RESUMO
The emerging occurrence of antibiotic-resistant bacterial pathogens leads to a recollection of bacteriophage as antimicrobial therapeutics. This article presents a short overview of the clinical phage application including their use in military medicine and discusses the genotypic and phenotypic properties of a potential "ideal" therapeutic phage. We describe current efforts to engineer phage for their improved usability in pathogen treatment. In addition, phage can be applied for pathogen detection, selective drug delivery, vaccine development, or food and surface decontamination. Instead of viable phage, (engineered) phage-derived enzymes, such as polysaccharide depolymerases or peptidoglycan-degrading enzymes, are considered as promising therapeutic candidates. Finally, we briefly summarize the use of phage for the detection and treatment of "Category A priority pathogens".
Assuntos
Infecções Bacterianas/terapia , Bacteriófagos/genética , Terapia por Fagos , Antibacterianos/efeitos adversos , Bactérias/patogenicidade , Bactérias/virologia , Infecções Bacterianas/patologia , Infecções Bacterianas/virologia , Biofilmes , HumanosRESUMO
Bacterial biofilms, highly resistant to the conventional antimicrobial therapy, remain an unresolved challenge pressing the medical community to investigate new and alternative strategies to fight chronic implant-associated infections. Recently, strictly lytic bacteriophages have been revalued as powerful agents to kill antibiotic-resistant bacteria even in biofilm. Here, the interaction of T3 bacteriophage and planktonic and biofilm Escherichia coli TG1, respectively, was evaluated using isothermal microcalorimetry. Microcalorimetry is a non-invasive and highly sensitive technique measuring growth-related heat production of microorganisms in real-time. Planktonic and biofilm E. coli TG1 were exposed to different titers of T3 bacteriophage, ranging from 102 to 107 PFU/ml. The incubation of T3 with E. coli TG1 showed a strong inhibition of heat production both in planktonic and biofilm already at lower bacteriophage titers (103 PFU/ml). This method could be used to screen and evaluate the antimicrobial potential of different bacteriophages, alone and in combination with antibiotics in order to improve the treatment success of biofilm-associated infections.
Assuntos
Antibacterianos/farmacologia , Bacteriófago T3/fisiologia , Biofilmes/crescimento & desenvolvimento , Escherichia coli/fisiologia , Escherichia coli/virologia , Bacteriófago T3/patogenicidade , Calorimetria/métodos , Sistemas Computacionais , Testes de Sensibilidade MicrobianaRESUMO
The nucleocapsid (N) protein of hantaviruses represents an impressive example of a viral multifunctional protein. It encompasses properties as diverse as genome packaging, RNA chaperoning, intracellular protein transport, DNA degradation, intervention in host translation, and restricting host immune responses. These functions all rely on the capability of N to interact with RNA and other viral and cellular proteins. We have compiled data on the N protein of different hantavirus species together with information of the recently published three-dimensional structural data of the protein. The array of diverse functional activities accommodated in the hantaviral N protein goes far beyond to be a static structural protein and makes it an interesting target in the development of antiviral therapeutics.
Assuntos
Proteínas do Nucleocapsídeo/metabolismo , Orthohantavírus/fisiologia , Proteínas do Nucleocapsídeo/química , Conformação Proteica , Montagem de Vírus , Replicação ViralRESUMO
We demonstrate that the nucleocapsid protein of Sin Nombre hantavirus (SNV-N) has a DNA-specific endonuclease activity. Upon incubation of SNV-N with DNA in the presence of magnesium or manganese, we observed DNA digestion in sequence-unspecific manner. In contrast, RNA was not affected under the same conditions. Moreover, pre-treatment of SNV-N with RNase before DNA cleavage increased the endonucleolytic activity. Structure-based protein fold prediction using known structures from the PDB database revealed that Asp residues in positions 88 and 103 of SNV-N show sequence similarity with the active site of the restriction endonuclease HindIII. Crystal structure of HindIII predicts that residues Asp93 and Asp108 are essential for coordination of the metal ions required for HindIII DNA cleavage. Therefore, we hypothesized that homologous residues in SNV-N, Asp88 and Asp103, may have a similar function. Replacing Asp88 and Asp103 by alanine led to an SNV-N protein almost completely abrogated for endonuclease activity.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Endodesoxirribonucleases/metabolismo , Metais/metabolismo , Proteínas do Nucleocapsídeo/metabolismo , Vírus Sin Nombre/fisiologia , Sequência de Aminoácidos , Cátions/metabolismo , Clonagem Molecular , Sequência Consenso , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/isolamento & purificação , Endodesoxirribonucleases/química , Endodesoxirribonucleases/genética , Endodesoxirribonucleases/isolamento & purificação , Ativação Enzimática , Expressão Gênica , Modelos Moleculares , Conformação Molecular , Mutação , Proteínas do Nucleocapsídeo/química , Proteínas do Nucleocapsídeo/genética , Proteínas do Nucleocapsídeo/isolamento & purificação , Ligação Proteica , Proteínas Recombinantes de Fusão , Ribonucleases/metabolismo , Especificidade por SubstratoRESUMO
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.
Assuntos
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 , 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 ProteínaRESUMO
For efficient DNA cleavage, the Type III restriction endonuclease EcoP15I communicates with two inversely oriented recognition sites in an ATP-dependent process. EcoP15I consists of methylation (Mod) and restriction (Res) subunits forming a multifunctional enzyme complex able to methylate or to cleave DNA. In this study, we determined by different analytical methods that EcoP15I contains a single Res subunit in a Mod(2)Res stoichiometry. The Res subunit comprises a translocase (Tr) domain carrying functional motifs of superfamily 2 helicases and an endonuclease domain with a PD..D/EXK motif. We show that the isolated Tr domain retains ATP-hydrolyzing activity and binds single- and double-stranded DNA in a sequence-independent manner. To localize the regions of DNA binding, we screened peptide arrays representing the entire Res sequence for their ability to interact with DNA. We discovered four DNA-binding regions in the Tr domain and two DNA-binding regions in the endonuclease domain. Modelling of the Tr domain shows that these multiple DNA-binding regions are located on the surface, free to interact with DNA. Interestingly, the positions of the DNA-binding regions are conserved among other Type III restriction endonucleases.
Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Desoxirribonucleases de Sítio Específico do Tipo III/química , Desoxirribonucleases de Sítio Específico do Tipo III/metabolismo , Adenosina Trifosfatases/química , Trifosfato de Adenosina/metabolismo , Cromatografia em Gel , Clonagem Molecular , DNA/metabolismo , Desoxirribonucleases de Sítio Específico do Tipo III/genética , Hidrólise , Estrutura Terciária de Proteína , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , DNA Metiltransferases Sítio Específica (Adenina-Específica)/química , DNA Metiltransferases Sítio Específica (Adenina-Específica)/metabolismoRESUMO
Two novel viral genomes and four plasmids were assembled from an environmental sample collected from a hot spring at Yellowstone National Park, USA, and maintained anaerobically in a bioreactor at 85°C and pH 6. The double-stranded DNA viral genomes are linear (22.7 kb) and circular (17.7 kb), and derive apparently from archaeal viruses HAV1 and HAV2. Genomic DNA was obtained from samples enriched in filamentous and tadpole-shaped virus-like particles respectively. They yielded few significant matches in public sequence databases reinforcing, further, the wide diversity of archaeal viruses. Several variants of HAV1 exhibit major genomic alterations, presumed to arise from viral adaptation to different hosts. They include insertions up to 350 bp, deletions up to 1.5 kb, and genes with extensively altered sequences. Some result from recombination events occurring at low complexity direct repeats distributed along the genome. In addition, a 33.8 kb archaeal plasmid pHA1 was characterized, encoding a possible conjugative apparatus, as well as three cryptic plasmids of thermophilic bacterial origin, pHB1 of 2.1 kb and two closely related variants pHB2a and pHB2b, of 5.2 and 4.8 kb respectively. Strategies are considered for assembling genomes of smaller genetic elements from complex environmental samples, and for establishing possible host identities on the basis of sequence similarity to host CRISPR immune systems.
Assuntos
Vírus de Archaea , Bases de Dados de Ácidos Nucleicos , Fontes Termais/microbiologia , Fontes Termais/virologia , Metagenômica , Plasmídeos , Archaea/genética , Vírus de Archaea/classificação , Vírus de Archaea/genética , Vírus de Archaea/isolamento & purificação , Vírus de Archaea/ultraestrutura , Bactérias/genética , Sequência de Bases , DNA Intergênico , Variação Genética , Genoma Viral , Temperatura Alta , Sequências Repetidas Invertidas , Microscopia Eletrônica , Mutagênese Insercional , Plasmídeos/classificação , Plasmídeos/genética , Plasmídeos/isolamento & purificação , Sequências Repetitivas de Ácido Nucleico , Análise de Sequência de DNA , Deleção de Sequência , WyomingRESUMO
Restriction endonucleases of the PD...D/EXK family need Mg(2+) for DNA cleavage. Whereas Mg(2+) (or Mn(2+)) promotes catalysis, Ca(2+) (without Mg(2+)) only supports DNA binding. The role of Mg(2+) in DNA cleavage by restriction endonucleases has elicited many hypotheses, differing mainly in the number of Mg(2+) involved in catalysis. To address this problem, we measured the Mg(2+) and Mn(2+) concentration dependence of DNA cleavage by BamHI, BglII, Cfr10I, EcoRI, EcoRII (catalytic domain), MboI, NgoMIV, PspGI, and SsoII, which were reported in co-crystal structure analyses to bind one (BglII and EcoRI) or two (BamHI and NgoMIV) Me(2+) per active site. DNA cleavage experiments were carried out at various Mg(2+) and Mn(2+) concentrations at constant ionic strength. All enzymes show a qualitatively similar Mg(2+) and Mn(2+) concentration dependence. In general, the Mg(2+) concentration optimum (between approximately 1 and 10 mM) is higher than the Mn(2+) concentration optimum (between approximately 0.1 and 1 mM). At still higher Mg(2+) or Mn(2+) concentrations, the activities of all enzymes tested are reduced but can be reactivated by Ca(2+). Based on these results, we propose that one Mg(2+) or Mn(2+) is critical for restriction enzyme activation, and binding of a second Me(2+) plays a role in modulating the activity. Steady-state kinetics carried out with EcoRI and BamHI suggest that binding of a second Mg(2+) or Mn(2+) mainly leads to an increase in K(m), such that the inhibitory effect of excess Mg(2+) or Mn(2+) can be overcome by increasing the substrate concentration. Our conclusions are supported by molecular dynamics simulations and are consistent with the structural observations of both one and two Me(2+) binding to these enzymes.
Assuntos
Cátions/farmacologia , Coenzimas/farmacologia , Clivagem do DNA , Enzimas de Restrição do DNA/metabolismo , Magnésio/farmacologia , Manganês/farmacologia , CinéticaRESUMO
Human cytomegalovirus (HCMV) is a ubiquitous pathogen with a predilection for dendritic cells (DCs). Latently infected myeloid progenitor cells develop into actively infected DCs with impaired functionality, allowing dissemination and transfer of virus throughout the body. However, the viral genes expressed in DCs and their effect on the cellular transcriptome are currently unknown. We investigated human DCs infected with HCMV by using SuperSAGE, allowing us to analyse the transcriptomes of both host and pathogen simultaneously. A small number of viral transcripts were expressed strongly and rapidly post-infection. However, only two were of the immediate-early class, including one with an unknown function. The viral genes expressed reflected the cellular milieu, with the majority having a known or suspected immune-evasion function. Several viral genes identified lack a known function and may fulfil specialized roles within DCs. The cellular response to infection included a strong interferon response, induction of cytokine and anti-apoptotic genes and alterations in genes involved in antigen presentation. We demonstrated the validity of our approach by showing that novel changes first seen in the transcriptome were reflected in the phenotype of HCMV-infected DCs. Delineation of the transcriptional changes underlying the phenotype of HCMV-infected DCs allows a better understanding of how a herpesvirus infects DCs and pinpoints linkages between phenotype and specific viral genes.
Assuntos
Infecções por Citomegalovirus/genética , Citomegalovirus/genética , Células Dendríticas/virologia , Interações Hospedeiro-Patógeno , Células Cultivadas , Citomegalovirus/imunologia , Infecções por Citomegalovirus/imunologia , Infecções por Citomegalovirus/virologia , Células Dendríticas/imunologia , Regulação Viral da Expressão Gênica , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas Virais/genética , Proteínas Virais/imunologiaRESUMO
Bacterial restriction endonuclease EcoRII requires two recognition sites to cleave DNA. Proteolysis of EcoRII revealed the existence of two stable domains, EcoRII-N and EcoRII-C. Reduction of the enzyme to its C-terminal domain, EcoRII-C, unleashed the enzyme activity; this truncated form no longer needed two recognition sites and cleaved DNA much more efficiently than EcoRII wild-type. The crystal structure of EcoRII showed that probably the N-terminal domain sterically occludes the catalytic site, thus apparently controlling the cleavage activity. Based on these data, EcoRII was the first restriction endonuclease for which an autoinhibition mechanism as regulatory strategy was proposed. In this study, we probed this assumption and searched for the inhibitory element that mediates autoinhibition. Here we show that repression of EcoRII-C is achieved by addition of the inhibitory domain EcoRII-N or by single soluble peptides thereof in trans. Moreover, we perturbed contacts between the N- and the C-terminal domain of EcoRII by site-directed mutagenesis and proved that beta-strand B1 and alpha-helix H2 are essential for autoinhibition; deletion of either secondary structural element completely relieved EcoRII autoinhibition. This potent regulation principle that keeps EcoRII enzyme activity controlled might protect bacteria against suicidal restriction of rare unmodified recognition sites in the cellular genome.
Assuntos
Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Sequência de Aminoácidos , Domínio Catalítico , Dicroísmo Circular , DNA/metabolismo , Desoxirribonucleases de Sítio Específico do Tipo II/genética , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Mutagênese Sítio-Dirigida , Estrutura Terciária de Proteína , Especificidade por SubstratoRESUMO
EcoP15I is a Type III restriction endonuclease requiring the interaction with two inversely oriented 5'-CAGCAG recognition sites for efficient DNA cleavage. Diverse models have been developed to explain how enzyme complexes bound to both sites move toward each other, DNA translocation, DNA looping and simple diffusion along the DNA. Conflicting data also exist about the impact of cofactor S-adenosyl-L-methionine (AdoMet), the AdoMet analogue sinefungin and the bases flanking the DNA recognition sequence on EcoP15I enzyme activity. To clarify the functional role of these questionable parameters on EcoP15I activity and to optimize the enzymatic reaction, we investigated the influence of cofactors, ionic conditions, bases flanking the recognition sequence and enzyme concentration. We found that AdoMet is not necessary for DNA cleavage. Moreover, the presence of AdoMet dramatically impaired DNA cleavage due to competing DNA methylation. Sinefungin neither had an appreciable effect on DNA cleavage by EcoP15I nor compensated for the second recognition site. Moreover, we discovered that adenine stretches on the 5' or 3' side of CAGCAG led to preferred cleavage of this site. The length of the adenine stretch was pivotal and had to be different on the two sides for most efficient cleavage. In the absence of AdoMet and with enzyme in molar excess over recognition sites, we observed minor cleavage at two communicating DNA sites simultaneously. These results could also be exploited in the high-throughput, quantitative transcriptome analysis method SuperSAGE to optimize the crucial EcoP15I digestion step.
Assuntos
Desoxirribonucleases de Sítio Específico do Tipo III/química , Desoxirribonucleases de Sítio Específico do Tipo III/metabolismo , Adenosina/análogos & derivados , Adenosina/farmacologia , Sequência de Bases , Sítios de Ligação/genética , Metilação de DNA , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Escherichia coli/enzimologia , Escherichia coli/genética , Perfilação da Expressão Gênica , S-Adenosilmetionina/metabolismo , Especificidade por SubstratoRESUMO
A newly characterized archaeal rudivirus Stygiolobus rod-shaped virus (SRV), which infects a hyperthermophilic Stygiolobus species, was isolated from a hot spring in the Azores, Portugal. Its virions are rod-shaped, 702 (+/- 50) by 22 (+/- 3) nm in size, and nonenveloped and carry three tail fibers at each terminus. The linear double-stranded DNA genome contains 28,096 bp and an inverted terminal repeat of 1,030 bp. The SRV shows morphological and genomic similarities to the other characterized rudiviruses Sulfolobus rod-shaped virus 1 (SIRV1), SIRV2, and Acidianus rod-shaped virus 1, isolated from hot acidic springs of Iceland and Italy. The single major rudiviral structural protein is shown to generate long tubular structures in vitro of similar dimensions to those of the virion, and we estimate that the virion constitutes a single, superhelical, double-stranded DNA embedded into such a protein structure. Three additional minor conserved structural proteins are also identified. Ubiquitous rudiviral proteins with assigned functions include glycosyl transferases and a S-adenosylmethionine-dependent methyltransferase, as well as a Holliday junction resolvase, a transcriptionally coupled helicase and nuclease implicated in DNA replication. Analysis of matches between known crenarchaeal chromosomal CRISPR spacer sequences, implicated in a viral defense system, and rudiviral genomes revealed that about 10% of the 3,042 unique acidothermophile spacers yield significant matches to rudiviral genomes, with a bias to highly conserved protein genes, consistent with the widespread presence of rudiviruses in hot acidophilic environments. We propose that the 12-bp indels which are commonly found in conserved rudiviral protein genes may be generated as a reaction to the presence of the host CRISPR defense system.
Assuntos
Rudiviridae/crescimento & desenvolvimento , Rudiviridae/genética , Sulfolobaceae/fisiologia , Sulfolobaceae/virologia , Açores , Cromossomos de Archaea , DNA Viral/química , DNA Viral/genética , Ordem dos Genes , Genes Virais , Fontes Termais , Mutação INDEL , Substâncias Macromoleculares , Microscopia Eletrônica de Transmissão , Modelos Moleculares , Dados de Sequência Molecular , Rudiviridae/isolamento & purificação , Rudiviridae/ultraestrutura , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Sintenia , Proteínas não Estruturais Virais/genética , Proteínas Estruturais Virais/química , Proteínas Estruturais Virais/genética , Vírion/ultraestruturaRESUMO
As a tool for high-throughput, quantitative gene expression analysis, serial analysis of gene expression (SAGE) is one of the most powerful techniques. However, the short size of tags (14 bp) has hindered the application of SAGE to a vast majority of eukaryotes without sufficient genomic resources, including expressed sequence tag and genome sequences. To overcome this problem, we developed SuperSAGE, which is based on 26-bp tags from complementary DNA (cDNA), using EcoP15I as a tagging enzyme. Because longer cDNA fragments can easily be recovered by 3'-rapid amplification of cDNA ends (RACE) PCR using primers corresponding to the 26-bp tag sequences in non-model organisms, SuperSAGE allows the identification of novel genes in all eukaryotic organisms, and recommends itself as a useful platform in various fields of biological studies. Here, we present an updated SuperSAGE protocol, which incorporates several modifications and some recommendations to avoid total failure, particularly in the EcoP15I digestion step.
Assuntos
Perfilação da Expressão Gênica/métodos , Sequência de Bases , Clonagem Molecular , DNA Complementar/biossíntese , DNA Complementar/genética , DNA Complementar/isolamento & purificação , Desoxirribonucleases de Sítio Específico do Tipo II , Escherichia coli/genética , Vetores Genéticos , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase , Transformação GenéticaRESUMO
The Type III restriction endonuclease EcoP15I forms a hetero-oligomeric enzyme complex that consists of two modification (Mod) subunits and two restriction (Res) subunits. Structural data on Type III restriction enzymes in general are lacking because of their remarkable size of more than 400 kDa and the laborious and low-yield protein purification procedures. We took advantage of the EcoP15I-overexpressing vector pQEP15 and affinity chromatography to generate a quantity of EcoP15I high enough for comprehensive proteolytic digestion studies and analyses of the proteolytic fragments by mass spectrometry. We show here that in the presence of specific DNA the entire Mod subunit is protected from trypsin digestion, whereas in the absence of DNA stable protein domains of the Mod subunit were not detected. In contrast, the Res subunit is comprised of two trypsin-resistant domains of approximately 77-79 kDa and 27-29 kDa, respectively. The cofactor ATP and the presence of DNA, either specific or unspecific, are important stabilizers of the Res subunit. The large N-terminal domain of Res contains numerous functional motifs that are predicted to be involved in ATP-binding and hydrolysis and/or DNA translocation. The C-terminal small domain harbours the catalytic center. Based on our data, we conclude that both structural Res domains are connected by a flexible linker region that spans 23 amino acid residues. To confirm this conclusion, we have investigated several EcoP15I enzyme mutants obtained by insertion mutagenesis in and around the predicted linker region within the Res subunit. All mutants tolerated the genetic manipulation and did not display loss of function or alteration of the DNA cleavage position.
Assuntos
Desoxirribonucleases de Sítio Específico do Tipo III/química , Espectrometria de Massas/métodos , Mutagênese Insercional/métodos , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Desoxirribonucleases de Sítio Específico do Tipo III/genética , Hidrólise , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação , Estrutura Secundária de Proteína , Estrutura Terciária de ProteínaRESUMO
The application of transcriptomics to study host-pathogen interactions has already brought important insights into the mechanisms of pathogenesis, and is expanding further keeping pace with the accumulation of genomic sequences of host organisms (human and economically important organisms such as food crops) and their pathogens (viruses, bacteria, fungi and protozoa). In this review, we introduce SuperSAGE, a substantially improved variant of serial analysis of gene expression (SAGE), as a potent tool for the transcriptomics of host-pathogen interactions. Notably, the generation of 26 bp tags in the SuperSAGE procedure allows to decipher the 'interaction transcriptome', i.e. the simultaneous monitoring of quantitative gene expression, of both a host and one of its eukaryotic pathogens. The potential of SuperSAGE tags for a rapid functional analysis of target genes is also discussed.
Assuntos
Perfilação da Expressão Gênica/métodos , Animais , Bases de Dados de Ácidos Nucleicos , Regulação da Expressão Gênica , Humanos , Infecções/microbiologia , Infecções/parasitologia , Infecções/virologiaRESUMO
The Type III restriction endonuclease EcoP15I is a multifunctional hetero-oligomeric enzyme that recognizes the non-symmetric DNA sequence 5'-CAGCAG. For efficient cleavage, EcoP15I needs the interaction with two copies of the recognition sequence that have to be inversely oriented in the DNA double strand. The enzyme cuts the upper DNA strand 25-26 bp and the lower DNA strand 27-28 bp, respectively, downstream of the recognition sequence-a distinct feature that makes the enzyme particularly valuable for gene expression profiling methods relying on the SAGE procedure (Matsumura et al., PNAS 100, 15718, 2003). Because the broader use of this transcriptome analysis method requires the availability of larger amounts of restriction endonuclease EcoP15I and the enzyme is not commercially available, we have cloned the genes coding for the EcoP15I restriction endonuclease into pQE-16 plasmid vector that provides the enzyme with a C-terminal 6xHis-tag. After Ni-NTA affinity chromatography and ion exchange chromatography on heparin sepharose, we obtained 5mg homogeneous EcoP15I per gram cell pellet within 1-2 day(s). Moreover, the C-terminally 6xHis-tagged EcoP15I restriction endonuclease shows comparable enzymatic activity as the untagged enzyme.
Assuntos
Cromatografia de Afinidade/métodos , Perfilação da Expressão Gênica/métodos , Proteoma/análise , Análise de Sequência de DNA/métodos , DNA Metiltransferases Sítio Específica (Adenina-Específica)/isolamento & purificação , DNA Metiltransferases Sítio Específica (Adenina-Específica)/metabolismo , Fatores de Transcrição/análise , Sequência de Aminoácidos , Enzimas de Restrição do DNA/genética , Enzimas de Restrição do DNA/isolamento & purificação , Enzimas de Restrição do DNA/metabolismo , Ativação Enzimática , Escherichia coli/genética , Escherichia coli/metabolismo , Dados de Sequência Molecular , Engenharia de Proteínas/métodos , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , DNA Metiltransferases Sítio Específica (Adenina-Específica)/químicaRESUMO
Type III restriction enzymes are multifunctional heterooligomeric enzymes that cleave DNA at a fixed position downstream of a non-symmetric recognition site. For effective DNA cleavage these restriction enzymes need the presence of two unmethylated, inversely oriented recognition sites in the DNA molecule. DNA cleavage was proposed to result from ATP-dependent DNA translocation, which is expected to induce DNA loop formation, and collision of two enzyme-DNA complexes. We used scanning force microscopy to visualise the protein interaction with linear DNA molecules containing two EcoP15I recognition sites in inverse orientation. In the presence of the cofactors ATP and Mg(2+), EcoP15I molecules were shown to bind specifically to the recognition sites and to form DNA loop structures. One of the origins of the protein-clipped DNA loops was shown to be located at an EcoP15I recognition site, the other origin had an unspecific position in between the two EcoP15I recognition sites. The data demonstrate for the first time DNA translocation by the Type III restriction enzyme EcoP15I using scanning force microscopy. Moreover, our study revealed differences in the DNA-translocation processes mediated by Type I and Type III restriction enzymes.
Assuntos
Adenosina Trifosfatases/metabolismo , DNA/metabolismo , Desoxirribonucleases de Sítio Específico do Tipo III/metabolismo , Escherichia coli/enzimologia , Microscopia de Força Atômica , Hidrólise , Cinética , Modelos Genéticos , Conformação de Ácido Nucleico , PlasmídeosRESUMO
EcoRII is a type IIE restriction endonuclease that interacts with two copies of the DNA recognition sequence 5'CCWGG, one being the actual target of cleavage, the other serving as the allosteric effector. The mode of enzyme activation by effector binding is unknown. To investigate the molecular basis of activation and cleavage mechanisms by EcoRII, the crystal structure of EcoRII mutant R88A has been solved at 2.1A resolution. The EcoRII monomer has two domains linked through a hinge loop. The N-terminal effector-binding domain has a novel DNA recognition fold with a prominent cleft. The C-terminal catalytic domain has a restriction endonuclease-like fold. Structure-based sequence alignment identified the putative catalytic site of EcoRII that is spatially blocked by the N-terminal domain. The structure together with the earlier characterized EcoRII enzyme activity enhancement in the absence of its N-terminal domain reveal an autoinhibition/activation mechanism of enzyme activity mediated by a novel effector-binding fold. This is the first case of autoinhibition, a mechanism described for many transcription factors and signal transducing proteins, of a restriction endonuclease.
Assuntos
Regulação Alostérica , Desoxirribonucleases de Sítio Específico do Tipo II/química , Sítio Alostérico , Sequência de Aminoácidos , Proteínas de Bactérias/química , Domínio Catalítico , Cristalografia por Raios X , Desoxirribonucleases de Sítio Específico do Tipo II/genética , Ativação Enzimática , Estrutura Molecular , Mutação de Sentido Incorreto , Conformação Proteica , Alinhamento de SequênciaRESUMO
The type III restriction endonuclease EcoP15I was used in isolating fragments of 26 bp from defined positions of cDNAs. We call this substantially improved variant to the conventional serial analysis of gene expression (SAGE) procedure "SuperSAGE." By applying SuperSAGE to Magnaporthe grisea (blast)-infected rice leaves, gene expression profiles of both the rice host and blast fungus were simultaneously monitored by making use of the fully sequenced genomes of both organisms, revealing that the hydrophobin gene is the most actively transcribed M. grisea gene in blast-infected rice leaves. Moreover, SuperSAGE was applied to study gene expression changes before the so-called hypersensitive response in INF1 elicitor-treated Nicotiana benthamiana, a "nonmodel" organism for which no DNA database is available. Again, SuperSAGE allowed rapid identification of genes up- or down-regulated by the elicitor. Surprisingly, many of the down-regulated genes coded for proteins involved in photosynthesis. SuperSAGE will be especially useful for transcriptome profiling of two or more interacting organisms like hosts and pathogens, and of organisms, for which no DNA database is available.