Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 241
Filtrar
2.
Structure ; 29(4): 371-384.e3, 2021 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-33306961

RESUMO

Proteins are known to undergo structural changes upon binding to partner proteins. However, the prevalence, extent, location, and function of change in protein dynamics due to transient protein-protein interactions is not well documented. Here, we have analyzed a dataset of 58 protein-protein complexes of known three-dimensional structure and structures of their corresponding unbound forms to evaluate dynamics changes induced by binding. Fifty-five percent of cases showed significant dynamics change away from the interfaces. This change is not always accompanied by an observed structural change. Binding of protein partner is found to alter inter-residue communication within the tertiary structure in about 90% of cases. Also, residue motions accessible to proteins in unbound form were not always maintained in the bound form. Further analyses revealed functional roles for the distant site where dynamics change was observed. Overall, the results presented here strongly suggest that alteration of protein dynamics due to binding of a partner protein commonly occurs.


Assuntos
Sítio Alostérico , Simulação de Dinâmica Molecular , Mapeamento de Interação de Proteínas/métodos , Ciclofilina A/química , Ciclofilina A/metabolismo , Desoxirribonucleases de Sítio Específico do Tipo I/química , Desoxirribonucleases de Sítio Específico do Tipo I/metabolismo , Humanos , Ligação Proteica
3.
Int J Antimicrob Agents ; 56(2): 106050, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32544567

RESUMO

Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae (KPC-KP) have disseminated worldwide and are a major threat to public health. The multidrug-resistant (MDR)-phenotype of KPC-KP are commonly associated with the presence of high molecular weight blaKPC plasmids. Restriction-modification (R-M) systems provide bacteria with innate defense against plasmids or other infectious gene elements. As blaKPC plasmids are favored by such MDR K. pneumoniae, it was of interest to examine the co-distribution of R-M and acquired blaKPC plasmids in KPC-KP. A total of 459 clinical K. pneumoniae isolates in China and 217 global whole-genome sequences in GenBank were collected to determine the prevalence of type I R-M systems. The type I R-M systems were scarce in the KPC-positive group and high-risk Klebsiella pneumoniae clonal group 258 (CG258). The polymorphisms of type I R-M observed in K. pneumoniae revealed the ubiquity of their recognition sequences in DNA; therefore, the type I R-M systems could attack most invading DNA elements, such as blaKPC genes. Overall, this work indicated the type I R-M systems may impact the acquisition of blaKPC genes in K. pneumoniae.


Assuntos
Proteínas de Bactérias/genética , Desoxirribonucleases de Sítio Específico do Tipo I/genética , Transferência Genética Horizontal , Klebsiella pneumoniae/genética , beta-Lactamases/genética , Farmacorresistência Bacteriana Múltipla/genética , Genoma Bacteriano , Humanos , Sequências Repetitivas Dispersas , Infecções por Klebsiella/microbiologia , Klebsiella pneumoniae/classificação , Plasmídeos/genética , Polimorfismo Genético , Sequenciamento Completo do Genoma
4.
Biochemistry (Mosc) ; 85(3): 318-325, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32564736

RESUMO

The antirestriction ArdB protein inhibits the endonuclease activity of type I restriction/modification (RM) systems in vivo; however, the mechanism of inhibition remains unknown. In this study, we showed that recombinant ArdB from Escherichia coli cells co-purified with DNA. When overexpressed in E. coli cells, a portion of ArdB protein formed insoluble DNA-free aggregates. Only native ArdB, but not the ArdBΔD141 mutant lacking the antirestriction activity, co-purified with DNA upon anion-exchange and affinity chromatography or total DNA isolation from formaldehyde-treated cells. These observations confirm the hypothesis that ArdB blocks DNA translocation via the R subunits of the R2M2S complex of type I RM enzymes.


Assuntos
DNA Bacteriano/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Cromatografia por Troca Iônica , Enzimas de Restrição-Modificação do DNA/metabolismo , Desoxirribonucleases de Sítio Específico do Tipo I/metabolismo , Formaldeído/química , Espectrometria de Massas , Mutação , Ligação Proteica , Proteínas Recombinantes/metabolismo
5.
Nat Microbiol ; 5(9): 1107-1118, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32483229

RESUMO

Type I restriction-modification (R-M) systems are widespread in prokaryotic genomes and provide robust protection against foreign DNA. They are multisubunit enzymes with methyltransferase, endonuclease and translocase activities. Despite extensive studies over the past five decades, little is known about the molecular mechanisms of these sophisticated machines. Here, we report the cryo-electron microscopy structures of the representative EcoR124I R-M system in different assemblies (R2M2S1, R1M2S1 and M2S1) bound to target DNA and the phage and mobile genetic element-encoded anti-restriction proteins Ocr and ArdA. EcoR124I can precisely regulate different enzymatic activities by adopting distinct conformations. The marked conformational transitions of EcoR124I are dependent on the intrinsic flexibility at both the individual-subunit and assembled-complex levels. Moreover, Ocr and ArdA use a DNA-mimicry strategy to inhibit multiple activities, but do not block the conformational transitions of the complexes. These structural findings, complemented by mutational studies of key intermolecular contacts, provide insights into assembly, operation and inhibition mechanisms of type I R-M systems.


Assuntos
Enzimas de Restrição-Modificação do DNA/química , Enzimas de Restrição-Modificação do DNA/metabolismo , Desoxirribonucleases de Sítio Específico do Tipo I/química , Desoxirribonucleases de Sítio Específico do Tipo I/metabolismo , Proteínas de Bactérias , Microscopia Crioeletrônica , DNA/química , DNA/metabolismo , Enzimas de Restrição-Modificação do DNA/genética , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Desoxirribonucleases de Sítio Específico do Tipo I/genética , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Modelos Moleculares , Mutação , Conformação Proteica , Proteínas Repressoras , Proteínas Virais
6.
FASEB J ; 34(1): 1038-1051, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31914596

RESUMO

Over recent years several examples of randomly switching methyltransferases, associated with Type III restriction-modification (R-M) systems, have been described in pathogenic bacteria. In every case examined, changes in simple DNA sequence repeats result in variable methyltransferase expression and result in global changes in gene expression, and differentiation of the bacterial cell into distinct phenotypes. These epigenetic regulatory systems are called phasevarions, phase-variable regulons, and are widespread in bacteria, with 17.4% of Type III R-M system containing simple DNA sequence repeats. A distinct, recombination-driven random switching system has also been described in Streptococci in Type I R-M systems that also regulate gene expression. Here, we interrogate the most extensive and well-curated database of R-M systems, REBASE, by searching for all possible simple DNA sequence repeats in the hsdRMS genes that encode Type I R-M systems. We report that 7.9% of hsdS, 2% of hsdM, and of 4.3% of hsdR genes contain simple sequence repeats that are capable of mediating phase variation. Phase variation of both hsdM and hsdS genes will lead to differential methyltransferase expression or specificity, and thereby the potential to control phasevarions. These data suggest that in addition to well characterized phasevarions controlled by Type III mod genes, and the previously described Streptococcal Type I R-M systems that switch via recombination, approximately 10% of all Type I R-M systems surveyed herein have independently evolved the ability to randomly switch expression via simple DNA sequence repeats.


Assuntos
Epigênese Genética , Repetições de Microssatélites , Regulon , Proteínas de Bactérias/genética , Biologia Computacional , DNA/análise , Metilação de DNA , Metilases de Modificação do DNA/metabolismo , Enzimas de Restrição-Modificação do DNA/genética , Enzimas de Restrição-Modificação do DNA/metabolismo , Desoxirribonucleases de Sítio Específico do Tipo I/genética , Fusobacterium nucleatum , Perfilação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica , Mannheimia haemolytica , Metiltransferases/metabolismo , Fenótipo , Pseudomonas aeruginosa , Salmonella enterica
7.
mBio ; 10(6)2019 12 17.
Artigo em Inglês | MEDLINE | ID: mdl-31848274

RESUMO

Staphylococcus epidermidis is a significant opportunistic pathogen of humans. Molecular studies in this species have been hampered by the presence of restriction-modification (RM) systems that limit introduction of foreign DNA. Here, we establish the complete genomes and methylomes for seven clinically significant, genetically diverse S. epidermidis isolates and perform the first systematic genomic analyses of the type I RM systems within both S. epidermidis and Staphylococcus aureus Our analyses revealed marked differences in the gene arrangement, chromosomal location, and movement of type I RM systems between the two species. Unlike S. aureus, S. epidermidis type I RM systems demonstrate extensive diversity even within a single genetic lineage. This is contrary to current assumptions and has important implications for approaching the genetic manipulation of S. epidermidis Using Escherichia coli plasmid artificial modification (PAM) to express S. epidermidishsdMS, we readily overcame restriction barriers in S. epidermidis and achieved electroporation efficiencies equivalent to those of modification-deficient mutants. With these functional experiments, we demonstrated how genomic data can be used to predict both the functionality of type I RM systems and the potential for a strain to be electroporation proficient. We outline an efficient approach for the genetic manipulation of S. epidermidis strains from diverse genetic backgrounds, including those that have hitherto been intractable. Additionally, we identified S. epidermidis BPH0736, a naturally restriction-defective, clinically significant, multidrug-resistant ST2 isolate, as an ideal candidate for molecular studies.IMPORTANCEStaphylococcus epidermidis is a major cause of hospital-acquired infections, especially those related to implanted medical devices. Understanding how S. epidermidis causes disease and devising ways to combat these infections have been hindered by an inability to genetically manipulate clinically significant hospital-adapted strains. Here, we provide the first comprehensive analyses of the barriers to the uptake of foreign DNA in S. epidermidis and demonstrate that these are distinct from those described for S. aureus Using these insights, we demonstrate an efficient approach for the genetic manipulation of S. epidermidis to enable the study of clinical isolates for the first time.


Assuntos
Biologia Computacional , Mineração de Dados , Desoxirribonucleases de Sítio Específico do Tipo I/genética , Epigenoma , Epigenômica , Perfilação da Expressão Gênica , Staphylococcus epidermidis/fisiologia , Mapeamento Cromossômico , Biologia Computacional/métodos , Elementos de DNA Transponíveis , Desoxirribonucleases de Sítio Específico do Tipo I/química , Desoxirribonucleases de Sítio Específico do Tipo I/metabolismo , Epigenômica/métodos , Evolução Molecular , Interações Hospedeiro-Patógeno , Humanos , Filogenia , Plasmídeos/genética , Plasmídeos/metabolismo , Fagos de Staphylococcus/genética , Staphylococcus epidermidis/classificação , Staphylococcus epidermidis/virologia
8.
Appl Environ Microbiol ; 85(2)2019 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-30389763

RESUMO

The gastrointestinal colonizer Enterococcus faecium is a leading cause of hospital-acquired infections. Multidrug-resistant (MDR) E. faecium isolates are particularly concerning for infection treatment. Previous comparative genomic studies revealed that subspecies referred to as clade A and clade B exist within E. faecium MDR E. faecium isolates belong to clade A, while clade B consists of drug-susceptible fecal commensal E. faecium isolates. Isolates from clade A are further grouped into two subclades, clades A1 and A2. In general, clade A1 isolates are hospital-epidemic isolates, whereas clade A2 isolates are isolates from animals and sporadic human infections. Such phylogenetic separation indicates that reduced gene exchange occurs between the clades. We hypothesize that endogenous barriers to gene exchange exist between E. faecium clades. Restriction-modification (R-M) systems are such barriers in other microbes. We utilized a bioinformatics analysis coupled with second-generation and third-generation deep-sequencing platforms to characterize the methylomes of two representative E. faecium strains, one from clade A1 and one from clade B. We identified a type I R-M system that is clade A1 specific, is active for DNA methylation, and significantly reduces the transformability of clade A1 E. faecium Based on our results, we conclude that R-M systems act as barriers to horizontal gene exchange in E. faecium and propose that R-M systems contribute to E. faecium subspecies separation.IMPORTANCEEnterococcus faecium is a leading cause of hospital-acquired infections around the world. Rising antibiotic resistance in certain E. faecium lineages leaves fewer treatment options. The overarching aim of this work was to determine whether restriction-modification (R-M) systems contribute to the structure of the E. faecium species, wherein hospital-epidemic and non-hospital-epidemic isolates have distinct evolutionary histories and highly resolved clade structures. R-M provides bacteria with a type of innate immunity to horizontal gene transfer (HGT). We identified a type I R-M system that is enriched in the hospital-epidemic clade and determined that it is active for DNA modification activity and significantly impacts HGT. Overall, this work is important because it provides a mechanism for the observed clade structure of E. faecium as well as a mechanism for facilitated gene exchange among hospital-epidemic E. faecium isolates.


Assuntos
Desoxirribonucleases de Sítio Específico do Tipo I/genética , Enterococcus faecium/genética , Evolução Molecular , Genoma Bacteriano/genética , Biologia Computacional , Desoxirribonucleases de Sítio Específico do Tipo I/metabolismo , Enterococcus faecium/metabolismo , Infecções por Bactérias Gram-Positivas/microbiologia , Sequenciamento de Nucleotídeos em Larga Escala , Hospitais
9.
Nucleic Acids Res ; 46(17): 9067-9080, 2018 09 28.
Artigo em Inglês | MEDLINE | ID: mdl-30165537

RESUMO

Restriction Modification (RM) systems prevent the invasion of foreign genetic material into bacterial cells by restriction and protect the host's genetic material by methylation. They are therefore important in maintaining the integrity of the host genome. RM systems are currently classified into four types (I to IV) on the basis of differences in composition, target recognition, cofactors and the manner in which they cleave DNA. Comparing the structures of the different types, similarities can be observed suggesting an evolutionary link between these different types. This work describes the 'deconstruction' of a large Type I RM enzyme into forms structurally similar to smaller Type II RM enzymes in an effort to elucidate the pathway taken by Nature to form these different RM enzymes. Based upon the ability to engineer new enzymes from the Type I 'scaffold', an evolutionary pathway and the evolutionary pressures required to move along the pathway from Type I RM systems to Type II RM systems are proposed. Experiments to test the evolutionary model are discussed.


Assuntos
DNA Bacteriano/metabolismo , Desoxirribonucleases de Sítio Específico do Tipo I/metabolismo , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Proteínas de Escherichia coli/metabolismo , Evolução Molecular , Modelos Genéticos , Sequência de Aminoácidos , Sítios de Ligação , DNA Bacteriano/química , DNA Bacteriano/genética , Desoxirribonucleases de Sítio Específico do Tipo I/química , Desoxirribonucleases de Sítio Específico do Tipo I/genética , Desoxirribonucleases de Sítio Específico do Tipo II/química , Desoxirribonucleases de Sítio Específico do Tipo II/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Cinética , Modelos Moleculares , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Engenharia de Proteínas , Domínios e Motivos de Interação entre Proteínas , Estrutura Terciária de Proteína , Homologia Estrutural de Proteína , Relação Estrutura-Atividade
10.
J Biol Chem ; 293(39): 15043-15054, 2018 09 28.
Artigo em Inglês | MEDLINE | ID: mdl-30054276

RESUMO

Although EcoR124 is one of the better-studied Type I restriction-modification enzymes, it still presents many challenges to detailed analyses because of its structural and functional complexity and missing structural information. In all available structures of its motor subunit HsdR, responsible for DNA translocation and cleavage, a large part of the HsdR C terminus remains unresolved. The crystal structure of the C terminus of HsdR, obtained with a crystallization chaperone in the form of pHluorin fusion and refined to 2.45 Å, revealed that this part of the protein forms an independent domain with its own hydrophobic core and displays a unique α-helical fold. The full-length HsdR model, based on the WT structure and the C-terminal domain determined here, disclosed a proposed DNA-binding groove lined by positively charged residues. In vivo and in vitro assays with a C-terminal deletion mutant of HsdR supported the idea that this domain is involved in complex assembly and DNA binding. Conserved residues identified through sequence analysis of the C-terminal domain may play a key role in protein-protein and protein-DNA interactions. We conclude that the motor subunit of EcoR124 comprises five structural and functional domains, with the fifth, the C-terminal domain, revealing a unique fold characterized by four conserved motifs in the IC subfamily of Type I restriction-modification systems. In summary, the structural and biochemical results reported here support a model in which the C-terminal domain of the motor subunit HsdR of the endonuclease EcoR124 is involved in complex assembly and DNA binding.


Assuntos
Proteínas de Ligação a DNA/química , Desoxirribonucleases de Sítio Específico do Tipo I/química , Proteínas de Escherichia coli/química , Escherichia coli/química , Sequência de Aminoácidos , Fenômenos Biofísicos , Cristalografia por Raios X , Proteínas de Ligação a DNA/genética , Desoxirribonucleases de Sítio Específico do Tipo I/genética , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Conformação Proteica , Domínios Proteicos/genética , Subunidades Proteicas/química , Subunidades Proteicas/genética
11.
J Mol Model ; 24(7): 176, 2018 Jun 26.
Artigo em Inglês | MEDLINE | ID: mdl-29943199

RESUMO

Type I restriction-modification enzymes differ significantly from the type II enzymes commonly used as molecular biology reagents. On hemi-methylated DNAs type I enzymes like the EcoR124I restriction-modification complex act as conventional adenine methylases at their specific target sequences, but unmethylated targets induce them to translocate thousands of base pairs through the stationary enzyme before cleaving distant sites nonspecifically. EcoR124I is a superfamily 2 DEAD-box helicase like eukaryotic double-strand DNA translocase Rad54, with two RecA-like helicase domains and seven characteristic sequence motifs that are implicated in translocation. In Rad54 a so-called extended region adjacent to motif III is involved in ATPase activity. Although the EcoR124I extended region bears sequence and structural similarities with Rad54, it does not influence ATPase or restriction activity as shown in this work, but mutagenesis of the conserved glycine residue of its motif III does alter ATPase and DNA cleavage activity. Through the lens of molecular dynamics, a full model of HsdR of EcoR124I based on available crystal structures allowed interpretation of functional effects of mutants in motif III and its extended region. The results indicate that the conserved glycine residue of motif III has a role in positioning the two helicase domains.


Assuntos
DNA Helicases/química , Desoxirribonucleases de Sítio Específico do Tipo I/química , Domínios e Motivos de Interação entre Proteínas , Subunidades Proteicas/química , Trifosfato de Adenosina/química , Sequência de Aminoácidos , DNA Helicases/genética , DNA Helicases/metabolismo , Desoxirribonucleases de Sítio Específico do Tipo I/genética , Desoxirribonucleases de Sítio Específico do Tipo I/metabolismo , Ativação Enzimática , Hidrólise , Simulação de Dinâmica Molecular , Complexos Multienzimáticos/química , Mutação , Análise de Componente Principal , Conformação Proteica , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo
12.
Clin Rheumatol ; 37(6): 1529-1537, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29468338

RESUMO

This study aimed to explore whether vitamin D receptor (VDR) polymorphisms are associated with susceptibility to systemic lupus erythematosus (SLE). Meta-analyses were conducted to determine the associations between the VDR FokI, TaqI, and ApaI polymorphisms and SLE in all subjects and each ethnic group. A total of 12 studies were considered in the meta-analysis, which involved 1974 patients and 2506 controls. Meta-analysis of the VDR FokI polymorphism showed no association between SLE and the FokI F allele in all study subjects (OR = 1.047, 95% CI = 0.781-1.403, p = 0.758). However, the meta-analysis showed a significant association between SLE and the VDR FokI F allele in the Arab population (OR = 1.721, 95% CI = 1.417-2.088, p < 0.001). Analysis using the recessive and dominant models and homozygote contrast showed the same pattern for the VDR FokI F allele in Europeans, Asians, and Arabs. This meta-analysis showed that polymorphisms in VDR FokI, TaqI, and ApaI are not associated with SLE susceptibility in overall, European and Asian populations. However, the VDR FokI polymorphism is associated with SLE susceptibility in the Arab population.


Assuntos
Lúpus Eritematoso Sistêmico/genética , Receptores de Calcitriol/genética , Estudos de Casos e Controles , Desoxirribonucleases de Sítio Específico do Tipo I , Predisposição Genética para Doença , Humanos , Polimorfismo de Fragmento de Restrição
13.
J Comput Aided Mol Des ; 31(12): 1063-1072, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-29177929

RESUMO

I-DmoI, from the hyperthermophilic archaeon Desulfurococcus mobilis, belongs to the LAGLIDADG homing endonuclease protein family. Its members are highly specific enzymes capable of recognizing long DNA target sequences, thus providing potential tools for genome manipulation. Working towards this particular application, many efforts have been made to generate mesophilic variants of I-DmoI that function at lower temperatures than the wild-type. Here, we report a structural and computational analysis of two I-DmoI mesophilic mutants. Despite very limited structural variations between the crystal structures of these variants and the wild-type, a different dynamical behaviour near the cleavage sites is observed. In particular, both the dynamics of the water molecules and the protein perturbation effect on the cleavage site correlate well with the changes observed in the experimental enzymatic activity.


Assuntos
Desoxirribonucleases de Sítio Específico do Tipo I , Modelos Moleculares , Proteínas Arqueais/química , Proteínas Arqueais/genética , Proteínas Arqueais/metabolismo , Simulação por Computador , Proteínas de Ligação a DNA/metabolismo , Desoxirribonucleases de Sítio Específico do Tipo I/química , Desoxirribonucleases de Sítio Específico do Tipo I/genética , Desoxirribonucleases de Sítio Específico do Tipo I/metabolismo , Desulfurococcaceae/enzimologia , Mutação , Ligação Proteica , Conformação Proteica
15.
Mol Biol (Mosk) ; 51(5): 831-835, 2017.
Artigo em Russo | MEDLINE | ID: mdl-29116070

RESUMO

Antirestriction proteins of the ArdB/KlcA family are specific inhibitors of restriction (endonuclease) activity of type-I restriction/modification enzymes. The effect of conserved amino acid residues on the antirestriction activity of the ArdB protein encoded by the transmissible R64 (IncI1) plasmid has been investigated. An analysis of the amino acid sequences of ArdB homologues demonstrated the presence of four groups of conserved residues ((1) R16, E32, and W51; (2) Y46 and G48; (3) S81, D83 and E132, and (4) N77, L(I)140, and D141) on the surface of the protein globule. Amino acid residues of the fourth group showed a unique localization pattern with the terminal residue protruding beyond the globule surface. The replacement of two conserved amino acids (D141 and N77) located in the close vicinity of each other on the globule surface showed that the C-terminal D141 is essential for the antirestriction activity of ArdB. The deletion of this residue, as well as replacement by a hydrophobic threonine residue (D141T), completely abolished the antirestriction activity of ArdB. The synonymous replacement of D141 by a glutamic acid residue (D141E) caused an approximately 30-fold decrease of the antirestriction activity of ArdB, and the point mutation N77A caused an approximately 20-fold decrease in activity. The residues D141 and N77 located on the surface of the protein globule are presumably essential for the formation of a contact between ArdB and a currently unknown factor that modulates the activity of type-I restriction/modification enzymes.


Assuntos
Escherichia coli K12/química , Proteínas de Escherichia coli/química , Substituição de Aminoácidos , Ácido Aspártico/química , Ácido Aspártico/genética , Desoxirribonucleases de Sítio Específico do Tipo I/química , Desoxirribonucleases de Sítio Específico do Tipo I/genética , Escherichia coli K12/genética , Proteínas de Escherichia coli/genética , Mutação de Sentido Incorreto , Domínios Proteicos
16.
Nucleic Acids Res ; 45(6): 3395-3406, 2017 04 07.
Artigo em Inglês | MEDLINE | ID: mdl-28180279

RESUMO

Staphylococcus aureus displays a clonal population structure in which horizontal gene transfer between different lineages is extremely rare. This is due, in part, to the presence of a Type I DNA restriction-modification (RM) system given the generic name of Sau1, which maintains different patterns of methylation on specific target sequences on the genomes of different lineages. We have determined the target sequences recognized by the Sau1 Type I RM systems present in a wide range of the most prevalent S. aureus lineages and assigned the sequences recognized to particular target recognition domains within the RM enzymes. We used a range of biochemical assays on purified enzymes and single molecule real-time sequencing on genomic DNA to determine these target sequences and their patterns of methylation. Knowledge of the main target sequences for Sau1 will facilitate the synthesis of new vectors for transformation of the most prevalent lineages of this 'untransformable' bacterium.


Assuntos
Metilases de Modificação do DNA/química , Metilases de Modificação do DNA/metabolismo , Desoxirribonucleases de Sítio Específico do Tipo I/química , Desoxirribonucleases de Sítio Específico do Tipo I/metabolismo , Staphylococcus aureus/enzimologia , Sequência de Aminoácidos , DNA/química , DNA/metabolismo , Domínios Proteicos , Análise de Sequência de DNA , Staphylococcus aureus/genética , Transformação Bacteriana
17.
Genes Cells ; 21(11): 1150-1163, 2016 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-27654959

RESUMO

Serum endonucleases are essential for degrading the chromatin released from dead cells and preventing autoimmune diseases such as systemic lupus erythematosus. Serum DNase I is known as the major endonuclease, but recently, another endonuclease, DNase γ/DNase I-like 3, gained attention. However, the precise role of each endonuclease, especially that of DNase γ, remains unclear. In this study, we distinguished the activities of DNase γ from those of DNase I in mouse serum and concluded that both cooperated in degrading DNA during necrosis: DNase γ functions as the primary chromatolytic activity, causing internucleosomal DNA fragmentation, and DNase I as the secondary one, causing random DNA digestion for its complete degradation. These results were confirmed by two in vivo experimental mouse models, in which necrosis was induced, acetaminophen-induced hepatic injury and streptozotocin-induced ß-cell necrosis models. We also determined that DNase γ functions as a backup endonuclease for caspase-activated DNase (CAD) in the secondary necrosis phase after γ-ray-induced apoptosis in vivo.


Assuntos
Degradação Necrótica do DNA , Desoxirribonucleases de Sítio Específico do Tipo I/sangue , Desoxirribonucleases/sangue , Endodesoxirribonucleases/sangue , Animais , Apoptose , Linhagem Celular Tumoral , Fragmentação do DNA , Feminino , Humanos , Fígado/metabolismo , Fígado/ultraestrutura , Masculino , Camundongos , Camundongos Knockout , Complexos Multienzimáticos , Necrose/sangue , Proteínas de Ligação a Poli-ADP-Ribose , Baço/metabolismo
18.
Acta Crystallogr F Struct Biol Commun ; 72(Pt 9): 672-6, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27599856

RESUMO

The HsdR subunit of the type I restriction-modification system EcoR124I is responsible for the translocation as well as the restriction activity of the whole complex consisting of the HsdR, HsdM and HsdS subunits, and while crystal structures are available for the wild type and several mutants, the C-terminal domain comprising approximately 150 residues was not resolved in any of these structures. Here, three fusion constructs with the GFP variant pHluorin developed to overexpress, purify and crystallize the C-terminal domain of HsdR are reported. The shortest of the three encompassed HsdR residues 887-1038 and yielded crystals that belonged to the orthorhombic space group C2221, with unit-cell parameters a = 83.42, b = 176.58, c = 126.03 Å, α = ß = γ = 90.00° and two molecules in the asymmetric unit (VM = 2.55 Å(3) Da(-1), solvent content 50.47%). X-ray diffraction data were collected to a resolution of 2.45 Å.


Assuntos
Desoxirribonucleases de Sítio Específico do Tipo I/química , Proteínas de Escherichia coli/química , Escherichia coli/genética , Proteínas de Fluorescência Verde/química , Subunidades Proteicas/química , Proteínas Recombinantes de Fusão/química , Sequência de Aminoácidos , Clonagem Molecular , Cristalização , Cristalografia por Raios X , Desoxirribonucleases de Sítio Específico do Tipo I/genética , Desoxirribonucleases de Sítio Específico do Tipo I/metabolismo , Escherichia coli/química , Escherichia coli/enzimologia , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Expressão Gênica , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Plasmídeos/química , Plasmídeos/metabolismo , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Difração de Raios X
19.
Biopolymers ; 105(12): 898-904, 2016 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-27506595

RESUMO

The conformational behavior of the wild-type endonucleases I-DmoI and two of its mutants has been studied in the presence and in the absence of DNA target sequences by means of extended molecular dynamics simulations. Our results show that in the absence of DNA, the three protein forms explore a similar essential conformational space, whereas when bound to the same DNA target sequence of 25 base pairs, they diversify and restrain the subspace explored. In addition, the differences in the essential subspaces explored by the residues near the catalytic site for both the bound and unbound forms are discussed in background of the experimental protein activity.


Assuntos
DNA/química , Desoxirribonucleases de Sítio Específico do Tipo I/química , Simulação de Dinâmica Molecular
20.
Sci Rep ; 6: 28579, 2016 06 29.
Artigo em Inglês | MEDLINE | ID: mdl-27353146

RESUMO

The methylation-dependent restriction endonuclease (REase) BisI (G(m5)C ↓ NGC) is found in Bacillus subtilis T30. We expressed and purified the BisI endonuclease and 34 BisI homologs identified in bacterial genomes. 23 of these BisI homologs are active based on digestion of (m5)C-modified substrates. Two major specificities were found among these BisI family enzymes: Group I enzymes cut GCNGC containing two to four (m5)C in the two strands, or hemi-methylated sites containing two (m5)C in one strand; Group II enzymes only cut GCNGC sites containing three to four (m5)C, while one enzyme requires all four cytosines to be modified for cleavage. Another homolog, Esp638I cleaves GCS ↓ SGC (relaxed specificity RCN ↓ NGY, containing at least four (m5)C). Two BisI homologs show degenerate specificity cleaving unmodified DNA. Many homologs are small proteins ranging from 150 to 190 amino acid (aa) residues, but some homologs associated with mobile genetic elements are larger and contain an extra C-terminal domain. More than 156 BisI homologs are found in >60 bacterial genera, indicating that these enzymes are widespread in bacteria. They may play an important biological function in restricting pre-modified phage DNA.


Assuntos
Bacillus subtilis/enzimologia , Proteínas de Bactérias , Metilação de DNA , DNA/química , Desoxirribonucleases de Sítio Específico do Tipo I , Expressão Gênica , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/química , Proteínas de Bactérias/isolamento & purificação , Desoxirribonucleases de Sítio Específico do Tipo I/biossíntese , Desoxirribonucleases de Sítio Específico do Tipo I/química , Desoxirribonucleases de Sítio Específico do Tipo I/isolamento & purificação , Domínios Proteicos , Homologia Estrutural de Proteína
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...