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  1 / 98 MEDLINE  
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[PMID]:26538601
[Au] Autor:Tóth J; Bollins J; Szczelkun MD
[Ad] Endereço:DNA-Protein Interactions Unit, School of Biochemistry, University of Bristol, Bristol BS8 1TD, UK.
[Ti] Título:Re-evaluating the kinetics of ATP hydrolysis during initiation of DNA sliding by Type III restriction enzymes.
[So] Source:Nucleic Acids Res;43(22):10870-81, 2015 Dec 15.
[Is] ISSN:1362-4962
[Cp] País de publicação:England
[La] Idioma:eng
[Ab] Resumo:DNA cleavage by the Type III restriction enzymes requires long-range protein communication between recognition sites facilitated by thermally-driven 1D diffusion. This 'DNA sliding' is initiated by hydrolysis of multiple ATPs catalysed by a helicase-like domain. Two distinct ATPase phases were observed using short oligoduplex substrates; the rapid consumption of ∼10 ATPs coupled to a protein conformation switch followed by a slower phase, the duration of which was dictated by the rate of dissociation from the recognition site. Here, we show that the second ATPase phase is both variable and only observable when DNA ends are proximal to the recognition site. On DNA with sites more distant from the ends, a single ATPase phase coupled to the conformation switch was observed and subsequent site dissociation required little or no further ATP hydrolysis. The overall DNA dissociation kinetics (encompassing site release, DNA sliding and escape via a DNA end) were not influenced by the second phase. Although the data simplifies the ATP hydrolysis scheme for Type III restriction enzymes, questions remain as to why multiple ATPs are hydrolysed to prepare for DNA sliding.
[Mh] Termos MeSH primário: Trifosfato de Adenosina/metabolismo
DNA/metabolismo
Desoxirribonucleases de Sítio Específico do Tipo III/metabolismo
[Mh] Termos MeSH secundário: Adenosina Trifosfatases/metabolismo
Clivagem do DNA
Hidrólise
Cinética
Ressonância de Plasmônio de Superfície
[Pt] Tipo de publicação:JOURNAL ARTICLE; RESEARCH SUPPORT, N.I.H., EXTRAMURAL; RESEARCH SUPPORT, NON-U.S. GOV'T
[Nm] Nome de substância:
8L70Q75FXE (Adenosine Triphosphate); 9007-49-2 (DNA); EC 3.1.21.- (DNA restriction enzyme EcoPI); EC 3.1.21.5 (Deoxyribonucleases, Type III Site-Specific); EC 3.6.1.- (Adenosine Triphosphatases)
[Em] Mês de entrada:1606
[Cu] Atualização por classe:161019
[Lr] Data última revisão:
161019
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:151106
[St] Status:MEDLINE
[do] DOI:10.1093/nar/gkv1154


  2 / 98 MEDLINE  
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[PMID]:25000732
[Au] Autor:Faidiuk IV; Tovkach EI
[Ti] Título:Phytopathogenic bacteria phenotype conversion as a result of their lysogenisation by coliphage P1.
[So] Source:Mikrobiol Z;76(2):59-66, 2014 Mar-Apr.
[Is] ISSN:1028-0987
[Cp] País de publicação:Ukraine
[La] Idioma:eng
[Ab] Resumo:A set of lysogenic strains of phytopathogenic bacteria Erwinia "horticola" and Erwinia amylovora associated with woody plants was obtained using bacteriophage P1 Cmc1ts100. The phenotype conversion from Cm(S) to Cm(R) was shown to be connected with introducing of authentic prophage DNA of 94.8 kb as a single-copy plasmid into the cells. Prophage state is unstable: P1 plasmid is spontaneously lost with high frequency by the cells. In lysogenic cells the prophage genes of type III restriction-modification complex EcoP1I are actively expressed. The system formed by E. "horticola" 450 and 60 as well as their lysogenic derivatives and specific bacteriophages provides an opportunity to divide the latter into three groups according to the level of restriction in the course of their interaction with the enzyme EcoP1I. The difference in phage responses to the endonuclease presence in a lysogenized host presumably correlates with the number of enzyme recognition sequences and the adsorption sites availability. After the prophage plasmid DNA curing the characteristic value of phage sensitivity of cells is changed. The lysogenic strains obtained in this work allow for the exploration of EcoP1I restriction-modification gene complex interaction with polyvalent phages able to grow not only on E. coli, but also on such phytopathogens as E. "horticola" and E. amylovora.
[Mh] Termos MeSH primário: Bacteriófago P1/genética
Erwinia amylovora/virologia
Erwinia/virologia
Genes Virais
Lisogenia/genética
Interações Microbianas/genética
[Mh] Termos MeSH secundário: DNA Viral
Desoxirribonucleases de Sítio Específico do Tipo III/genética
Desoxirribonucleases de Sítio Específico do Tipo III/metabolismo
Genótipo
Metiltransferases/genética
Metiltransferases/metabolismo
Fenótipo
Plantas/microbiologia
Plasmídeos
Prófagos/genética
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (DNA, Viral); EC 2.1.1.- (Methyltransferases); EC 3.1.21.- (endodeoxyribonuclease EcoP1); EC 3.1.21.5 (Deoxyribonucleases, Type III Site-Specific)
[Em] Mês de entrada:1407
[Cu] Atualização por classe:140708
[Lr] Data última revisão:
140708
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:140709
[St] Status:MEDLINE


  3 / 98 MEDLINE  
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[PMID]:24974681
[Au] Autor:Nakajima T; Ono K; Tazumi A; Misawa N; Moore JE; Millar BC; Matsuda M
[Ti] Título:Molecular characterisation of a type III restriction-modification system in Campylobacter upsaliensis.
[So] Source:Br J Biomed Sci;71(2):66-72, 2014.
[Is] ISSN:0967-4845
[Cp] País de publicação:England
[La] Idioma:eng
[Ab] Resumo:Two examples of Campylobacter upsaliensis RM3195 and JV21 strains are shown to carry putative type III restriction (res)-modification (mod) enzyme gene clusters, following genome sequence analyses. It is suggested that the cluster is composed of at least three structural genes, res, internal methylase gene and mod, in the strains, based on the nucleotide sequence information. A ribosome binding site, a putative promoter consisting of a consensus sequence at the -10-like structure and a semiconserved T-rich region and a putative intrinsic p-independent transcriptional terminator were identified for the gene cluster in the two strains. Using two primer pairs, f-/r-res and f-/r-mod, 34 of 41 C. upsaliensis isolates generated two expected amplicons of the res and mod gene segments, and using another primer pair, the same number of isolates also generated an amplicon of the res and mod gene segments cluster, including the third internal methylase gene. Thus, C. upsaliensis isolates frequently carried putative type III R-M gene clusters, encoding the three enzymes. Interestingly, two possible overlaps were identified within the three tandem structural genes. In addition, the type III R-M gene cluster loci appear to be very similar among the C. upsaliensis isolates and very different from other thermophilic campylobacters.
[Mh] Termos MeSH primário: Campylobacter upsaliensis/enzimologia
Desoxirribonucleases de Sítio Específico do Tipo III/química
[Mh] Termos MeSH secundário: Sequência de Aminoácidos
Animais
Sequência de Bases
Campylobacter upsaliensis/genética
Campylobacter upsaliensis/isolamento & purificação
Clonagem Molecular
Desoxirribonucleases de Sítio Específico do Tipo III/genética
Desoxirribonucleases de Sítio Específico do Tipo III/isolamento & purificação
Dados de Sequência Molecular
[Pt] Tipo de publicação:JOURNAL ARTICLE; RESEARCH SUPPORT, NON-U.S. GOV'T
[Nm] Nome de substância:
EC 3.1.21.5 (Deoxyribonucleases, Type III Site-Specific)
[Em] Mês de entrada:1408
[Cu] Atualização por classe:140630
[Lr] Data última revisão:
140630
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:140701
[St] Status:MEDLINE


  4 / 98 MEDLINE  
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[PMID]:24510100
[Au] Autor:Butterer A; Pernstich C; Smith RM; Sobott F; Szczelkun MD; Tóth J
[Ad] Endereço:Biomolecular & Analytical Mass Spectrometry and Center for Proteomics (CFP-CeProMa), Department of Chemistry, University of Antwerp, Antwerp 2020, Belgium and DNA-Protein Interactions Unit, School of Biochemistry, University of Bristol, Bristol BS8 1TD, UK.
[Ti] Título:Type III restriction endonucleases are heterotrimeric: comprising one helicase-nuclease subunit and a dimeric methyltransferase that binds only one specific DNA.
[So] Source:Nucleic Acids Res;42(8):5139-50, 2014 Apr.
[Is] ISSN:1362-4962
[Cp] País de publicação:England
[La] Idioma:eng
[Ab] Resumo:Fundamental aspects of the biochemistry of Type III restriction endonucleases remain unresolved despite being characterized by numerous research groups in the past decades. One such feature is the subunit stoichiometry of these hetero-oligomeric enzyme complexes, which has important implications for the reaction mechanism. In this study, we present a series of results obtained by native mass spectrometry and size exclusion chromatography with multi-angle light scattering consistent with a 1:2 ratio of Res to Mod subunits in the EcoP15I, EcoPI and PstII complexes as the main holoenzyme species and a 1:1 stoichiometry of specific DNA (sDNA) binding by EcoP15I and EcoPI. Our data are also consistent with a model where ATP hydrolysis activated by recognition site binding leads to release of the enzyme from the site, dissociation from the substrate via a free DNA end and cleavage of the DNA. These results are discussed critically in the light of the published literature, aiming to resolve controversies and discuss consequences in terms of the reaction mechanism.
[Mh] Termos MeSH primário: Metilases de Modificação do DNA/metabolismo
DNA/metabolismo
Desoxirribonucleases de Sítio Específico do Tipo III/química
Desoxirribonucleases de Sítio Específico do Tipo III/metabolismo
[Mh] Termos MeSH secundário: Trifosfato de Adenosina/metabolismo
Metilases de Modificação do DNA/química
Holoenzimas/metabolismo
Multimerização Proteica
Subunidades Proteicas/química
Subunidades Proteicas/metabolismo
DNA Metiltransferases Sítio Específica (Adenina-Específica)/metabolismo
[Pt] Tipo de publicação:JOURNAL ARTICLE; RESEARCH SUPPORT, NON-U.S. GOV'T
[Nm] Nome de substância:
0 (Holoenzymes); 0 (Protein Subunits); 8L70Q75FXE (Adenosine Triphosphate); 9007-49-2 (DNA); EC 2.1.1.- (DNA Modification Methylases); EC 2.1.1.- (DNA modification methylase EcoP15I); EC 2.1.1.- (DNA modification methylase EcoPI); EC 2.1.1.72 (Site-Specific DNA-Methyltransferase (Adenine-Specific)); EC 3.1.21.- (DNA restriction enzyme EcoPI); EC 3.1.21.- (endodeoxyribonuclease EcoP15I); EC 3.1.21.5 (Deoxyribonucleases, Type III Site-Specific)
[Em] Mês de entrada:1407
[Cu] Atualização por classe:170220
[Lr] Data última revisão:
170220
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:140211
[St] Status:MEDLINE
[do] DOI:10.1093/nar/gku122


  5 / 98 MEDLINE  
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[PMID]:24423871
[Au] Autor:Ghosh A; Passaris I; Tesfazgi Mebrhatu M; Rocha S; Vanoirbeek K; Hofkens J; Aertsen A
[Ad] Endereço:Department of Microbial and Molecular Systems (M2S), Laboratory of Food Microbiology, KU Leuven, B-3001 Leuven, Belgium and Department of Chemistry, KU Leuven, B-3001 Leuven, Belgium.
[Ti] Título:Cellular localization and dynamics of the Mrr type IV restriction endonuclease of Escherichia coli.
[So] Source:Nucleic Acids Res;42(6):3908-18, 2014 Apr.
[Is] ISSN:1362-4962
[Cp] País de publicação:England
[La] Idioma:eng
[Ab] Resumo:In this study, we examined the intracellular whereabouts of Mrr, a cryptic type IV restriction endonuclease of Escherichia coli K12, in response to different conditions. In absence of stimuli triggering its activity, Mrr was found to be strongly associated with the nucleoid as a number of discrete foci, suggesting the presence of Mrr hotspots on the chromosome. Previously established elicitors of Mrr activity, such as exposure to high (hydrostatic) pressure (HP) or expression of the HhaII methyltransferase, both caused nucleoid condensation and an unexpected coalescence of Mrr foci. However, although the resulting Mrr/nucleoid complex was stable when triggered with HhaII, it tended to be only short-lived when elicited with HP. Moreover, HP-mediated activation of Mrr typically led to cellular blebbing, suggesting a link between chromosome and cellular integrity. Interestingly, Mrr variants could be isolated that were specifically compromised in either HhaII- or HP-dependent activation, underscoring a mechanistic difference in the way both triggers activate Mrr. In general, our results reveal that Mrr can take part in complex spatial distributions on the nucleoid and can be engaged in distinct modes of activity.
[Mh] Termos MeSH primário: Enzimas de Restrição do DNA/análise
Enzimas de Restrição do DNA/metabolismo
Proteínas de Escherichia coli/análise
Proteínas de Escherichia coli/metabolismo
[Mh] Termos MeSH secundário: Enzimas de Restrição do DNA/genética
Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo
Desoxirribonucleases de Sítio Específico do Tipo III
Proteínas de Escherichia coli/genética
Pressão Hidrostática
Mutação
[Pt] Tipo de publicação:JOURNAL ARTICLE; RESEARCH SUPPORT, NON-U.S. GOV'T
[Nm] Nome de substância:
0 (Escherichia coli Proteins); EC 3.1.21.- (DNA Restriction Enzymes); EC 3.1.21.- (Mrr protein, E coli); EC 3.1.21.4 (Deoxyribonucleases, Type II Site-Specific); EC 3.1.21.4 (GANTC-specific type II deoxyribonucleases); EC 3.1.21.5 (Deoxyribonucleases, Type III Site-Specific)
[Em] Mês de entrada:1406
[Cu] Atualização por classe:150515
[Lr] Data última revisão:
150515
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:140116
[St] Status:MEDLINE
[do] DOI:10.1093/nar/gkt1370


  6 / 98 MEDLINE  
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[PMID]:24141096
[Au] Autor:Loenen WA; Dryden DT; Raleigh EA; Wilson GG; Murray NE
[Ad] Endereço:Leiden University Medical Center, Leiden, the Netherlands, EaStChemSchool of Chemistry, University of Edinburgh, West Mains Road, Edinburgh EH9 3JJ, Scotland, UK and New England Biolabs, Inc., 240 County Road, Ipswich, MA 01938, USA.
[Ti] Título:Highlights of the DNA cutters: a short history of the restriction enzymes.
[So] Source:Nucleic Acids Res;42(1):3-19, 2014 Jan.
[Is] ISSN:1362-4962
[Cp] País de publicação:England
[La] Idioma:eng
[Ab] Resumo:In the early 1950's, 'host-controlled variation in bacterial viruses' was reported as a non-hereditary phenomenon: one cycle of viral growth on certain bacterial hosts affected the ability of progeny virus to grow on other hosts by either restricting or enlarging their host range. Unlike mutation, this change was reversible, and one cycle of growth in the previous host returned the virus to its original form. These simple observations heralded the discovery of the endonuclease and methyltransferase activities of what are now termed Type I, II, III and IV DNA restriction-modification systems. The Type II restriction enzymes (e.g. EcoRI) gave rise to recombinant DNA technology that has transformed molecular biology and medicine. This review traces the discovery of restriction enzymes and their continuing impact on molecular biology and medicine.
[Mh] Termos MeSH primário: Enzimas de Restrição do DNA/história
[Mh] Termos MeSH secundário: Metilases de Modificação do DNA/história
Desoxirribonucleases de Sítio Específico do Tipo I/história
Desoxirribonucleases de Sítio Específico do Tipo II/história
Desoxirribonucleases de Sítio Específico do Tipo III/história
História do Século XX
[Pt] Tipo de publicação:HISTORICAL ARTICLE; JOURNAL ARTICLE
[Nm] Nome de substância:
EC 2.1.1.- (DNA Modification Methylases); EC 3.1.21.- (DNA Restriction Enzymes); EC 3.1.21.3 (Deoxyribonucleases, Type I Site-Specific); EC 3.1.21.4 (Deoxyribonucleases, Type II Site-Specific); EC 3.1.21.5 (Deoxyribonucleases, Type III Site-Specific)
[Em] Mês de entrada:1402
[Cu] Atualização por classe:170220
[Lr] Data última revisão:
170220
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:131022
[St] Status:MEDLINE
[do] DOI:10.1093/nar/gkt990


  7 / 98 MEDLINE  
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[PMID]:23863841
[Au] Autor:Rao DN; Dryden DT; Bheemanaik S
[Ad] Endereço:Department of Biochemistry, Indian Institute of Science, Bangalore 560 012, India and School of Chemistry, The King's Buildings, The University of Edinburgh, Edinburgh EH9 3JJ, Scotland, UK.
[Ti] Título:Type III restriction-modification enzymes: a historical perspective.
[So] Source:Nucleic Acids Res;42(1):45-55, 2014 Jan.
[Is] ISSN:1362-4962
[Cp] País de publicação:England
[La] Idioma:eng
[Ab] Resumo:Restriction endonucleases interact with DNA at specific sites leading to cleavage of DNA. Bacterial DNA is protected from restriction endonuclease cleavage by modifying the DNA using a DNA methyltransferase. Based on their molecular structure, sequence recognition, cleavage position and cofactor requirements, restriction-modification (R-M) systems are classified into four groups. Type III R-M enzymes need to interact with two separate unmethylated DNA sequences in inversely repeated head-to-head orientations for efficient cleavage to occur at a defined location (25-27 bp downstream of one of the recognition sites). Like the Type I R-M enzymes, Type III R-M enzymes possess a sequence-specific ATPase activity for DNA cleavage. ATP hydrolysis is required for the long-distance communication between the sites before cleavage. Different models, based on 1D diffusion and/or 3D-DNA looping, exist to explain how the long-distance interaction between the two recognition sites takes place. Type III R-M systems are found in most sequenced bacteria. Genome sequencing of many pathogenic bacteria also shows the presence of a number of phase-variable Type III R-M systems, which play a role in virulence. A growing number of these enzymes are being subjected to biochemical and genetic studies, which, when combined with ongoing structural analyses, promise to provide details for mechanisms of DNA recognition and catalysis.
[Mh] Termos MeSH primário: Desoxirribonucleases de Sítio Específico do Tipo III/metabolismo
[Mh] Termos MeSH secundário: Colífagos/enzimologia
Clivagem do DNA
Metilases de Modificação do DNA/genética
Desoxirribonucleases de Sítio Específico do Tipo III/química
Desoxirribonucleases de Sítio Específico do Tipo III/genética
Desoxirribonucleases de Sítio Específico do Tipo III/história
História do Século XX
História do Século XXI
[Pt] Tipo de publicação:HISTORICAL ARTICLE; JOURNAL ARTICLE; RESEARCH SUPPORT, NON-U.S. GOV'T; REVIEW
[Nm] Nome de substância:
EC 2.1.1.- (DNA Modification Methylases); EC 3.1.21.5 (Deoxyribonucleases, Type III Site-Specific)
[Em] Mês de entrada:1402
[Cu] Atualização por classe:150708
[Lr] Data última revisão:
150708
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:130719
[St] Status:MEDLINE
[do] DOI:10.1093/nar/gkt616


  8 / 98 MEDLINE  
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[PMID]:23599494
[Au] Autor:Schwarz FW; Tóth J; van Aelst K; Cui G; Clausing S; Szczelkun MD; Seidel R
[Ad] Endereço:DNA motors group, Biotechnology Center, Technische Universität Dresden, 01062 Dresden, Germany.
[Ti] Título:The helicase-like domains of type III restriction enzymes trigger long-range diffusion along DNA.
[So] Source:Science;340(6130):353-6, 2013 Apr 19.
[Is] ISSN:1095-9203
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:Helicases are ubiquitous adenosine triphosphatases (ATPases) with widespread roles in genome metabolism. Here, we report a previously undescribed functionality for ATPases with helicase-like domains; namely, that ATP hydrolysis can trigger ATP-independent long-range protein diffusion on DNA in one dimension (1D). Specifically, using single-molecule fluorescence microscopy we show that the Type III restriction enzyme EcoP15I uses its ATPase to switch into a distinct structural state that diffuses on DNA over long distances and long times. The switching occurs only upon binding to the target site and requires hydrolysis of ~30 ATPs. We define the mechanism for these enzymes and show how ATPase activity is involved in DNA target site verification and 1D signaling, roles that are common in DNA metabolism: for example, in nucleotide excision and mismatch repair.
[Mh] Termos MeSH primário: Trifosfato de Adenosina/metabolismo
Clivagem do DNA
DNA Helicases/metabolismo
DNA/metabolismo
Desoxirribonucleases de Sítio Específico do Tipo III/metabolismo
[Mh] Termos MeSH secundário: DNA/química
DNA Helicases/química
Desoxirribonucleases de Sítio Específico do Tipo III/química
Hidrólise
Microscopia de Fluorescência/métodos
Conformação de Ácido Nucleico
Estrutura Terciária de Proteína
[Pt] Tipo de publicação:JOURNAL ARTICLE; RESEARCH SUPPORT, NON-U.S. GOV'T
[Nm] Nome de substância:
8L70Q75FXE (Adenosine Triphosphate); 9007-49-2 (DNA); EC 3.1.21.- (endodeoxyribonuclease EcoP15I); EC 3.1.21.5 (Deoxyribonucleases, Type III Site-Specific); EC 3.6.4.- (DNA Helicases)
[Em] Mês de entrada:1304
[Cu] Atualização por classe:170922
[Lr] Data última revisão:
170922
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:130420
[St] Status:MEDLINE
[do] DOI:10.1126/science.1231122


  9 / 98 MEDLINE  
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[PMID]:23220200
[Au] Autor:Mackeldanz P; Alves J; Möncke-Buchner E; Wyszomirski KH; Krüger DH; Reuter M
[Ad] Endereço:Institute of Medical Virology, Helmut-Ruska-Haus, Charité - Universitätsmedizin Berlin, Charitéplatz 1, 10117 Berlin, Germany.
[Ti] Título:Functional consequences of mutating conserved SF2 helicase motifs in the Type III restriction endonuclease EcoP15I translocase domain.
[So] Source:Biochimie;95(4):817-23, 2013 Apr.
[Is] ISSN:1638-6183
[Cp] País de publicação:France
[La] Idioma:eng
[Ab] Resumo:For efficient DNA hydrolysis, Type III restriction endonuclease EcoP15I interacts with two inversely oriented recognition sites in an ATP-dependent process. EcoP15I consists of two methylation (Mod) subunits and a single restriction (Res) subunit yielding a multifunctional enzyme complex able to methylate or to hydrolyse DNA. Comprehensive sequence alignments, limited proteolysis and mass spectroscopy suggested that the Res subunit is a fusion of a motor or translocase (Tr) domain of superfamily II helicases and an endonuclease domain with a catalytic PD…EXK motif. In the Tr domain, seven predicted helicase motifs (I, Ia, II-VI), a recently discovered Q-tip motif and three additional regions (IIIa, IVa, Va) conserved among Type III restriction enzymes have been identified that are predicted to be involved in DNA binding and ATP hydrolysis. Because DNA unwinding activity for EcoP15I (as for bona fide helicases) has never been found and EcoP15I ATPase rates are only low, the functional importance of the helicase motifs and regions was questionable and has never been probed systematically. Therefore, we mutated all helicase motifs and conserved regions predicted in Type III restriction enzyme EcoP15I and examined the functional consequences on EcoP15I enzyme activity and the structural integrity of the variants by CD spectroscopy. The resulting eleven enzyme variants all, except variant IVa, are properly folded showing the same secondary structure distribution as the wild-type enzyme. Classical helicase motifs I-VI are important for ATP and DNA cleavage by EcoP15I and mutations therein led to complete loss of ATPase and cleavage activity. Among the catalytically inactive enzyme variants three preserved the ability to bind ATP. In contrast, newly assigned motifs Q-tip, Ia and Va are not essential for EcoP15I activity and the corresponding enzyme variants were still catalytically active. DNA binding was only marginally reduced (2-7 fold) in all enzyme variants tested.
[Mh] Termos MeSH primário: Sequência Conservada
DNA Helicases/química
Desoxirribonucleases de Sítio Específico do Tipo III/química
Desoxirribonucleases de Sítio Específico do Tipo III/metabolismo
Proteínas Mutantes/química
Proteínas Mutantes/metabolismo
Mutação
[Mh] Termos MeSH secundário: Motivos de Aminoácidos
Sequência de Aminoácidos
Desoxirribonucleases de Sítio Específico do Tipo III/genética
Modelos Moleculares
Dados de Sequência Molecular
Mutagênese
Proteínas Mutantes/genética
Estrutura Terciária de Proteína
[Pt] Tipo de publicação:JOURNAL ARTICLE; RESEARCH SUPPORT, NON-U.S. GOV'T
[Nm] Nome de substância:
0 (Mutant Proteins); EC 3.1.21.5 (Deoxyribonucleases, Type III Site-Specific); EC 3.6.4.- (DNA Helicases)
[Em] Mês de entrada:1308
[Cu] Atualização por classe:131121
[Lr] Data última revisão:
131121
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:121211
[St] Status:MEDLINE


  10 / 98 MEDLINE  
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[PMID]:22523084
[Au] Autor:Tóth J; van Aelst K; Salmons H; Szczelkun MD
[Ad] Endereço:DNA-Protein Interactions Unit, School of Biochemistry, Medical Sciences Building, University of Bristol, Bristol, BS8 1TD, UK.
[Ti] Título:Dissociation from DNA of Type III Restriction-Modification enzymes during helicase-dependent motion and following endonuclease activity.
[So] Source:Nucleic Acids Res;40(14):6752-64, 2012 Aug.
[Is] ISSN:1362-4962
[Cp] País de publicação:England
[La] Idioma:eng
[Ab] Resumo:DNA cleavage by the Type III Restriction-Modification (RM) enzymes requires the binding of a pair of RM enzymes at two distant, inversely orientated recognition sequences followed by helicase-catalysed ATP hydrolysis and long-range communication. Here we addressed the dissociation from DNA of these enzymes at two stages: during long-range communication and following DNA cleavage. First, we demonstrated that a communicating species can be trapped in a DNA domain without a recognition site, with a non-specific DNA association lifetime of ∼ 200 s. If free DNA ends were present the lifetime became too short to measure, confirming that ends accelerate dissociation. Secondly, we observed that Type III RM enzymes can dissociate upon DNA cleavage and go on to cleave further DNA molecules (they can 'turnover', albeit inefficiently). The relationship between the observed cleavage rate and enzyme concentration indicated independent binding of each site and a requirement for simultaneous interaction of at least two enzymes per DNA to achieve cleavage. In light of various mechanisms for helicase-driven motion on DNA, we suggest these results are most consistent with a thermally driven random 1D search model (i.e. 'DNA sliding').
[Mh] Termos MeSH primário: Clivagem do DNA
Desoxirribonucleases de Sítio Específico do Tipo III/metabolismo
[Mh] Termos MeSH secundário: DNA/metabolismo
DNA Helicases/metabolismo
Ensaios Enzimáticos/métodos
Cinética
Movimento (Física)
[Pt] Tipo de publicação:JOURNAL ARTICLE; RESEARCH SUPPORT, NON-U.S. GOV'T
[Nm] Nome de substância:
9007-49-2 (DNA); EC 3.1.21.- (DNA restriction enzyme EcoPI); EC 3.1.21.5 (Deoxyribonucleases, Type III Site-Specific); EC 3.6.4.- (DNA Helicases)
[Em] Mês de entrada:1211
[Cu] Atualização por classe:161019
[Lr] Data última revisão:
161019
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:120424
[St] Status:MEDLINE
[do] DOI:10.1093/nar/gks328



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