RESUMO
The conserved TREX complex has multiple functions in gene expression such as transcription elongation, 3' end processing, mRNP assembly and nuclear mRNA export as well as the maintenance of genomic stability. In Saccharomyces cerevisiae, TREX is composed of the pentameric THO complex, the DEAD-box RNA helicase Sub2, the nuclear mRNA export adaptor Yra1, and the SR-like proteins Gbp2 and Hrb1. Here, we present the structural analysis of the endogenous TREX complex of S. cerevisiae purified from its native environment. To this end, we used cross-linking mass spectrometry to gain structural information on regions of the complex that are not accessible to classical structural biology techniques. We also used negative-stain electron microscopy to investigate the organization of the cross-linked complex used for XL-MS by comparing our endogenous TREX complex with recently published structural models of recombinant THO-Sub2 complexes. According to our analysis, the endogenous yeast TREX complex preferentially assembles into a dimer.
Assuntos
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , RNA Mensageiro/genética , Transporte de RNA , Transcrição Gênica , Proteínas de Transporte Nucleocitoplasmático/genética , Proteínas de Ligação a Poli(A)/genéticaRESUMO
Urban green spaces, like community gardens, received increased attention during the COVID-19 pandemic. Drawing from an ethnographic study on participating in community garden activities in Edmonton, Canada and inputs from 194 gardeners and 21 garden coordinators, this paper captures the experiences of creating community during a pandemic. Garden coordinators had to rethink and rework their operating styles in keeping participants physically apart but socially connected. Participants confirmed that garden activities provided respite from the pandemic restrictions. Findings also indicate that some participants missed group activities like work bees and potlucks while others were able to re-create community in digital spaces and in chanced and informal interactions. This study draws from and subsequently contributes to the existing literature on social resilience provided by community gardens during and after a crisis event. It also provides policy recommendations on how the city administration can help facilitate garden activities during times of disruptions.
RESUMO
A bacterial strain 2H isolated from soil and identified as Thermoactinomyces vulgaris produce a potent Type II restriction endonuclease activity that has been extracted by a PEG/dextran aqueous two-phase system. Optimal temperature for the restriction endonuclease activity was 55-65°C. Specific DNA cleavage was obtained at pH range 7-10 and 10-20mM MgCl2. Restriction cleavage analysis followed by sequencing confirms GG^CC as the recognition sequence. This enzyme, named Tvu2HI, is a thermostable isoschizomer of the mesophilic prototype restriction endonuclease HaeIII. Sequencing of the complete Thermoactinomyces vulgaris 2H genome revealed the presence of two adjacent ORFs coding for the restriction endonuclease Tvu2HI and the corresponding methyltransferase; an ORF coding for a putative Vsr nicking enzyme was found close to those coding for the Tvu2HI restriction-modification system. Phylogenetic analysis based on sequence alignment suggests a common origin of Tvu2HI R-M system with HaeIII-like R-M systems. This is the first investigation dealing with a Type II restriction endonuclease identified in a natural isolate of the genus Thermoactinomyces.
Assuntos
Desoxirribonucleases de Sítio Específico do Tipo II/genética , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Thermoactinomyces/classificação , Sequenciamento Completo do Genoma/métodos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Desoxirribonucleases de Sítio Específico do Tipo II/química , Estabilidade Enzimática , Evolução Molecular , Concentração de Íons de Hidrogênio , Fases de Leitura Aberta , Filogenia , Microbiologia do Solo , Especificidade por Substrato , Thermoactinomyces/enzimologia , Thermoactinomyces/genética , Thermoactinomyces/isolamento & purificação , TermodinâmicaRESUMO
mRNA is the "hermes" of gene expression as it carries the information of a protein-coding gene to the ribosome. Already during its synthesis, the mRNA is bound by mRNA-binding proteins that package the mRNA into a messenger ribonucleoprotein particle (mRNP). This mRNP assembly is important for mRNA stability and nuclear mRNA export. It also often regulates later steps in the mRNA lifetime such as translation and mRNA degradation in the cytoplasm. Thus, mRNP composition and accordingly the assembly of nuclear mRNA-binding proteins onto the mRNA are of crucial importance for correct gene expression. Here, we review our current knowledge of the mechanism of co-transcriptional mRNP assembly and nuclear mRNA export. We introduce the proteins involved and elaborate on what is known about their functions so far. In addition, we discuss the importance of regulated mRNP assembly in changing environmental conditions, especially during stress. Furthermore, we examine how defects in mRNP assembly cause diseases and how viruses exploit the host's nuclear mRNA export pathway. Finally, we summarize the questions that need to be answered in the future.
Assuntos
Transporte de RNA , Núcleo Celular/metabolismo , RNA Mensageiro/metabolismo , RibonucleoproteínasRESUMO
Common genome-editing strategies are either based on non-homologous end joining (NHEJ) or, in the presence of a template DNA, based on homologous recombination with long (homology-directed repair [HDR]) or short (microhomology-mediated end joining [MMEJ]) homologous sequences. In the current study, we aim to develop a model system to test the activity of MMEJ after CRISPR/Cas9-mediated cleavage in cell culture. Following successful proof of concept in an episomally based reporter system, we tested template plasmids containing a promoter-less luciferase gene flanked by microhomologous sequences (mhs) of different length (5, 10, 15, 20, 30, and 50 bp) that are complementary to the mouse retinitis pigmentosa GTPase regulator (RPGR)-ORF15, which is under the control of a CMV promoter stably integrated into a HEK293 cell line. Luciferase signal appearance represented successful recombination events and was highest when the mhs were 5 bp long, while longer mhs revealed lower luciferase signal. In addition, presence of Csy4 RNase was shown to increase luciferase signaling. The luciferase reporter system is a valuable tool to study the input of the different DNA repair mechanisms in the replacement of large DNA sequences by mhs.
RESUMO
In vivo genome editing represents an emerging field in the treatment of monogenic disorders, as it may constitute a solution to the current hurdles in classic gene addition therapy, which are the low levels and limited duration of transgene expression. Following the introduction of a double strand break (DSB) at the mutational site by highly specific endonucleases, such as TALENs (transcription activator like effector nucleases) or RNA based nucleases (clustered regulatory interspaced short palindromic repeats - CRISPR-Cas), the cell's own DNA repair machinery restores integrity to the DNA strand and corrects the mutant sequence, thus allowing the cell to produce protein levels as needed. The DNA repair happens either through the error prone non-homologous end-joining (NHEJ) pathway or with high fidelity through homology directed repair (HDR) in the presence of a DNA donor template. A third pathway called microhomology mediated endjoining (MMEJ) has been recently discovered. In this review, the authors focus on the different DNA repair mechanisms, the current state of the art tools for genome editing and the particularities of the retina and photoreceptors with regard to in vivo therapeutic approaches. Finally, current attempts in the field of retinal in vivo genome editing are discussed and future directions of research identified.
Assuntos
Edição de Genes/métodos , Terapia Genética/métodos , Genoma , Guias de Prática Clínica como Assunto , Distrofias Retinianas , Animais , Humanos , Distrofias Retinianas/genética , Distrofias Retinianas/metabolismo , Distrofias Retinianas/terapiaRESUMO
BACKGROUND: Nicking endonucleases are enzymes that recognize specific sites in double-stranded DNA and cleave only one strand at a predetermined position. These enzymes are involved in DNA replication and repair; they can also function as subunits of bacterial heterodimeric restriction endonucleases. One example of such a proteins is the restriction endonuclease BspD6I (R.BspD6I) from Bacillus species strain D6, which consists of the large subunit - nicking endonuclease BspD6I (Nt.BspD6I), and the small subunit (ss.BspD6I). Nt.BspD6I can function independently. Similar enzymes are now widely used in numerous biotechnological applications. The aim of this study was to investigate the fundamental properties of two subunits of R.BspD6I and their interdependence in the course of R.BspD6I activity. METHODS: The binding and hydrolysis of DNA duplexes by R.BspD6I are primary analyzed by gel electrophoresis. To elucidate the difference between Nt.BspD6I interaction with the substrate and product of hydrolysis, the thickness shear mode acoustic method is used. RESULTS AND CONCLUSIONS: The thermodynamic and kinetic parameters of the Nt.BspD6I interaction with DNA are determined. For the first time we demonstrated that Nt.BspD6I bends the DNA during complex formation. Nt.BspD6I is able to form complexes with the product nicked in the top strand and ss.BspD6I cleaves the bottom strand of the DNA consecutively. Furthermore, the influence of dA methylation in the R.BspD6I recognition site on ss.BspD6I activity is analyzed. GENERAL SIGNIFICANCE: The obtained results provide evidence that Nt.BspD6I coordinates the activity of R.BspD6I by strictly coupling of the bottom strand cleavage by ss.BspD6I to the top strand cleavage.
Assuntos
DNA/química , Desoxirribonuclease I/química , Subunidades Proteicas/química , Bacillus/química , Sítios de Ligação , Clonagem Molecular , DNA/metabolismo , Desoxirribonuclease I/genética , Desoxirribonuclease I/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Hidrólise , Cinética , Ligação Proteica , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , TermodinâmicaRESUMO
BACKGROUND: Mutations in the EDA-EDAR-EDARADD genes and more recently, mutations in the WNT10A gene have been described as the cause of syndromic and nonsyndromic tooth agenesis concomitant with diverse abnormalities of ectodermally derived tissues. AIM: In the present investigation, two brothers presenting severe tooth agenesis (oligodontia) concomitant with subtle signs of ectodermal dysplasia (ED) symptoms, as well as six family relatives were analyzed for a causative mutation. METHODS: Genomic DNA was isolated from saliva, and genetic screening performed via direct sequencing of PCR fragments covering the entire coding regions and the intron-exon junctions of the EDA, EDAR, EDARADD as well as the WNT10A genes. Mutation analysis was conducted using the Mutation Surveyor(®) Software. RESULTS AND CONCLUSION: We identified a novel G > A mutation located on exon 7 at nucleotide position c.866 in the EDA gene in both patients. The nucleotide change results in a substitution of arginine by histidine (p.Arg289His). According to the programs MutationTaster and PolyPhen2, this mutation is pathogenic. Based on a computerized protein structure analysis, we suggest that the change p.Arg289His in EDA impairs protein stabilization and thus might possibly be involved in the development of oligodontia concomitant with a mild ED phenotype.
Assuntos
Anodontia/diagnóstico , Anodontia/genética , Ectodisplasinas/genética , Mutação de Sentido Incorreto/genética , Polimorfismo de Nucleotídeo Único/genética , Marcadores Genéticos/genética , Predisposição Genética para Doença/genética , Alemanha , Humanos , Masculino , IrmãosRESUMO
This article continues the series of Surveys and Summaries on restriction endonucleases (REases) begun this year in Nucleic Acids Research. Here we discuss 'Type II' REases, the kind used for DNA analysis and cloning. We focus on their biochemistry: what they are, what they do, and how they do it. Type II REases are produced by prokaryotes to combat bacteriophages. With extreme accuracy, each recognizes a particular sequence in double-stranded DNA and cleaves at a fixed position within or nearby. The discoveries of these enzymes in the 1970s, and of the uses to which they could be put, have since impacted every corner of the life sciences. They became the enabling tools of molecular biology, genetics and biotechnology, and made analysis at the most fundamental levels routine. Hundreds of different REases have been discovered and are available commercially. Their genes have been cloned, sequenced and overexpressed. Most have been characterized to some extent, but few have been studied in depth. Here, we describe the original discoveries in this field, and the properties of the first Type II REases investigated. We discuss the mechanisms of sequence recognition and catalysis, and the varied oligomeric modes in which Type II REases act. We describe the surprising heterogeneity revealed by comparisons of their sequences and structures.
Assuntos
Desoxirribonucleases de Sítio Específico do Tipo II/química , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , DNA/química , DNA/metabolismo , Desoxirribonucleases de Sítio Específico do Tipo II/genética , Desoxirribonucleases de Sítio Específico do Tipo II/história , Evolução Molecular , História do Século XX , História do Século XXI , Engenharia de Proteínas , Mapeamento por RestriçãoRESUMO
Nitrogen mustards are an important class of bifunctional alkylating agents routinely used in chemotherapy. They react with DNA as electrophiles through the formation of highly reactive aziridinium ion intermediates. The antibiotic 593A, with potential antitumor activity, can be considered a naturally occurring piperidine mustard containing a unique 3-chloropiperidine ring. However, the total synthesis of this antibiotic proved to be rather challenging. With the aim of designing simplified analogues of this natural product, we developed an efficient bidirectional synthetic route to bis-3-chloropiperidines joined by flexible, conformationally restricted, or rigid diamine linkers. The key step involves an iodide-catalyzed double cyclization of unsaturated bis-N-chloroamines to simultaneously generate both piperidine rings. Herein we describe the synthesis and subsequent evaluation of a series of novel nitrogen-bridged bis-3-chloropiperidines, enabling the study of the impact of the linker structure on DNA alkylation properties. Our studies reveal that the synthesized compounds possess DNA alkylating abilities and induce strand cleavage, with a strong preference for guanine residues.
Assuntos
Alquilantes/síntese química , Alquilantes/farmacologia , Antineoplásicos Alquilantes/síntese química , Antineoplásicos Alquilantes/farmacologia , Clivagem do DNA/efeitos dos fármacos , Compostos de Mostarda Nitrogenada/síntese química , Compostos de Mostarda Nitrogenada/farmacologia , Piperidinas/síntese química , Piperidinas/farmacologia , Alquilação , Ciclização , Conformação Molecular , Piperazinas/síntese química , Piperazinas/farmacologia , Plasmídeos/efeitos dos fármacosRESUMO
Zinc finger nucleases (ZFNs) consist of zinc fingers as DNA-binding module and the non-specific DNA-cleavage domain of the restriction endonuclease FokI as DNA-cleavage module. This architecture is also used by TALE nucleases (TALENs), in which the DNA-binding modules of the ZFNs have been replaced by DNA-binding domains based on transcription activator like effector (TALE) proteins. Both TALENs and ZFNs are programmable nucleases which rely on the dimerization of FokI to induce double-strand DNA cleavage at the target site after recognition of the target DNA by the respective DNA-binding module. TALENs seem to have an advantage over ZFNs, as the assembly of TALE proteins is easier than that of ZFNs. Here, we present evidence that variant TALENs can be produced by replacing the catalytic domain of FokI with the restriction endonuclease PvuII. These fusion proteins recognize only the composite recognition site consisting of the target site of the TALE protein and the PvuII recognition sequence (addressed site), but not isolated TALE or PvuII recognition sites (unaddressed sites), even at high excess of protein over DNA and long incubation times. In vitro, their preference for an addressed over an unaddressed site is > 34,000-fold. Moreover, TALE-PvuII fusion proteins are active in cellula with minimal cytotoxicity.
Assuntos
DNA-Citosina Metilases/metabolismo , Proteínas de Homeodomínio/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Sítios de Ligação , Linhagem Celular , Clivagem do DNA , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , DNA-Citosina Metilases/genética , Marcação de Genes/métodos , Proteínas de Homeodomínio/genética , Humanos , Modelos Moleculares , Conformação Proteica , Multimerização Proteica , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/toxicidade , Especificidade por SubstratoRESUMO
Dense coverage of DNA by proteins is a ubiquitous feature of cellular processes such as DNA organization, replication and repair. We present a single-molecule approach capable of visualizing individual DNA-binding proteins on densely covered DNA and in the presence of high protein concentrations. Our approach combines optical tweezers with multicolor confocal and stimulated emission depletion (STED) fluorescence microscopy. Proteins on DNA are visualized at a resolution of 50 nm, a sixfold resolution improvement over that of confocal microscopy. High temporal resolution (<50 ms) is ensured by fast one-dimensional beam scanning. Individual trajectories of proteins translocating on DNA can thus be distinguished and tracked with high precision. We demonstrate our multimodal approach by visualizing the assembly of dense nucleoprotein filaments with unprecedented spatial resolution in real time. Experimental access to the force-dependent kinetics and motility of DNA-associating proteins at biologically relevant protein densities is essential for linking idealized in vitro experiments with the in vivo situation.
Assuntos
Proteínas de Ligação a DNA/análise , DNA/metabolismo , Microscopia de Fluorescência/instrumentação , Microscopia de Fluorescência/métodos , Imagem Molecular/métodos , Pinças Ópticas , DNA/análise , Proteínas de Ligação a DNA/metabolismo , Desenho de Equipamento , Processamento de Imagem Assistida por Computador , Microscopia Confocal/métodos , Nanotecnologia/métodos , Nucleoproteínas/análise , Nucleoproteínas/metabolismoRESUMO
Targeted genome engineering requires nucleases that introduce a highly specific double-strand break in the genome that is either processed by homology-directed repair in the presence of a homologous repair template or by non-homologous end-joining (NHEJ) that usually results in insertions or deletions. The error-prone NHEJ can be efficiently suppressed by 'nickases' that produce a single-strand break rather than a double-strand break. Highly specific nickases have been produced by engineering of homing endonucleases and more recently by modifying zinc finger nucleases (ZFNs) composed of a zinc finger array and the catalytic domain of the restriction endonuclease FokI. These ZF-nickases work as heterodimers in which one subunit has a catalytically inactive FokI domain. We present two different approaches to engineer highly specific nickases; both rely on the sequence-specific nicking activity of the DNA mismatch repair endonuclease MutH which we fused to a DNA-binding module, either a catalytically inactive variant of the homing endonuclease I-SceI or the DNA-binding domain of the TALE protein AvrBs4. The fusion proteins nick strand specifically a bipartite recognition sequence consisting of the MutH and the I-SceI or TALE recognition sequences, respectively, with a more than 1000-fold preference over a stand-alone MutH site. TALE-MutH is a programmable nickase.
Assuntos
Quebras de DNA de Cadeia Simples , Proteínas de Ligação a DNA/metabolismo , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Endodesoxirribonucleases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Clivagem do DNA , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Desoxirribonucleases de Sítio Específico do Tipo II/química , Desoxirribonucleases de Sítio Específico do Tipo II/genética , Endodesoxirribonucleases/química , Endodesoxirribonucleases/genética , Engenharia de Proteínas , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Sequências Repetitivas de Aminoácidos , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Especificidade por SubstratoRESUMO
Enoyl-acyl carrier protein reductase (ENR; the product of the fabI gene) is an important enzyme that is involved in the type II fatty-acid-synthesis pathway of bacteria, plants, apicomplexan protozoa and mitochondria. Harmful pathogens such as Mycobacterium tuberculosis and Plasmodium falciparum use the type II fatty-acid-synthesis system, but not mammals or fungi, which contain a type I fatty-acid-synthesis pathway consisting of one or two multifunctional enzymes. For this reason, specific inhibitors of ENR are attractive antibiotic candidates. Triclosan, a broad-range antibacterial agent, binds to ENR, inhibiting fatty-acid synthesis. As humans do not have an ENR enzyme, they are not affected. Here, high-resolution structures of Thermus thermophilus (Tth) ENR in the apo form, bound to NAD(+) and bound to NAD(+) plus triclosan are reported. Differences from and similarities to other known ENR structures are reported; in general, the structures are very similar. The cofactor-binding site is also very similar to those of other ENRs and, as reported for other species, triclosan leads to greater ordering of the loop that covers the cofactor-binding site, which, together with the presence of triclosan itself, presumably provides tight binding of the dinucleotide, preventing cycling of the cofactor. Differences between the structures of Tth ENR and other ENRs are the presence of an additional ß-sheet at the N-terminus and a larger number of salt bridges and side-chain hydrogen bonds. These features may be related to the high thermal stability of Tth ENR.
Assuntos
Enoil-(Proteína de Transporte de Acila) Redutase (NADH)/química , NAD/química , Thermus thermophilus/enzimologia , Triclosan/química , Sequência de Aminoácidos , Apoenzimas/química , Apoenzimas/metabolismo , Enoil-(Proteína de Transporte de Acila) Redutase (NADH)/metabolismo , Estabilidade Enzimática , Modelos Moleculares , Dados de Sequência Molecular , NAD/metabolismo , Ligação Proteica , Estrutura Quaternária de Proteína , Alinhamento de Sequência , Homologia Estrutural de Proteína , Triclosan/metabolismoRESUMO
The restriction endonuclease PvuII has been introduced as a sequence-specific cleavage module in highly-specific nucleases for gene targeting. Here, a structural reorganization of the single-chain variant of PvuII (scPvuII) was performed by circular permutation as a proof-of-concept in order to find out whether the relocated, new termini next to structural elements important for DNA recognition and catalysis could be used for the fusion with other regulatory protein domains. Three circularly permuted variants of scPvuII were obtained that all maintain the specific endonucleolytic activity of scPvuII.
Assuntos
Proteínas de Bactérias/química , Enzimas de Restrição do DNA/química , Engenharia de Proteínas , Sequência de Aminoácidos , Substituição de Aminoácidos , Proteínas de Bactérias/genética , Clivagem do DNA , Enzimas de Restrição do DNA/genética , Cinética , Oligonucleotídeos/química , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genéticaRESUMO
It has been proposed that certain type II restriction enzymes (REs), such as EcoRV, track the helical pitch of DNA as they diffuse along DNA, a so-called rotation-coupled sliding. As of yet, there is no direct experimental observation of this phenomenon, but mounting indirect evidence gained from single-molecule imaging of RE-DNA complexes support the hypothesis. We address this issue by conjugating fluorescent labels of varying size (organic dyes, proteins and quantum dots) to EcoRV, and by fusing it to the engineered Rop protein scRM6. Single-molecule imaging of these modified EcoRVs sliding along DNA provides us with their linear diffusion constant (D(1)), revealing a significant size dependency. To account for the dependence of D(1) on the size of the EcoRV label, we have developed four theoretical models describing different types of motion along DNA and find that our experimental results are best described by rotation-coupled sliding of the protein. The similarity of EcoRV to other type II REs and DNA binding proteins suggests that this type of motion could be widely preserved in other biological contexts.
Assuntos
Desoxirribonucleases de Sítio Específico do Tipo II/química , DNA/química , Desoxirribonucleases de Sítio Específico do Tipo II/genética , Difusão , Corantes Fluorescentes , Modelos Moleculares , Movimento (Física) , Proteínas Recombinantes de Fusão/química , RotaçãoRESUMO
Zinc-finger nucleases and TALE nucleases are produced by combining a specific DNA-binding module and a non-specific DNA-cleavage module, resulting in nucleases able to cleave DNA at a unique sequence. Here a new approach for creating highly specific nucleases was pursued by fusing a catalytically inactive variant of the homing endonuclease I-SceI, as DNA binding-module, to the type IIP restriction enzyme PvuII, as cleavage module. The fusion enzymes were designed to recognize a composite site comprising the recognition site of PvuII flanked by the recognition site of I-SceI. In order to reduce activity on PvuII sites lacking the flanking I-SceI sites, the enzymes were optimized so that the binding of I-SceI to its sites positions PvuII for cleavage of the composite site. This was achieved by optimization of the linker and by introducing amino acid substitutions in PvuII which decrease its activity or disturb its dimer interface. The most specific variant showed a more than 1000-fold preference for the addressed composite site over an unaddressed PvuII site. These results indicate that using a specific restriction enzyme, such as PvuII, as cleavage module, offers an alternative to the otherwise often used catalytic domain of FokI, which by itself does not contribute to the specificity of the engineered nuclease.
Assuntos
Desoxirribonucleases de Sítio Específico do Tipo II/química , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Endodesoxirribonucleases/metabolismo , Substituição de Aminoácidos , Biocatálise , Clivagem do DNA , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Desoxirribonucleases de Sítio Específico do Tipo II/genética , Endodesoxirribonucleases/genética , Engenharia de Proteínas , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Especificidade por SubstratoRESUMO
Zinc-finger nucleases (ZFNs) typically consist of three to four zinc fingers (ZFs) and the non-specific DNA-cleavage domain of the restriction endonuclease FokI. In this configuration, the ZFs constitute the binding module and the FokI domain the cleavage module. Whereas new binding modules, e.g. TALE sequences, have been considered as alternatives to ZFs, no efforts have been undertaken so far to replace the catalytic domain of FokI as the cleavage module in ZFNs. Here, we have fused a three ZF array to the restriction endonuclease PvuII to generate an alternative ZFN. While PvuII adds an extra element of specificity when combined with ZFs, ZF-PvuII constructs must be designed such that only PvuII sites with adjacent ZF-binding sites are cleaved. To achieve this, we introduced amino acid substitutions into PvuII that alter K(m) and k(cat) and increase fidelity. The optimized ZF-PvuII fusion constructs cleave DNA at addressed sites with a >1000-fold preference over unaddressed PvuII sites in vitro as well as in cellula. In contrast to the 'analogous' ZF-FokI nucleases, neither excess of enzyme over substrate nor prolonged incubation times induced unaddressed cleavage in vitro. These results present the ZF-PvuII platform as a valid alternative to conventional ZFNs.