RESUMO
Restriction endonucleases (REs) cleave DNA at specific site in presence of Mg2+ ion. Experiments further emphasize the role of hydration in metal ion specificity and sequence specificity of DNA cleavage. However, the relation between hydration and specificity has not been understood till date. This leads us to study via all-atom molecular dynamics (MD) simulations how the hydration around the scissile phosphate group changes in presence of Mg2+ and Ca2+ and depend on the DNA sequence. We observe the least number of hydrogen bonds around the scissile phosphate group in presence of Mg2+ ion. We further find that the hydrogen bonds decrease at the scissile phosphate on mutating one base pair in the cleavage region of the DNA in Mg2+ loaded EcoRI-DNA complex. We also perform steered MD simulations and observe that the rate of decrease of fraction of hydrogen bonds is slower in the mutated complex than the unmutated complex.
Assuntos
DNA , Silício , Desoxirribonuclease EcoRI/química , Desoxirribonuclease EcoRI/genética , Desoxirribonuclease EcoRI/metabolismo , Sítios de Ligação , Sequência de Bases , DNA/química , Fosfatos , Especificidade por Substrato , CinéticaRESUMO
We sought to examine whether dietary intakes may affect the relationship between ApoB EcoRI and lipid profile, as well as serum inflammatory markers, in patients with type 2 diabetes (T2DM). This current study consisted of 648 diabetic patients. Dietary intake was calculated by a food frequency questionnaire. Biochemical markers (high-density lipoprotein (HDL), total cholesterol (TC), LDL, TG, CRP, IL-18, PGF2α) were measured based on standard protocols. Genotyping of the Apo-B polymorphisms (rs1042031) was conducted by the PCR-RFLP method. The gene-diet interactions were evaluated using GLMs. In comparison to GG homozygotes, A-allele carriers with above the median -CHO intake (≥ 54 percent of total energy) had considerably greater TC and PGF2a concentrations. Furthermore, as compared to GG homozygotes, A-allele carriers with above the median protein intake (≥ 14 percent of total energy) had higher serum levels of TG (P = 0.001), CRP (P = 0.02), TG/HDL (P = 0.005), and LDL/HDL (P = 0.04) ratios. Moreover, A-allele carriers with above the median total fat intake (≥ 35 percent of total calories) had significantly higher TC level (P = 0.04) and LDL/HDL (P = 0.04) ratios compared to GG homozygotes. Furthermore, when compared to GG homozygotes, A-allele carriers who consumed above the median cholesterol (> 196 mg) had greater TG (P = 0.04), TG/HDL (P = 0.01) ratio, and IL-18 (P = 0.02). Furthermore, diabetic patients with the GA, AA genotype who consume above the median cholesterol had lower ghrelin levels (P = 0.01). In terms of LDL/HDL ratio, ApoB EcoRI and dietary intakes of specific fatty acids (≥ 9 percent for SFA and ≥ 12 percent for MUFA) had significant interaction. LDL/HDL ratio is greater in A-allele carriers with above the median SFA intake (P = 0.04), also when they consumed above the median MUFA this association was inverse (P = 0.04). Our study showed that plasma lipid levels in participants carrying the (AA or AG) genotype were found to be more responsive to increasing the percentage of energy derived from dietary fat, CHO, protein, SFA, and cholesterol consumption. Therefore, patients with a higher genetic susceptibility (AA or AG) seemed to have greater metabolic markers with a higher percentage of macronutrient consumption. Also, ApoB EcoRI correlations with metabolic markers might be attenuated with above the median MUFA consumption.
Assuntos
Apolipoproteínas B , Diabetes Mellitus Tipo 2 , Dieta , Nutrientes , Apolipoproteína B-100 , Apolipoproteínas B/genética , Biomarcadores/sangue , Colesterol/sangue , Desoxirribonuclease EcoRI/genética , Diabetes Mellitus Tipo 2/sangue , Diabetes Mellitus Tipo 2/genética , Genótipo , HumanosRESUMO
Restriction-modification systems (R-M) are one of the antiviral defense tools used by bacteria, and those of the Type II family are composed of a restriction endonuclease (REase) and a DNA methyltransferase (MTase). Most entering DNA molecules are usually cleaved by the REase before they can be methylated by MTase, although the observed level of fragmented DNA may vary significantly. Using a model EcoRI R-M system, we report that the balance between DNA methylation and cleavage may be severely affected by transcriptional signals coming from outside the R-M operon. By modulating the activity of the promoter, we obtained a broad range of restriction phenotypes for the EcoRI R-M system that differed by up to 4 orders of magnitude in our biological assays. Surprisingly, we found that high expression levels of the R-M proteins were associated with reduced restriction of invading bacteriophage DNA. Our results suggested that the regulatory balance of cleavage and methylation was highly sensitive to fluctuations in transcriptional signals both up- and downstream of the R-M operon. Our data provided further insights into Type II R-M system maintenance and the potential conflict within the host bacterium.
Assuntos
Colífagos/metabolismo , Desoxirribonuclease EcoRI/metabolismo , Escherichia coli/enzimologia , Escherichia coli/virologia , DNA Metiltransferases Sítio Específica (Adenina-Específica)/metabolismo , Bacteriófago lambda/genética , Bacteriófago lambda/metabolismo , Colífagos/genética , Clivagem do DNA , Metilação de DNA/genética , Desoxirribonuclease EcoRI/genética , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Óperon/genética , Plasmídeos/genética , DNA Metiltransferases Sítio Específica (Adenina-Específica)/genéticaRESUMO
Restriction enzymes recognize and bind to specific sequences on invading bacteriophage DNA. Like a key in a lock, these proteins require many contacts to specify the correct DNA sequence. Using information theory we develop an equation that defines the number of independent contacts, which is the dimensionality of the binding. We show that EcoRI, which binds to the sequence GAATTC, functions in 24 dimensions. Information theory represents messages as spheres in high dimensional spaces. Better sphere packing leads to better communications systems. The densest known packing of hyperspheres occurs on the Leech lattice in 24 dimensions. We suggest that the single protein EcoRI molecule employs a Leech lattice in its operation. Optimizing density of sphere packing explains why 6 base restriction enzymes are so common.
Assuntos
Enzimas de Restrição do DNA/genética , Proteínas de Ligação a DNA/genética , DNA/genética , Desoxirribonuclease EcoRI/genética , Bacteriófagos/genética , Sequência de Bases , DNA/química , Enzimas de Restrição do DNA/química , Proteínas de Ligação a DNA/química , Desoxirribonuclease EcoRI/química , Modelos MolecularesRESUMO
Helicases catalyse DNA and RNA strand separation. Proteins bound to the nucleic acid must also be displaced in order to unwind DNA. This is exemplified by accessory helicases that clear protein barriers from DNA ahead of advancing replication forks. How helicases catalyse DNA unwinding is increasingly well understood but how protein displacement is achieved is unclear. Escherichia coli Rep accessory replicative helicase lacking one of its four subdomains, 2B, has been shown to be hyperactivated for DNA unwinding in vitro but we show here that RepΔ2B is, in contrast, deficient in displacing proteins from DNA. This defect correlates with an inability to promote replication of protein-bound DNA in vitro and lack of accessory helicase function in vivo. Defective protein displacement is manifested on double-stranded and single-stranded DNA. Thus binding and distortion of duplex DNA by the 2B subdomain ahead of the helicase is not the missing function responsible for this deficiency. These data demonstrate that protein displacement from DNA is not simply achieved by helicase translocation alone. They also imply that helicases may have evolved different specific features to optimise DNA unwinding and protein displacement, both of which are now recognised as key functions in all aspects of nucleic acid metabolism.
Assuntos
DNA Helicases/química , DNA Bacteriano/química , DNA de Cadeia Simples/química , DNA/química , Proteínas de Escherichia coli/química , Escherichia coli/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , DNA/genética , DNA/metabolismo , DNA Helicases/genética , DNA Helicases/metabolismo , DNA Polimerase III/genética , DNA Polimerase III/metabolismo , DNA Primase/genética , DNA Primase/metabolismo , Replicação do DNA , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Desoxirribonuclease EcoRI/genética , Desoxirribonuclease EcoRI/metabolismo , DnaB Helicases/genética , DnaB Helicases/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Expressão Gênica , Modelos Moleculares , Conformação de Ácido Nucleico , Plasmídeos/química , Plasmídeos/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre ProteínasRESUMO
RNA folding during transcription directs an order of folding that can determine RNA structure and function. However, the experimental study of cotranscriptional RNA folding has been limited by the lack of easily approachable methods that can interrogate nascent RNA structure at nucleotide resolution. To address this, we previously developed cotranscriptional selective 2Î-hydroxyl acylation analyzed by primer extension sequencing (SHAPE-Seq) to simultaneously probe all intermediate RNA transcripts during transcription by stalling elongation complexes at catalytically dead EcoRIE111Q roadblocks. While effective, the distribution of elongation complexes using EcoRIE111Q requires laborious PCR using many different oligonucleotides for each sequence analyzed. Here, we improve the broad applicability of cotranscriptional SHAPE-Seq by developing a sequence-independent biotin-streptavidin (SAv) roadblocking strategy that simplifies the preparation of roadblocking DNA templates. We first determine the properties of biotin-SAv roadblocks. We then show that randomly distributed biotin-SAv roadblocks can be used in cotranscriptional SHAPE-Seq experiments to identify the same RNA structural transitions related to a riboswitch decision-making process that we previously identified using EcoRIE111Q. Lastly, we find that EcoRIE111Q maps nascent RNA structure to specific transcript lengths more precisely than biotin-SAv and propose guidelines to leverage the complementary strengths of each transcription roadblock in cotranscriptional SHAPE-Seq.
Assuntos
Biotina/química , Técnicas de Química Analítica , Dobramento de RNA , RNA/química , Estreptavidina/química , Transcrição Gênica , Acilação , Pareamento de Bases , Sequência de Bases , Biotina/genética , Primers do DNA/química , Primers do DNA/genética , Desoxirribonuclease EcoRI/química , Desoxirribonuclease EcoRI/genética , Hidróxidos/química , Conformação de Ácido Nucleico , RNA/biossíntese , RNA/genética , Riboswitch , Análise de Sequência de RNA , Estreptavidina/genéticaRESUMO
We have developed a simple system for tagging and purifying proteins. Recent experiments have demonstrated that RTX (Repeat in Toxin) motifs from the adenylate cyclase toxin gene (CyaA) of B. pertussis undergo a conformational change upon binding calcium, resulting in precipitation of fused proteins and making this method a viable alternative for bioseparation. We have designed an iGEM Biobrick comprised of an RTX tag that can be easily fused to any protein of interest. In this paper, we detail the process of creating an RTX tagged version of the restriction enzyme EcoRI and describe a method for expression and purification of the functional enzyme.
Assuntos
Motivos de Aminoácidos/genética , Cálcio/metabolismo , Desoxirribonuclease EcoRI/genética , Engenharia Genética/métodos , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Toxina Adenilato Ciclase/genética , Cálcio/química , Clonagem Molecular , Conformação Proteica , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genéticaRESUMO
Novel mdfA gene variants were identified simultaneously from 3 of 13 positive isolates of PCR amplification in Escherichia coli from patients. These 13 positive isolates showed resistance to chloramphenicol, tetracycline, and erythromycin. The 3 mdfA gene variants were of the same genotype and all the 13 positive isolates were investigated by conjugation experiment, EcoRI restriction, and gene mapping. Conjugation experiments demonstrated that the novel mdfA variant and mdfA genes were located on plasmids that were restricted by EcoRI for â¼8.2 kb-length, which was also validated by gene mapping. Further study indicated three types of genetic structures (A, B, and C) in the recombinant plasmids harboring mdfA and surrounding genes, and structure B was first reported in the article. Structure A comprises two partial-length and six full-length genes, including the mdfA gene variant in the recombinant plasmid; structure B comprises four full-length genes, the mdfA, ybjG, dacC, and ybjI; structure C comprises two full-length genes, the mdfA and dacC. These results suggested that the mdfA gene can function as transporter responsible for multidrug resistance and also mediated the synergistic function with its surrounding genes in conjugative plasmids.
Assuntos
Conjugação Genética , Farmacorresistência Bacteriana Múltipla/genética , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Proteínas de Membrana Transportadoras/genética , Antibacterianos/farmacologia , Transporte Biológico , Cloranfenicol/farmacologia , Desoxirribonuclease EcoRI/genética , Desoxirribonuclease EcoRI/metabolismo , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Eritromicina/farmacologia , Escherichia coli/efeitos dos fármacos , Escherichia coli/isolamento & purificação , Escherichia coli/metabolismo , Infecções por Escherichia coli/tratamento farmacológico , Infecções por Escherichia coli/microbiologia , Proteínas de Escherichia coli/metabolismo , Genótipo , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Plasmídeos/química , Plasmídeos/metabolismo , D-Ala-D-Ala Carboxipeptidase Tipo Serina/genética , D-Ala-D-Ala Carboxipeptidase Tipo Serina/metabolismo , Tetraciclina/farmacologiaRESUMO
Fluorescent sensing systems based on the quenching of fluorophores have found wide applications in bioassays. An efficient quencher will endow the sensing system a high sensitivity. The frequently used quenchers are based on organic molecules or nanomaterials, which usually need tedious synthesizing and modifying steps, and exhibit different quenching efficiencies to different fluorophores. In this work, we for the first time report that aggregated perylene derivative can serve as a broad-spectrum and label-free quencher that is able to efficiently quench a variety of fluorophores, such as green, red and far red dyes labeled on DNA. By choosing nucleases as model biomolecules, such a broad-spectrum quencher was then employed to construct a multiplexed bioassay platform through a label-free manner. Due to the high quenching efficiency of the aggregated perylene, the proposed platform could detect nuclease with high sensitivity, with a detection limit of 0.03U/mL for EcoRV, and 0.05U/mL for EcoRI. The perylene quencher does not affect the activity of nuclease, which makes it possible to design post-addition type bioassay platform. Moreover, the proposed platform allows simultaneous and multicolor analysis of nucleases in homogeneous solution, demonstrating its value of potential application in rapid screening of multiple bio-targets.
Assuntos
Bioensaio/instrumentação , Técnicas Biossensoriais/instrumentação , DNA/química , Desoxirribonuclease EcoRI/análise , Perileno/química , Espectrometria de Fluorescência/instrumentação , DNA/genética , Desoxirribonuclease EcoRI/química , Desoxirribonuclease EcoRI/genética , Desenho de Equipamento , Análise de Falha de Equipamento , Coloração e RotulagemRESUMO
To investigate the effect of sugar pucker conformation on DNA-protein interactions, we used 2'-O-methyl nucleotide (2'-OMeN) to modify the EcoRI recognition sequence -TGAATTCT-, and monitored the enzymatic cleavage process using FRET method. The 2'-O-methyl nucleotide has a C3'-endo sugar pucker conformation different from the C2'-endo sugar pucker conformation of native DNA nucleotides. The initial reaction velocities were measured and the kinetic parameters, Km and Vmax were derived using Michaelis-Menten equation. Experimental results showed that 2'-OMeN substitutions for the EcoRI recognition sequence decreased the cleavage efficiency for A2, A3 and T4 substitutions significantly, and 2'-OMeN substitution for T5 residue inhibited the enzymatic activity completely. In contrast, substitutions for G1 and C6 could maintain the original activity. 2'-fluoro nucleic acid (2'-FNA) and locked nucleic acid (LNA) having similar C3'-endo sugar pucker conformation also demonstrated similar enzymatic results. This position-dependent enzymatic cleavage property might be attributed to the phosphate backbone distortion caused by the switch from C2'-endo to C3'-endo sugar pucker conformation, and was interpreted on the basis of the DNA-EcoRI structure. These 2'-modified nucleotides could behave as a regulatory element to modulate the enzymatic activity in vitro, and this property will have potential applications in genetic engineering and biomedicine.
Assuntos
Clivagem do DNA , DNA/metabolismo , Desoxirribonuclease EcoRI/genética , Desoxirribonuclease EcoRI/metabolismo , Sequência de Bases/genética , Transferência Ressonante de Energia de Fluorescência , Cinética , Oligonucleotídeos/genéticaRESUMO
OBJECTIVE: To describe the hearing loss in facioscapulohumeral muscular dystrophy (FSHD) and examine the relationship to genotype. METHODS: Medical records of all individuals with FSHD seen at the University of Iowa neuromuscular clinic between July 2006 and July 2012 (n = 59) were reviewed. Eleven had significant hearing loss and no non-FSHD cause. All available audiology records for these individuals were analyzed. The relationship between the FSHD mutation (EcoRI/BlnI fragment size) and hearing loss was evaluated using a logistic regression analysis. RESULTS: In patients with hearing loss, recalled age at onset of facial weakness ranged from birth to 5 years and shoulder weakness was 3 to 15 years. The age at diagnosis of hearing loss ranged from birth to 7 years. Only 2 were identified by newborn hearing screen. Most audiograms demonstrated a bilateral, sloping, high-frequency sensorineural hearing loss. Of the 4 patients with more than 5 years of data, 3 had progression of hearing loss. Logistic regression showed statistically significant negative association between the presence of hearing loss and EcoRI/BlnI fragment size (p = 0.0207). CONCLUSIONS: FSHD with a small EcoRI/BlnI fragment is associated with a bilateral, progressive, sloping, high-frequency hearing loss with onset in childhood. Patients with FSHD and small EcoRI/BlnI fragment sizes should have hearing screened, even if the child passed newborn hearing screening.
Assuntos
Perda Auditiva de Alta Frequência/fisiopatologia , Distrofia Muscular Facioescapuloumeral/fisiopatologia , Adolescente , Adulto , Idade de Início , Audiometria , Criança , Desoxirribonuclease EcoRI/genética , Progressão da Doença , Perda Auditiva de Alta Frequência/complicações , Perda Auditiva de Alta Frequência/genética , Humanos , Modelos Logísticos , Pessoa de Meia-Idade , Distrofia Muscular Facioescapuloumeral/complicações , Distrofia Muscular Facioescapuloumeral/genética , Fenótipo , Sistema de Registros , Estudos Retrospectivos , Adulto JovemRESUMO
Facioscapulohumeral muscular dystrophy (FSHD) is characterised by weakness and atrophy of the facial and shoulder girdle muscles. The FSHD phenotype segregates as an autosomal dominant trait and is caused by a deletion of an integral number of 3.3 kilobase pair (kb) repeat units on chromosome 4q35. Haplotype and Southern blot analyses of chromosome 4 resulted in the detection of two BlnI resistant deletion fragments, of 24 kb and 34 kb respectively, in a single individual from a South African FSHD family. The patient had moderate facial weakness and marked winging and high-riding of the scapulae with prominent pectoral and proximal arm muscle atrophy and weakness. Quadriceps and anterior tibial muscles were weak and the patient had bilateral foot drop. Although none of his children were symptomatic yet and only two showed very mild clinical signs, one had inherited the 24 kb deletion fragment, while the other two had the 34 kb deletion fragment. Molecular analysis conclusively identified the first compound heterozygous case in the South African FSHD population. However, in accordance with other studies of compound heterozygotes and clinical findings, no direct correlation between the clinical severity of this patient and the number of deletion fragments was observed.
Assuntos
Cromossomos Humanos Par 4/genética , Deleção de Genes , Heterozigoto , Distrofia Muscular Facioescapuloumeral/etnologia , Distrofia Muscular Facioescapuloumeral/genética , Desoxirribonuclease EcoRI/genética , Desoxirribonucleases de Sítio Específico do Tipo II/genética , Haplótipos/genética , Humanos , Masculino , Pessoa de Meia-Idade , Linhagem , África do SulRESUMO
Restriction-modification systems consist of a modification enzyme that methylates a specific DNA sequence and a restriction endonuclease that cleaves DNA lacking this epigenetic signature. Their gene expression should be finely regulated because their potential to attack the host bacterial genome needs to be controlled. In the EcoRI system, where the restriction gene is located upstream of the modification gene in the same orientation, we previously identified intragenic reverse promoters affecting gene expression. In the present work, we identified a small (88 nt) antisense RNA (Rna0) transcribed from a reverse promoter (P(REV0)) at the 3' end of the restriction gene. Its antisense transcription, as measured by transcriptional gene fusion, appeared to be terminated by the P(M1,M2) promoter. P(M1,M2) promoter-initiated transcription, in turn, appeared to be inhibited by P(REV0). Mutational inactivation of P(REV0) increased expression of the restriction gene. The biological significance of this antisense transcription is 2-fold. First, a mutation in P(REV0) increased restriction of incoming DNA. Second, the presence of the antisense RNA gene (ecoRIA) in trans alleviated cell killing after loss of the EcoRI plasmid (post-segregational killing). Taken together, these results strongly suggested the involvement of an antisense RNA in the biological regulation of this restriction-modification system.
Assuntos
Desoxirribonuclease EcoRI/genética , Regulação Bacteriana da Expressão Gênica , RNA Antissenso/fisiologia , DNA Metiltransferases Sítio Específica (Adenina-Específica)/genética , Mutação , Regiões Promotoras Genéticas , RNA Antissenso/biossíntese , RNA Antissenso/genética , Transcrição GênicaRESUMO
Removal by the light: The photochemical regulation of restriction endonucleases, which are important enzymes in molecular biology, has been investigated. Photolabile protecting groups have been installed on DNA substrates and have been demonstrated to inhibit restriction endonuclease activity until removed by UV light irradiation. Interestingly, these groups do not appear to dramatically affect initial binding of the enzyme to the DNA substrate, but rather prevent recognition of the specific cleavage site.
Assuntos
Benzodioxóis/química , Enzimas de Restrição do DNA/metabolismo , DNA/química , Timidina/análogos & derivados , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , DNA/efeitos da radiação , Clivagem do DNA , Enzimas de Restrição do DNA/genética , Desoxirribonuclease BamHI/genética , Desoxirribonuclease BamHI/metabolismo , Desoxirribonuclease EcoRI/genética , Desoxirribonuclease EcoRI/metabolismo , Desoxirribonucleases de Sítio Específico do Tipo II/genética , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Especificidade por Substrato , Timidina/química , Raios UltravioletaRESUMO
Previous studies based on case descriptions and neuroradiological findings have suggested central nervous system (CNS) involvement in facioscapulohumeral dystrophy. The aim of this work is to explore the relationship between cognitive/personality pattern and the underlying molecular defect for this muscular dystrophy. We performed a wide-ranging neuropsychological assessment of 34 molecularly confirmed facioscapulohumeral dystrophy patients and 49 control subjects, all of whom also received the Millon-II Multiaxial Clinical Inventory (MCMI-II). Patients and controls show mild learning-level differences in the neuropsychological profile, and only the hysteriform scale is statistically higher in patients than controls. The patients' intelligence quotient (IQ) is related to the size of the deleted fragment but not to the degree of muscular impairment. The results of this study indicate a cut-off point and two distinct cognitive profiles in facioscapulohumeral dystrophy, depending on the patients' molecular defect: patients with a fragment size > 24 kb show a relatively normal cognitive pattern, whereas those with a fragment size < or = 24 kb show a significantly reduced IQ and difficulties with verbal function and visuo-constructive tasks. This work provides more evidence for the involvement of the CNS in facioscapulohumeral dystrophy and suggests that the fragment size should be taken into account in the clinical management of facioscapulohumeral dystrophy as it has a predictive value on the cognitive phenotype.
Assuntos
Distrofias Musculares/genética , Distrofias Musculares/psicologia , Adolescente , Adulto , Idoso , Cognição/fisiologia , DNA/genética , Desoxirribonuclease EcoRI/genética , Feminino , Frequência do Gene , Genótipo , Humanos , Inteligência/genética , Modelos Lineares , Masculino , Pessoa de Meia-Idade , Testes Neuropsicológicos , Personalidade/fisiologia , Testes de Personalidade , Fenótipo , Análise de Regressão , Aprendizagem Verbal , Escalas de Wechsler , Adulto JovemRESUMO
Eukaryotic cells devoid of mitochondrial DNA (rho0 cells) were originally generated under artificial growth conditions utilizing ethidium bromide. The chemical is known to intercalate preferentially with the mitochondrial double-stranded DNA thereby interfering with enzymes of the replication machinery. Rho0 cell lines are highly valuable tools to study human mitochondrial disorders because they can be utilized in cytoplasmic transfer experiments. However, mutagenic effects of ethidium bromide onto the nuclear DNA cannot be excluded. To foreclose this mutagenic character during the development of rho0 cell lines, we developed an extremely mild, reliable and timesaving method to generate rho0 cell lines within 3-5 days based on an enzymatic approach. Utilizing the genes for the restriction endonuclease EcoRI and the fluorescent protein EGFP that were fused to a mitochondrial targeting sequence, we developed a CMV-driven expression vector that allowed the temporal expression of the resulting fusion enzyme in eukaryotic cells. Applied on the human cell line 143B.TK- the active protein localized to mitochondria and induced the complete destruction of endogenous mtDNA. Mouse and rat rho0 cell lines were also successfully created with this approach. Furthermore, the newly established 143B.TK- rho0 cell line was characterized in great detail thereby releasing interesting insights into the morphology and ultra structure of human rho0 mitochondria.
Assuntos
Linhagem Celular , DNA Mitocondrial/metabolismo , Desoxirribonuclease EcoRI/genética , Animais , Linhagem Celular Tumoral , Meios de Cultura , Desoxirribonuclease EcoRI/metabolismo , Corantes Fluorescentes , Proteínas de Fluorescência Verde/genética , Humanos , Camundongos , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Mitocôndrias/química , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Sinais Direcionadores de Proteínas , Ratos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Promiscuous mutant EcoRI endonucleases bind to the canonical site GAATTC more tightly than does the wild-type endonuclease, yet cleave variant (EcoRI(*)) sites more rapidly than does wild-type. The crystal structure of the A138T promiscuous mutant homodimer in complex with a GAATTC site is nearly identical to that of the wild-type complex, except that the Thr138 side chains make packing interactions with bases in the 5'-flanking regions outside the recognition hexanucleotide while excluding two bound water molecules seen in the wild-type complex. Molecular dynamics simulations confirm exclusion of these waters. The structure and simulations suggest possible reasons why binding of the A138T protein to the GAATTC site has DeltaS degrees more favorable and DeltaH degrees less favorable than for wild-type endonuclease binding. The interactions of Thr138 with flanking bases may permit A138T, unlike wild-type enzyme, to form complexes with EcoRI(*) sites that structurally resemble the specific wild-type complex with GAATTC.
Assuntos
DNA/metabolismo , Desoxirribonuclease EcoRI/química , Desoxirribonuclease EcoRI/genética , Mutação , Termodinâmica , Sequência de Bases , Sítios de Ligação , Cristalografia por Raios X , Desoxirribonuclease EcoRI/metabolismo , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Estrutura Terciária de ProteínaRESUMO
A nucleotide sequence was established for the full-length Sporosarcina species 9D operon coding for enzymes of type II restriction-modification system Sse9I. These enzymes recognize the tetranucleotide DNA sequence 5'-AATT-3'. The operon was shown to consist of three genes that are situated with the order: sse9IC-sse9IR-sse9IM and are transcribed in the same direction. These genes encode the control protein (C.Sse9I), restriction endonuclease (R.Sse9I) and DNA-methyltransferase (M.Sse9I), respectively. A specific DNA sequence (C-box) presumably recognized by C-protein was found immediately upstream of sse9IC gene. The comparative analysis of amino acid sequences of C.Sse9I and R.Sse9I with those of relative proteins has been done. It was found that R.Sse9I revealed the most homology with the segments of R.MunI (5'-CAATTG-3') and R.EcoRI (5'-GAATTC-3'), where amino acid residues, responsible for recogniton of AATT core sequence are located. The sse9IR gene was cloned into the temperature-inducible expression vector, and recombinant Sse9I restriction endonuclease preparation was isolated.
Assuntos
Enzimas de Restrição-Modificação do DNA/metabolismo , Óperon , DNA Metiltransferases Sítio Específica (Adenina-Específica)/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Enzimas de Restrição-Modificação do DNA/genética , Desoxirribonuclease EcoRI/genética , Desoxirribonucleases de Sítio Específico do Tipo II/genética , Bactérias Gram-Positivas Formadoras de Endosporo/enzimologia , Dados de Sequência Molecular , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , DNA Metiltransferases Sítio Específica (Adenina-Específica)/genética , DNA Metiltransferases Sítio Específica (Adenina-Específica)/isolamento & purificaçãoRESUMO
The hemimethylated d(GATC) sequence that directs Escherichia coli mismatch repair can reside on either side of a mismatch at a separation distance of 1,000 bp or more. Initiation of repair involves the mismatch-, MutS-, and MutL-dependent activation of MutH endonuclease, which incises the unmethylated strand at the d(GATC) sequence, with the ensuing strand break serving as the loading site for the appropriate 3'-to-5' or 5'-to-3' excision system. However, the mechanism responsible for the coordinated recognition of the mismatch and a hemimodified d(GATC) site is uncertain. We show that a protein roadblock (EcoRI(E111Q), a hydrolytically defective form of EcoRI endonuclease) placed on the helix between the two DNA sites inhibits MutH activation by 70-80% and that events that escape inhibition are attributable, at least in part, to diffusion of EcoRI(E111Q) away from its recognition site. We also demonstrate that a double-strand break located within the shorter path linking the mismatch and a d(GATC) site in a circular heteroduplex abolishes MutH activation, whereas a double-strand break within the longer path is without effect. These findings support the idea that initiation of mismatch repair involves signaling along the helix contour.
Assuntos
Reparo de Erro de Pareamento de DNA , DNA/química , Desoxirribonuclease EcoRI/metabolismo , Sequência de Bases , DNA Helicases/metabolismo , Desoxirribonuclease EcoRI/genética , Ativação Enzimática , Ácido Glutâmico/genética , Ácido Glutâmico/metabolismo , Mutação/genética , Especificidade por SubstratoRESUMO
Type II restriction-modification systems are expected to possess mechanisms for tight regulation of their expression to suppress the potential of lethal attack on their host bacteria when they establish and maintain themselves within them. Although the EcoRI restriction enzyme has been well characterized, regulation of its expression is still poorly understood. In this study, mutational analysis with lacZ gene fusion and primer extension assay identified a promoter for the transcription of the ecoRIR gene. Further analyses revealed that an intragenic region containing two overlapping reverse promoter-like elements acted as a negative regulator for ecoRIR gene expression. The activity of these putative reverse promoters was verified by transcriptional gene fusion, primer extension and in vitro transcription. Mutations in these reverse promoters resulted in increased gene expression in both translational and transcriptional gene fusions. An RNase protection assay revealed that the transcript level of the wild type relative to that of the reverse promoter mutant at the downstream regions was much lower than the level at the upstream regions. This suggests that these reverse promoter-like elements affect their downstream transcript level. The possible mechanisms of this kind of negative regulation, in addition to their possible biological roles, are discussed.