Exodesoxirribonucleases/química
Exodesoxirribonucleases/genética
Taq Polimerase/química
Taq Polimerase/genética
[Mh] Termos MeSH secundário: Engenharia Genética/métodos
Fosforilação
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
9007-49-2 (DNA); EC 2.7.7.- (Taq Polymerase); EC 3.1.- (Exodeoxyribonucleases)
[Em] Mês de entrada:1710
[Cu] Atualização por classe:171030
[Lr] Data última revisão:
171030
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:170407
[St] Status:MEDLINE
[do] DOI:10.1080/21655979.2017.1308986


  5 / 1726 MEDLINE  
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[PMID]:28222630
[Au] Autor:Liu S; Cai H; Cheng W; Zhang H; Pan Z; Wang D
[Ad] Endereço:1 Department of Urology Surgery, Taizhou University Affiliated Taizhou Municipal Hospital, Taizhou, China.
[Ti] Título:Association of VDR polymorphisms ( Taq I and Bsm I) with prostate cancer: a new meta-analysis.
[So] Source:J Int Med Res;45(1):3-10, 2017 Feb.
[Is] ISSN:1473-2300
[Cp] País de publicação:England
[La] Idioma:eng
[Ab] Resumo:Objective Prostate cancer is a malignant tumour that poses a serious risk to human health. Epidemiological studies suggest that it may be associated with vitamin D receptor gene ( VDR) polymorphisms. Previous work investigated potential risks between Taq I (rs731236) and Bsm I (rs1544410) VDR polymorphisms with prostate cancer in humans; however, results are inconsistent. Methods We conducted a meta-analysis to retrieve genetic association analyses of rs731236 and rs1544410 polymorphisms with prostate cancer from studies published between 2006-2016. Pooled odds ratios with 95% confidence intervals were used to assess genetic associations, and heterogeneity was assessed by Q and I statistics. Results Our findings suggest a significant association between rs731236 and prostate cancer risk in Asians and African Americans, but rs1544410 was not associated with prostate cancer under three genetic models. Conclusion Future studies including larger sample sizes and the analysis of gene functions are needed to help develop prostate cancer treatment.
[Mh] Termos MeSH primário: Desoxirribonucleases de Sítio Específico do Tipo II/química
Predisposição Genética para Doença
Polimorfismo de Nucleotídeo Único
Neoplasias da Próstata/genética
Receptores de Calcitriol/genética
Taq Polimerase/química
[Mh] Termos MeSH secundário: Grupo com Ancestrais do Continente Africano
Grupo com Ancestrais do Continente Asiático
Grupo com Ancestrais do Continente Europeu
Expressão Gênica
Seres Humanos
Masculino
Modelos Genéticos
Razão de Chances
Neoplasias da Próstata/diagnóstico
Neoplasias da Próstata/etnologia
Neoplasias da Próstata/patologia
Risco
[Pt] Tipo de publicação:JOURNAL ARTICLE; META-ANALYSIS
[Nm] Nome de substância:
0 (Receptors, Calcitriol); 0 (VDR protein, human); EC 2.7.7.- (Taq Polymerase); EC 3.1.21.- (endodeoxyribonuclease BsmI); EC 3.1.21.4 (Deoxyribonucleases, Type II Site-Specific)
[Em] Mês de entrada:1705
[Cu] Atualização por classe:171004
[Lr] Data última revisão:
171004
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:170223
[St] Status:MEDLINE
[do] DOI:10.1177/0300060516668939


  6 / 1726 MEDLINE  
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[PMID]:28160372
[Au] Autor:Rosenblum SL; Weiden AG; Lewis EL; Ogonowsky AL; Chia HE; Barrett SE; Liu MD; Leconte AM
[Ad] Endereço:W. M. Keck Science Department, Claremont McKenna, Pitzer, and Scripps Colleges, Claremont, CA, 91711, USA.
[Ti] Título:Design and Discovery of New Combinations of Mutant DNA Polymerases and Modified DNA Substrates.
[So] Source:Chembiochem;18(8):816-823, 2017 Apr 18.
[Is] ISSN:1439-7633
[Cp] País de publicação:Germany
[La] Idioma:eng
[Ab] Resumo:Chemical modifications can enhance the properties of DNA by imparting nuclease resistance and generating more-diverse physical structures. However, native DNA polymerases generally cannot synthesize significant lengths of DNA with modified nucleotide triphosphates. Previous efforts have identified a mutant of DNA polymerase I from Thermus aquaticus DNA (SFM19) as capable of synthesizing a range of short, 2'-modified DNAs; however, it is limited in the length of the products it can synthesize. Here, we rationally designed and characterized ten mutants of SFM19. From this, we identified enzymes with substantially improved activity for the synthesis of 2'F-, 2'OH-, 2'OMe-, and 3'OMe-modified DNA as well as for reverse transcription of 2'OMe DNA. We also evaluated mutant DNA polymerases previously only tested for synthesis for 2'OMe DNA and showed that they are capable of an expanded range of modified DNA synthesis. This work significantly expands the known combinations of modified DNA and Taq DNA polymerase mutants.
[Mh] Termos MeSH primário: DNA Polimerase I/química
DNA/síntese química
Taq Polimerase/química
[Mh] Termos MeSH secundário: DNA/química
DNA Polimerase I/genética
Manganês/química
Mutação
Engenharia de Proteínas
RNA/síntese química
Transcrição Reversa
Taq Polimerase/genética
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
42Z2K6ZL8P (Manganese); 63231-63-0 (RNA); 9007-49-2 (DNA); EC 2.7.7.- (DNA Polymerase I); EC 2.7.7.- (Taq Polymerase)
[Em] Mês de entrada:1709
[Cu] Atualização por classe:170919
[Lr] Data última revisão:
170919
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:170205
[St] Status:MEDLINE
[do] DOI:10.1002/cbic.201600701


  7 / 1726 MEDLINE  
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[PMID]:28157593
[Au] Autor:Milelli A; Marchetti C; Greco ML; Moraca F; Costa G; Turrini E; Catanzaro E; Betari N; Calcabrini C; Sissi C; Alcaro S; Fimognari C; Tumiatti V; Minarini A
[Ad] Endereço:Department for Life Quality Studies, Alma Mater Studiorum-University of Bologna, 47921 Rimini, Italy. Electronic address: andrea.milelli3@unibo.it.
[Ti] Título:Naphthalene diimide-polyamine hybrids as antiproliferative agents: Focus on the architecture of the polyamine chains.
[So] Source:Eur J Med Chem;128:107-122, 2017 Mar 10.
[Is] ISSN:1768-3254
[Cp] País de publicação:France
[La] Idioma:eng
[Ab] Resumo:Naphthalene diimides (NDIs) have been widely used as scaffold to design DNA-directed agents able to target peculiar DNA secondary arrangements endowed with relevant biochemical roles. Recently, we have reported disubstituted linear- and macrocyclic-NDIs that bind telomeric and non-telomeric G-quadruplex with high degree of affinity and selectivity. Herein, the synthesis, biological evaluation and molecular modelling studies of a series of asymmetrically substituted NDIs are reported. Among these, compound 9 emerges as the most interesting of the series being able to bind telomeric G-quadruplex (ΔTm = 29 °C at 2.5 µM), to inhibit the activity of DNA processing enzymes, such as topoisomerase II and TAQ-polymerase, and to exert antiproliferative effects in the NCI panel of cancer cell lines with GI values in the micro-to nanomolar concentration range (i.e. SR cell line, GI = 76 nM). Molecular mechanisms of cell death have been investigated and molecular modelling studies have been performed in order to shed light on the antiproliferative and DNA-recognition processes.
[Mh] Termos MeSH primário: Antineoplásicos/farmacologia
Sobrevivência Celular/efeitos dos fármacos
Imidas/química
Naftalenos/química
Fenantrolinas/farmacologia
Poliaminas/química
[Mh] Termos MeSH secundário: Ciclo Celular/efeitos dos fármacos
DNA/química
DNA Topoisomerases Tipo II/química
Ensaios de Seleção de Medicamentos Antitumorais
Citometria de Fluxo
Quadruplex G
Seres Humanos
Células Jurkat
Modelos Moleculares
Neoplasias/tratamento farmacológico
Neoplasias/patologia
Taq Polimerase/antagonistas & inibidores
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (2-(3-((4-aminobutyl )amino)propyl )-7-(3-((2-methoxybenzyl)amino)propyl)benzo(lmn)(3,8)phenanthroline-1,3,6,8(2H,7H)-tetraone); 0 (Antineoplastic Agents); 0 (Imides); 0 (Naphthalenes); 0 (Phenanthrolines); 0 (Polyamines); 22291-04-9 (naphthalenediimide); 9007-49-2 (DNA); EC 2.7.7.- (Taq Polymerase); EC 5.99.1.3 (DNA Topoisomerases, Type II)
[Em] Mês de entrada:1704
[Cu] Atualização por classe:170418
[Lr] Data última revisão:
170418
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:170204
[St] Status:MEDLINE


  8 / 1726 MEDLINE  
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[PMID]:28127668
[Au] Autor:Zoccola R; Mazzei M; Carrozza ML; Ricci E; Forzan M; Pizzurro F; Giammarioli M; Bandecchi P; Tolari F
[Ad] Endereço:Department of Veterinary Sciences, University of Pisa, 56124, Pisa, Italy.
[Ti] Título:A newly developed BVDV-1 RT-qPCR Taqman assay based on Italian isolates: evaluation as a diagnostic tool.
[So] Source:Folia Microbiol (Praha);62(4):279-286, 2017 Jul.
[Is] ISSN:1874-9356
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:A single-step TaqMan® RT-qPCR was developed for the detection of bovine viral diarrhea virus type 1 (BVDV-1), an important pathogen of cattle worldwide. The assay was based on conserved 5'UTR sequences of Italian BVDV-1 isolates. In order to establish a diagnostic protocol which simplifies sample collection and processing, the assay was tested on a variety of biological specimens collected from persistently infected calves. The samples analyzed included PBMCs, plasma, dry blood, ear notch and hair bulb. Time and costs required for the analysis of each type of specimen were compared. The RT-qPCR, whose lower limit of detection was 100 copies of viral RNA (1 TCID50), correctly identified all PI animals, irrespective of the type of specimen. The highest copy numbers were obtained from the RNAs extracted from PBMCs, ear notches and hair bulbs. Hair bulb-supernatants directly used as a template allowed identification of all PI animals. In conclusion, based on time and cost evaluation, the most effective and efficient protocol was the one based on the direct analysis of hair bulb-supernatants, avoiding the RNA extraction step.
[Mh] Termos MeSH primário: Doença das Mucosas por Vírus da Diarreia Viral Bovina/diagnóstico
Doença das Mucosas por Vírus da Diarreia Viral Bovina/virologia
Vírus da Diarreia Viral Bovina Tipo 1/isolamento & purificação
Reação em Cadeia da Polimerase em Tempo Real/métodos
[Mh] Termos MeSH secundário: Animais
Bovinos
Vírus da Diarreia Viral Bovina Tipo 1/classificação
Vírus da Diarreia Viral Bovina Tipo 1/genética
Itália
RNA Viral/genética
Taq Polimerase/genética
Taq Polimerase/metabolismo
[Pt] Tipo de publicação:EVALUATION STUDIES; JOURNAL ARTICLE
[Nm] Nome de substância:
0 (RNA, Viral); EC 2.7.7.- (Taq Polymerase)
[Em] Mês de entrada:1708
[Cu] Atualização por classe:171109
[Lr] Data última revisão:
171109
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:170128
[St] Status:MEDLINE
[do] DOI:10.1007/s12223-017-0497-8


  9 / 1726 MEDLINE  
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[PMID]:28115632
[Au] Autor:Lei C; Li SY; Liu JK; Zheng X; Zhao GP; Wang J
[Ad] Endereço:Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China.
[Ti] Título:The CCTL (Cpf1-assisted Cutting and Taq DNA ligase-assisted Ligation) method for efficient editing of large DNA constructs in vitro.
[So] Source:Nucleic Acids Res;45(9):e74, 2017 May 19.
[Is] ISSN:1362-4962
[Cp] País de publicação:England
[La] Idioma:eng
[Ab] Resumo:As Cpf1 cleaves double-stranded DNA in a staggered way, it can be used in DNA assembly. However, the Cpf1 cleavage was found to be inaccurate, which may cause errors in DNA assembly. Here, the Cpf1 cleavage sites were precisely characterized, where the cleavage site on the target strand was around the 22nd base relative to the protospacer adjacent motif site, but the cleavage on the non-target strand was affected by the spacer length. When the spacer length was 20 nt or longer, Cpf1 mainly cleaved around the 14th and the 18th bases on the non-target strand; otherwise, with a shorter spacer (i.e. 17-19 nt), Cpf1 mainly cleaved after the 14th base, generating 8-nt sticky ends. With this finding, Cpf1 with a 17-nt spacer crRNA were employed for in vitro substitution of the actII-orf4 promoter in the actinorhodin biosynthetic cluster with a constitutively expressing promoter. The engineered cluster yielded more actinorhodin and produced actinorhodin from an earlier phase. Moreover, Taq DNA ligase was further employed to increase both the ligation efficiency and the ligation accuracy of the method. We expect this CCTL (Cpf1-assisted Cutting and Taq DNA ligase-mediated Ligation) method can be widely used in in vitro editing of large DNA constructs.
[Mh] Termos MeSH primário: Proteínas Associadas a CRISPR/metabolismo
DNA/metabolismo
Taq Polimerase/metabolismo
[Mh] Termos MeSH secundário: Francisella/enzimologia
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (CRISPR-Associated Proteins); 9007-49-2 (DNA); EC 2.7.7.- (Taq Polymerase)
[Em] Mês de entrada:1708
[Cu] Atualização por classe:170829
[Lr] Data última revisão:
170829
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:170125
[St] Status:MEDLINE
[do] DOI:10.1093/nar/gkx018


  10 / 1726 MEDLINE  
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[PMID]:28063932
[Au] Autor:Hikida Y; Kimoto M; Hirao I; Yokoyama S
[Ad] Endereço:RIKEN Systems and Structural Biology Center, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan; Department of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan; RIKEN Structural Biology Laboratory, 1-7-22 Suehiro-cho,
[Ti] Título:Crystal structure of Deep Vent DNA polymerase.
[So] Source:Biochem Biophys Res Commun;483(1):52-57, 2017 Jan 29.
[Is] ISSN:1090-2104
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:DNA polymerases are useful tools in various biochemical experiments. We have focused on the DNA polymerases involved in DNA replication including the unnatural base pair between 7-(2-thienyl)imidazo[4,5-b]pyridine (Ds) and 2-nitro-4-propynylpyrrole (Px). Many reports have described the different combinations between unnatural base pairs and DNA polymerases. As an example, for the replication of the Ds-Px pair, Deep Vent DNA polymerase exhibits high efficiency and fidelity, but Taq DNA polymerase shows much lower efficiency and fidelity. In the present study, we determined the crystal structure of Deep Vent DNA polymerase in the apo form at 2.5 Å resolution. Using this structure, we constructed structural models of Deep Vent DNA polymerase complexes with DNA containing an unnatural or natural base in the replication position. The models revealed that the unnatural Ds base in the template-strand DNA clashes with the side-chain oxygen of Thr664 in Taq DNA polymerase, but not in Deep Vent DNA polymerase.
[Mh] Termos MeSH primário: DNA Polimerase Dirigida por DNA/química
[Mh] Termos MeSH secundário: Proteínas Arqueais/química
Pareamento de Bases
Sítios de Ligação
Cristalografia por Raios X
DNA/química
Modelos Moleculares
Pyrococcus/enzimologia
Homologia Estrutural de Proteína
Taq Polimerase/química
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Archaeal Proteins); 9007-49-2 (DNA); EC 2.7.7.- (Deep Vent DNA polymerase); EC 2.7.7.- (Taq Polymerase); EC 2.7.7.7 (DNA-Directed DNA Polymerase)
[Em] Mês de entrada:1706
[Cu] Atualização por classe:170605
[Lr] Data última revisão:
170605
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:170109
[St] Status:MEDLINE



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BIREME/OPAS/OMS - Centro Latino-Americano e do Caribe de Informação em Ciências da Saúde