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  1 / 922 MEDLINE  
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[PMID]:28968392
[Au] Autor:Sinha AK; Durand A; Desfontaines JM; Iurchenko I; Auger H; Leach DRF; Barre FX; Michel B
[Ad] Endereço:Bacterial DNA stability, Genome biology department, Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Université Paris-Sud, Université Paris-Saclay, Gif-sur-Yvette, France.
[Ti] Título:Division-induced DNA double strand breaks in the chromosome terminus region of Escherichia coli lacking RecBCD DNA repair enzyme.
[So] Source:PLoS Genet;13(10):e1006895, 2017 Oct.
[Is] ISSN:1553-7404
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:Marker frequency analysis of the Escherichia coli recB mutant chromosome has revealed a deficit of DNA in a specific zone of the terminus, centred on the dif/TerC region. Using fluorescence microscopy of a marked chromosomal site, we show that the dif region is lost after replication completion, at the time of cell division, in one daughter cell only, and that the phenomenon is transmitted to progeny. Analysis by marker frequency and microscopy shows that the position of DNA loss is not defined by the replication fork merging point since it still occurs in the dif/TerC region when the replication fork trap is displaced in strains harbouring ectopic Ter sites. Terminus DNA loss in the recB mutant is also independent of dimer resolution by XerCD at dif and of Topo IV action close to dif. It occurs in the terminus region, at the point of inversion of the GC skew, which is also the point of convergence of specific sequence motifs like KOPS and Chi sites, regardless of whether the convergence of GC skew is at dif (wild-type) or a newly created sequence. In the absence of FtsK-driven DNA translocation, terminus DNA loss is less precisely targeted to the KOPS convergence sequence, but occurs at a similar frequency and follows the same pattern as in FtsK+ cells. Importantly, using ftsIts, ftsAts division mutants and cephalexin treated cells, we show that DNA loss of the dif region in the recB mutant is decreased by the inactivation of cell division. We propose that it results from septum-induced chromosome breakage, and largely contributes to the low viability of the recB mutant.
[Mh] Termos MeSH primário: Cromossomos Bacterianos/genética
Quebras de DNA de Cadeia Dupla
Proteínas de Escherichia coli/genética
Escherichia coli/genética
Exodesoxirribonuclease V/genética
[Mh] Termos MeSH secundário: Divisão Celular
Reparo do DNA
Replicação do DNA
DNA Bacteriano/genética
Proteínas de Escherichia coli/metabolismo
Exodesoxirribonuclease V/metabolismo
Análise de Sequência de DNA
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (DNA, Bacterial); 0 (Escherichia coli Proteins); EC 3.1.11.5 (Exodeoxyribonuclease V); EC 3.1.11.5 (exodeoxyribonuclease V, E coli)
[Em] Mês de entrada:1710
[Cu] Atualização por classe:171031
[Lr] Data última revisão:
171031
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:171003
[St] Status:MEDLINE
[do] DOI:10.1371/journal.pgen.1006895


  2 / 922 MEDLINE  
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[PMID]:28475211
[Au] Autor:Marshall R; Maxwell CS; Collins SP; Beisel CL; Noireaux V
[Ad] Endereço:School of Physics and Astronomy, University of Minnesota, Minneapolis, Minnesota 55455.
[Ti] Título:Short DNA containing χ sites enhances DNA stability and gene expression in E. coli cell-free transcription-translation systems.
[So] Source:Biotechnol Bioeng;114(9):2137-2141, 2017 Sep.
[Is] ISSN:1097-0290
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:Escherichia coli cell-free transcription-translation (TXTL) systems offer versatile platforms for advanced biomanufacturing and for prototyping synthetic biological parts and devices. Production and testing could be accelerated with the use of linear DNA, which can be rapidly and cheaply synthesized. However, linear DNA is efficiently degraded in TXTL preparations from E. coli. Here, we show that double-stranded DNA encoding χ sites-eight base-pair sequences preferentially bound by the RecBCD recombination machinery-stabilizes linear DNA and greatly enhances the TXTL-based expression and activity of a fluorescent reporter gene, simple regulatory cascades, and T7 bacteriophage particles. The χ-site DNA and the DNA-binding λ protein Gam yielded similar enhancements, and DNA with as few as four χ sites was sufficient to ensure robust gene expression in TXTL. Given the affordability and scalability of producing the short χ-site DNA, this generalized strategy is expected to advance the broad use of TXTL systems across its many applications. Biotechnol. Bioeng. 2017;114: 2137-2141. © 2017 Wiley Periodicals, Inc.
[Mh] Termos MeSH primário: DNA Bacteriano/genética
Escherichia coli/genética
Exodesoxirribonuclease V/genética
Regulação Bacteriana da Expressão Gênica/genética
Engenharia Genética/métodos
Biossíntese de Proteínas/genética
Transcrição Genética/genética
[Mh] Termos MeSH secundário: Sistema Livre de Células/fisiologia
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (DNA, Bacterial); EC 3.1.11.5 (Exodeoxyribonuclease V)
[Em] Mês de entrada:1711
[Cu] Atualização por classe:171106
[Lr] Data última revisão:
171106
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:170506
[St] Status:MEDLINE
[do] DOI:10.1002/bit.26333


  3 / 922 MEDLINE  
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[PMID]:28369478
[Au] Autor:Laureti L; Lee L; Philippin G; Pagès V
[Ad] Endereço:Team DNA Damage Tolerance, Cancer Research Center of Marseille, CRCM, Aix Marseille univ, CNRS, inserm, institut Paoli-Calmettes, 13009 Marseille, France.
[Ti] Título:A non-catalytic role of RecBCD in homology directed gap repair and translesion synthesis.
[So] Source:Nucleic Acids Res;45(10):5877-5886, 2017 Jun 02.
[Is] ISSN:1362-4962
[Cp] País de publicação:England
[La] Idioma:eng
[Ab] Resumo:The RecBCD complex is a key factor in DNA metabolism. This protein complex harbors a processive nuclease and two helicases activities that give it the ability to process duplex DNA ends. These enzymatic activities make RecBCD a major player in double strand break repair, conjugational recombination and degradation of linear DNA. In this work, we unravel a new role of the RecBCD complex in the processing of DNA single-strand gaps that are generated at DNA replication-blocking lesions. We show that independently of its nuclease or helicase activities, the entire RecBCD complex is required for recombinational repair of the gap and efficient translesion synthesis. Since none of the catalytic functions of RecBCD are required for those processes, we surmise that the complex acts as a structural element that stabilizes the blocked replication fork, allowing efficient DNA damage tolerance.
[Mh] Termos MeSH primário: Replicação do DNA
Proteínas de Escherichia coli/genética
Escherichia coli/genética
Exodesoxirribonuclease V/genética
Reparo de DNA por Recombinação
[Mh] Termos MeSH secundário: Quebras de DNA de Cadeia Dupla
DNA Bacteriano/genética
DNA Bacteriano/metabolismo
DNA de Cadeia Simples/genética
DNA de Cadeia Simples/metabolismo
Escherichia coli/metabolismo
Proteínas de Escherichia coli/metabolismo
Exodesoxirribonuclease V/metabolismo
Deleção de Genes
Domínios Proteicos
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (DNA, Bacterial); 0 (DNA, Single-Stranded); 0 (Escherichia coli Proteins); EC 3.1.11.5 (Exodeoxyribonuclease V); EC 3.1.11.5 (exodeoxyribonuclease V, E coli)
[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:170404
[St] Status:MEDLINE
[do] DOI:10.1093/nar/gkx217


  4 / 922 MEDLINE  
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[PMID]:28202049
[Au] Autor:Hülter N; Sørum V; Borch-Pedersen K; Liljegren MM; Utnes AL; Primicerio R; Harms K; Johnsen PJ
[Ad] Endereço:Genomic Microbiology, Institute of Microbiology, Christian-Albrechts-University Kiel, Am Botanischen Garten 11, 24118, Kiel, Germany.
[Ti] Título:Costs and benefits of natural transformation in Acinetobacter baylyi.
[So] Source:BMC Microbiol;17(1):34, 2017 Feb 15.
[Is] ISSN:1471-2180
[Cp] País de publicação:England
[La] Idioma:eng
[Ab] Resumo:BACKGROUND: Natural transformation enables acquisition of adaptive traits and drives genome evolution in prokaryotes. Yet, the selective forces responsible for the evolution and maintenance of natural transformation remain elusive since taken-up DNA has also been hypothesized to provide benefits such as nutrients or templates for DNA repair to individual cells. RESULTS: We investigated the immediate effects of DNA uptake and recombination on the naturally competent bacterium Acinetobacter baylyi in both benign and genotoxic conditions. In head-to-head competition experiments between DNA uptake-proficient and -deficient strains, we observed a fitness benefit of DNA uptake independent of UV stress. This benefit was found with both homologous and heterologous DNA and was independent of recombination. Recombination with taken-up DNA reduced survival of transformed cells with increasing levels of UV-stress through interference with nucleotide excision repair, suggesting that DNA strand breaks occur during recombination attempts with taken-up DNA. Consistent with this, we show that absence of RecBCD and RecFOR recombinational DNA repair pathways strongly decrease natural transformation. CONCLUSIONS: Our data show a physiological benefit of DNA uptake unrelated to recombination. In contrast, recombination during transformation is a strand break inducing process that represents a previously unrecognized cost of natural transformation.
[Mh] Termos MeSH primário: Acinetobacter/genética
Acinetobacter/efeitos da radiação
Evolução Biológica
Análise Custo-Benefício
Transformação Bacteriana/genética
Transformação Bacteriana/efeitos da radiação
[Mh] Termos MeSH secundário: Acinetobacter/enzimologia
Acinetobacter/metabolismo
Proteínas de Bactérias/genética
Proteínas de Bactérias/metabolismo
Proteínas de Bactérias/efeitos da radiação
Dano ao DNA/efeitos da radiação
Reparo do DNA/fisiologia
Reparo do DNA/efeitos da radiação
DNA Bacteriano/genética
DNA Bacteriano/efeitos da radiação
Exodesoxirribonuclease V/metabolismo
Exodesoxirribonuclease V/efeitos da radiação
Deleção de Genes
Transferência Genética Horizontal/genética
Transferência Genética Horizontal/efeitos da radiação
Genes Bacterianos/genética
Genes Bacterianos/efeitos da radiação
Proteínas de Membrana/genética
Proteínas de Membrana/efeitos da radiação
Mutação/genética
Mutação/efeitos da radiação
Fenótipo
Recombinação Genética/efeitos da radiação
Estresse Fisiológico
Sobrevida
Raios Ultravioleta/efeitos adversos
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Bacterial Proteins); 0 (DNA, Bacterial); 0 (DprA protein, bacteria); 0 (Membrane Proteins); EC 3.1.11.5 (Exodeoxyribonuclease V)
[Em] Mês de entrada:1709
[Cu] Atualização por classe:170925
[Lr] Data última revisão:
170925
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:170217
[St] Status:MEDLINE
[do] DOI:10.1186/s12866-017-0953-2


  5 / 922 MEDLINE  
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[PMID]:27999939
[Au] Autor:Dawoud TM; Khatiwara A; Park SH; Davis ML; Baker CA; Ricke SC; Kwon YM
[Ad] Endereço:Cell and Molecular Biology Program, University of Arkansas, Fayetteville, AR, 72701, USA.
[Ti] Título:Heat Survival and Phenotype Microarray Profiling of Salmonella Typhimurium Mutants.
[So] Source:Curr Microbiol;74(2):257-267, 2017 Feb.
[Is] ISSN:1432-0991
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:Contamination of food products by pathogenic microorganisms continues to be a major public health and food industry concern. Non-typhoidal Salmonella species have led to numerous outbreaks associated with various foods. A wide variety of methods have been applied and introduced for treatment of fresh foods to eliminate pathogenic as well as spoilage microorganisms. Salmonella can become exposed to elevated temperatures while associated with hosts such as poultry. In addition, heat treatment is also applied at various stages of processing to retain the shelf life of food products. Despite this, these microorganisms may overcome exposure to such treatments through the efficient expression of stress response mechanisms and result in illness following consumption. Thermal stress induces a range of destructive exposures to bacterial cells such as protein damage and DNA damage caused by reactive oxygen species. In this study, we chose three genes (∆recD, ∆STM14_5307, and ∆aroD) associated with conditionally essential genes required for different aspects of optimal growth at 42 °C and evaluated the responses of wild type and mutant Salmonella Typhimurium strains to uncover potential mechanisms that may enable survival and resistance under thermal stress. The RecBCD complex that initiates repair of double-stranded DNA breaks through homologous recombination. STM14_5307 is a transcriptional regulator involved in stationary phase growth and inositol metabolism. The gene aroD is involved in metabolism and stationary phase growth. These strains were characterized via high throughput phenotypic profiling in response to two different growth temperatures (37 °C (human host temperature) and 42 °C (poultry host temperature)). The ∆aroD strain exhibited the highest sensitivity to the various temperatures followed by the ∆recD and ∆STM14_5307 strains, respectively. Achieving more understanding of the molecular mechanisms of heat survival may lead to the development of more effective strategies to limit Salmonella in food products through thermal treatment by developing interventions that specifically target the pathways these genes are involved in.
[Mh] Termos MeSH primário: Viabilidade Microbiana/efeitos da radiação
Fenótipo
Salmonella typhimurium/fisiologia
Salmonella typhimurium/efeitos da radiação
Temperatura Ambiente
[Mh] Termos MeSH secundário: Proteínas de Bactérias/genética
Exodesoxirribonuclease V/genética
Deleção de Genes
Genes Reguladores
Seres Humanos
Salmonella typhimurium/genética
Estresse Fisiológico
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Bacterial Proteins); EC 3.1.11.5 (Exodeoxyribonuclease V)
[Em] Mês de entrada:1702
[Cu] Atualização por classe:170926
[Lr] Data última revisão:
170926
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:161222
[St] Status:MEDLINE
[do] DOI:10.1007/s00284-016-1170-1


  6 / 922 MEDLINE  
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[PMID]:28009252
[Au] Autor:Wilkinson M; Troman L; Wan Nur Ismah WA; Chaban Y; Avison MB; Dillingham MS; Wigley DB
[Ad] Endereço:Department of Medicine, Section of Structural Biology, Imperial College London, London, United Kingdom.
[Ti] Título:Structural basis for the inhibition of RecBCD by Gam and its synergistic antibacterial effect with quinolones.
[So] Source:Elife;5, 2016 Dec 23.
[Is] ISSN:2050-084X
[Cp] País de publicação:England
[La] Idioma:eng
[Ab] Resumo:Our previous paper (Wilkinson , 2016) used high-resolution cryo-electron microscopy to solve the structure of the RecBCD complex, which acts in both the repair of double-stranded DNA breaks and the degradation of bacteriophage DNA. To counteract the latter activity, bacteriophage λ encodes a small protein inhibitor called Gam that binds to RecBCD and inactivates the complex. Here, we show that Gam inhibits RecBCD by competing at the DNA-binding site. The interaction surface is extensive and involves molecular mimicry of the DNA substrate. We also show that expression of Gam in or increases sensitivity to fluoroquinolones; antibacterials that kill cells by inhibiting topoisomerases and inducing double-stranded DNA breaks. Furthermore, fluoroquinolone-resistance in clinical isolates is reversed by expression of Gam. Together, our data explain the synthetic lethality observed between topoisomerase-induced DNA breaks and the RecBCD gene products, suggesting a new co-antibacterial strategy.
[Mh] Termos MeSH primário: Antibacterianos/farmacologia
Proteínas de Ligação a DNA/metabolismo
Sinergismo Farmacológico
Escherichia coli/enzimologia
Exodesoxirribonuclease V/antagonistas & inibidores
Klebsiella pneumoniae/enzimologia
Quinolonas/farmacologia
Proteínas Virais/metabolismo
[Mh] Termos MeSH secundário: Bacteriófago lambda/enzimologia
Proteínas de Ligação a DNA/genética
Escherichia coli/efeitos dos fármacos
Escherichia coli/genética
Klebsiella pneumoniae/efeitos dos fármacos
Klebsiella pneumoniae/genética
Proteínas Virais/genética
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Anti-Bacterial Agents); 0 (DNA-Binding Proteins); 0 (Quinolones); 0 (Viral Proteins); 0 (gam protein, Coliphage); EC 3.1.11.5 (Exodeoxyribonuclease V)
[Em] Mês de entrada:1710
[Cu] Atualização por classe:171010
[Lr] Data última revisão:
171010
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:161224
[St] Status:MEDLINE


  7 / 922 MEDLINE  
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[PMID]:27711223
[Au] Autor:Darrigo C; Guillemet E; Dervyn R; Ramarao N
[Ad] Endereço:Micalis Institute, INRA, AgroParisTech, Université Paris-Saclay, 78350 Jouy-en-Josas, France.
[Ti] Título:The Bacterial Mfd Protein Prevents DNA Damage Induced by the Host Nitrogen Immune Response in a NER-Independent but RecBC-Dependent Pathway.
[So] Source:PLoS One;11(10):e0163321, 2016.
[Is] ISSN:1932-6203
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:Production of reactive nitrogen species is an important component of the host immune defence against bacteria. Here, we show that the bacterial protein Mfd (Mutation frequency decline), a highly conserved and ubiquitous bacterial protein involved in DNA repair, confers bacterial resistance to the eukaryotic nitrogen response produced by macrophage cells and during mice infection. In addition, we show that RecBC is also necessary to survive this stress. The inactivation of recBC and mfd genes is epistatic showing that Mfd follows the RecBC repair pathway to protect the bacteria against the genotoxic effect of nitrite. Surprisingly given the role of Mfd in transcription-coupled repair, UvrA is not necessary to survive the nitrite response. Taken together, our data reveal that during the eukaryotic nitrogen response, Mfd is required to maintain bacterial genome integrity in a NER-independent but RecBC-dependent pathway.
[Mh] Termos MeSH primário: Proteínas de Bactérias/metabolismo
Dano ao DNA
Reparo do DNA/efeitos dos fármacos
Exodesoxirribonuclease V/metabolismo
Interações Hospedeiro-Patógeno/genética
Interações Hospedeiro-Patógeno/imunologia
Nitrogênio/farmacologia
Fatores de Transcrição/metabolismo
[Mh] Termos MeSH secundário: Animais
Bacillus cereus/efeitos dos fármacos
Bacillus cereus/genética
Bacillus cereus/fisiologia
Proteínas de Bactérias/genética
Células HeLa
Seres Humanos
Camundongos
Viabilidade Microbiana
Mutação
Óxido Nítrico/metabolismo
Células RAW 264.7
Fatores de Transcrição/deficiência
Fatores de Transcrição/genética
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Bacterial Proteins); 0 (Transcription Factors); 0 (transcription repair coupling factor protein, Bacteria); 31C4KY9ESH (Nitric Oxide); EC 3.1.11.5 (Exodeoxyribonuclease V); N762921K75 (Nitrogen)
[Em] Mês de entrada:1706
[Cu] Atualização por classe:170620
[Lr] Data última revisão:
170620
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:161007
[St] Status:MEDLINE
[do] DOI:10.1371/journal.pone.0163321


  8 / 922 MEDLINE  
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[PMID]:27401752
[Au] Autor:Amundsen SK; Sharp JW; Smith GR
[Ad] Endereço:Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, Washington 98109.
[Ti] Título:RecBCD Enzyme "Chi Recognition" Mutants Recognize Chi Recombination Hotspots in the Right DNA Context.
[So] Source:Genetics;204(1):139-52, 2016 Sep.
[Is] ISSN:1943-2631
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:RecBCD enzyme is a complex, three-subunit protein machine essential for the major pathway of DNA double-strand break repair and homologous recombination in Escherichia coli Upon encountering a Chi recombination-hotspot during DNA unwinding, RecBCD nicks DNA to produce a single-stranded DNA end onto which it loads RecA protein. Conformational changes that regulate RecBCD's helicase and nuclease activities are induced upon its interaction with Chi, defined historically as 5' GCTGGTGG 3'. Chi is thought to be recognized as single-stranded DNA passing through a tunnel in RecC. To define the Chi recognition-domain in RecC and thus the mechanism of the RecBCD-Chi interaction, we altered by random mutagenesis eight RecC amino acids lining the tunnel. We screened for loss of Chi activity with Chi at one site in bacteriophage λ. The 25 recC mutants analyzed thoroughly had undetectable or strongly reduced Chi-hotspot activity with previously reported Chi sites. Remarkably, most of these mutants had readily detectable, and some nearly wild-type, activity with Chi at newly generated Chi sites. Like wild-type RecBCD, these mutants had Chi activity that responded dramatically (up to fivefold, equivalent to Chi's hotspot activity) to nucleotide changes flanking 5' GCTGGTGG 3'. Thus, these and previously published RecC mutants thought to be Chi-recognition mutants are actually Chi context-dependence mutants. Our results fundamentally alter the view that Chi is a simple 8-bp sequence recognized by the RecC tunnel. We propose that Chi hotspots have dual nucleotide sequence interactions, with both the RecC tunnel and the RecB nuclease domain.
[Mh] Termos MeSH primário: DNA/genética
Exodesoxirribonuclease V/genética
[Mh] Termos MeSH secundário: Sequência de Bases
Sítios de Ligação
DNA/metabolismo
Quebras de DNA de Cadeia Dupla
DNA Helicases/genética
DNA Helicases/metabolismo
Reparo do DNA
DNA Bacteriano/genética
DNA Bacteriano/metabolismo
Endonucleases/metabolismo
Escherichia coli/genética
Escherichia coli/metabolismo
Proteínas de Escherichia coli/genética
Proteínas de Escherichia coli/metabolismo
Exodesoxirribonuclease V/química
Exodesoxirribonuclease V/metabolismo
Modelos Moleculares
Recombinases Rec A/genética
Recombinação Genética
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (DNA, Bacterial); 0 (Escherichia coli Proteins); 9007-49-2 (DNA); EC 2.7.7.- (Rec A Recombinases); EC 3.1.- (Endonucleases); EC 3.1.11.5 (Exodeoxyribonuclease V); EC 3.6.4.- (DNA Helicases)
[Em] Mês de entrada:1706
[Cu] Atualização por classe:170902
[Lr] Data última revisão:
170902
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:160713
[St] Status:MEDLINE
[do] DOI:10.1534/genetics.116.191056


  9 / 922 MEDLINE  
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[PMID]:27330137
[Au] Autor:Taylor AF; Amundsen SK; Smith GR
[Ad] Endereço:Division of Basic Sciences, Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue North, Seattle, WA 98109, USA.
[Ti] Título:Unexpected DNA context-dependence identifies a new determinant of Chi recombination hotspots.
[So] Source:Nucleic Acids Res;44(17):8216-28, 2016 Sep 30.
[Is] ISSN:1362-4962
[Cp] País de publicação:England
[La] Idioma:eng
[Ab] Resumo:Homologous recombination occurs especially frequently near special chromosomal sites called hotspots. In Escherichia coli, Chi hotspots control RecBCD enzyme, a protein machine essential for the major pathway of DNA break-repair and recombination. RecBCD generates recombinogenic single-stranded DNA ends by unwinding DNA and cutting it a few nucleotides to the 3' side of 5' GCTGGTGG 3', the sequence historically equated with Chi. To test if sequence context affects Chi activity, we deep-sequenced the products of a DNA library containing 10 random base-pairs on each side of the Chi sequence and cut by purified RecBCD. We found strongly enhanced cutting at Chi with certain preferred sequences, such as A or G at nucleotides 4-7, on the 3' flank of the Chi octamer. These sequences also strongly increased Chi hotspot activity in E. coli cells. Our combined enzymatic and genetic results redefine the Chi hotspot sequence, implicate the nuclease domain in Chi recognition, indicate that nicking of one strand at Chi is RecBCD's biologically important reaction in living cells, and enable more precise analysis of Chi's role in recombination and genome evolution.
[Mh] Termos MeSH primário: DNA/genética
Recombinação Genética
[Mh] Termos MeSH secundário: Sequência de Bases
Escherichia coli/efeitos dos fármacos
Escherichia coli/genética
Proteínas de Escherichia coli/metabolismo
Exodesoxirribonuclease V/metabolismo
Íons
Magnésio/farmacologia
Modelos Genéticos
Nucleotídeos/genética
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Escherichia coli Proteins); 0 (Ions); 0 (Nucleotides); 9007-49-2 (DNA); EC 3.1.11.5 (Exodeoxyribonuclease V); I38ZP9992A (Magnesium)
[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:160623
[St] Status:MEDLINE
[do] DOI:10.1093/nar/gkw541


  10 / 922 MEDLINE  
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[PMID]:27220465
[Au] Autor:Carter AR; Seaberg MH; Fan HF; Sun G; Wilds CJ; Li HW; Perkins TT
[Ad] Endereço:Department of Physics, Amherst College, Amherst, MA 01002, USA.
[Ti] Título:Sequence-dependent nanometer-scale conformational dynamics of individual RecBCD-DNA complexes.
[So] Source:Nucleic Acids Res;44(12):5849-60, 2016 Jul 08.
[Is] ISSN:1362-4962
[Cp] País de publicação:England
[La] Idioma:eng
[Ab] Resumo:RecBCD is a multifunctional enzyme that possesses both helicase and nuclease activities. To gain insight into the mechanism of its helicase function, RecBCD unwinding at low adenosine triphosphate (ATP) (2-4 µM) was measured using an optical-trapping assay featuring 1 base-pair (bp) precision. Instead of uniformly sized steps, we observed forward motion convolved with rapid, large-scale (∼4 bp) variations in DNA length. We interpret this motion as conformational dynamics of the RecBCD-DNA complex in an unwinding-competent state, arising, in part, by an enzyme-induced, back-and-forth motion relative to the dsDNA that opens and closes the duplex. Five observations support this interpretation. First, these dynamics were present in the absence of ATP. Second, the onset of the dynamics was coupled to RecBCD entering into an unwinding-competent state that required a sufficiently long 5' strand to engage the RecD helicase. Third, the dynamics were modulated by the GC-content of the dsDNA. Fourth, the dynamics were suppressed by an engineered interstrand cross-link in the dsDNA that prevented unwinding. Finally, these dynamics were suppressed by binding of a specific non-hydrolyzable ATP analog. Collectively, these observations show that during unwinding, RecBCD binds to DNA in a dynamic mode that is modulated by the nucleotide state of the ATP-binding pocket.
[Mh] Termos MeSH primário: DNA Bacteriano/química
DNA/química
Proteínas de Escherichia coli/química
Escherichia coli/genética
Exodesoxirribonuclease V/química
[Mh] Termos MeSH secundário: Difosfato de Adenosina/análogos & derivados
Difosfato de Adenosina/química
Difosfato de Adenosina/metabolismo
Trifosfato de Adenosina/química
Trifosfato de Adenosina/metabolismo
Adenilil Imidodifosfato/química
Adenilil Imidodifosfato/metabolismo
Sítios de Ligação
DNA/genética
DNA/metabolismo
DNA Bacteriano/genética
DNA Bacteriano/metabolismo
Escherichia coli/metabolismo
Proteínas de Escherichia coli/genética
Proteínas de Escherichia coli/metabolismo
Exodesoxirribonuclease V/genética
Exodesoxirribonuclease V/metabolismo
Expressão Gênica
Cinética
Conformação de Ácido Nucleico
Ligação Proteica
Conformação Proteica
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (DNA, Bacterial); 0 (Escherichia coli Proteins); 1492-61-1 (2-fluoro-ADP); 25612-73-1 (Adenylyl Imidodiphosphate); 61D2G4IYVH (Adenosine Diphosphate); 8L70Q75FXE (Adenosine Triphosphate); 9007-49-2 (DNA); EC 3.1.11.5 (Exodeoxyribonuclease V); EC 3.1.11.5 (exodeoxyribonuclease V, E coli)
[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:160526
[St] Status:MEDLINE
[do] DOI:10.1093/nar/gkw445



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