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2.
Nucleic Acids Res ; 46(12): 6229-6237, 2018 07 06.
Artigo em Inglês | MEDLINE | ID: mdl-29846668

RESUMO

Endonucleolytic cleavage of DNA by Type III restriction-modification (RM) enzymes requires long-range communication between at least two recognition sites in inverted orientation. This results in convergence of two nuclease domains, one each from the enzymes loaded at the recognition sites with one still bound to the site. The nucleases catalyze scission of the single-strands leading to double-strand DNA break. An obscure feature of the Type III RM enzymes EcoP1I and EcoP15I is their ability to cleave DNA having a single recognition site under certain conditions. Here we demonstrate that single-site cleavage is the result of cooperation between an enzyme bound to the recognition site in cis and one in trans. DNA cleavage is catalyzed by converging nucleases that are activated by hydrolysis-competent ATPase in presence of their respective DNA substrates. Furthermore, a single activated nuclease cannot nick a strand on its own, and requires the partner. Based on the commonalities in the features of single-site and two-site cleavage derived from this study, we propose that their mechanism is similar. Furthermore, the products of two-site cleavage can act as substrates and activators of single-site cleavage. The difference in the two modes lies in how the two cooperating enzymes converge, which in case of single-site cleavage appears to be via 3D diffusion.


Assuntos
Adenosina Trifosfatases/metabolismo , Desoxirribonucleases de Sítio Específico do Tipo III/metabolismo , Trifosfato de Adenosina/metabolismo , DNA/química , DNA/metabolismo , Clivagem do DNA , Desoxirribonucleases de Sítio Específico do Tipo III/genética , Mutação
3.
ACS Synth Biol ; 7(1): 98-106, 2018 01 19.
Artigo em Inglês | MEDLINE | ID: mdl-28968490

RESUMO

Scarless genetic manipulation of genomes is an essential tool for biological research. The restriction-modification (R-M) system is a defense system in bacteria that protects against invading genomes on the basis of its ability to distinguish foreign DNA from self DNA. Here, we designed an R-M system-mediated genome editing (RMGE) technique for scarless genetic manipulation in different microorganisms. For bacteria with Type IV REase, an RMGE technique using the inducible DNA methyltransferase gene, bceSIIM (RMGE-bceSIIM), as the counter-selection cassette was developed to edit the genome of Escherichia coli. For bacteria without Type IV REase, an RMGE technique based on a restriction endonuclease (RMGE-mcrA) was established in Bacillus subtilis. These techniques were successfully used for gene deletion and replacement with nearly 100% counter-selection efficiencies, which were higher and more stable compared to conventional methods. Furthermore, precise point mutation without limiting sites was achieved in E. coli using RMGE-bceSIIM to introduce a single base mutation of A128C into the rpsL gene. In addition, the RMGE-mcrA technique was applied to delete the CAN1 gene in Saccharomyces cerevisiae DAY414 with 100% counter-selection efficiency. The effectiveness of the RMGE technique in E. coli, B. subtilis, and S. cerevisiae suggests the potential universal usefulness of this technique for microbial genome manipulation.


Assuntos
Bacillus subtilis/genética , Enzimas de Restrição-Modificação do DNA/genética , Escherichia coli/genética , Edição de Genes/métodos , Genoma Bacteriano , Sistemas de Transporte de Aminoácidos Básicos/deficiência , Sistemas de Transporte de Aminoácidos Básicos/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Enzimas de Restrição do DNA/genética , Enzimas de Restrição-Modificação do DNA/metabolismo , Desoxirribonucleases de Sítio Específico do Tipo III/genética , Proteínas de Escherichia coli , Plasmídeos/genética , Plasmídeos/metabolismo , Mutação Puntual , Proteína S9 Ribossômica , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética
4.
Nucleic Acids Res ; 43(22): 10870-81, 2015 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-26538601

RESUMO

DNA cleavage by the Type III restriction enzymes requires long-range protein communication between recognition sites facilitated by thermally-driven 1D diffusion. This 'DNA sliding' is initiated by hydrolysis of multiple ATPs catalysed by a helicase-like domain. Two distinct ATPase phases were observed using short oligoduplex substrates; the rapid consumption of ∼10 ATPs coupled to a protein conformation switch followed by a slower phase, the duration of which was dictated by the rate of dissociation from the recognition site. Here, we show that the second ATPase phase is both variable and only observable when DNA ends are proximal to the recognition site. On DNA with sites more distant from the ends, a single ATPase phase coupled to the conformation switch was observed and subsequent site dissociation required little or no further ATP hydrolysis. The overall DNA dissociation kinetics (encompassing site release, DNA sliding and escape via a DNA end) were not influenced by the second phase. Although the data simplifies the ATP hydrolysis scheme for Type III restriction enzymes, questions remain as to why multiple ATPs are hydrolysed to prepare for DNA sliding.


Assuntos
Trifosfato de Adenosina/metabolismo , DNA/metabolismo , Desoxirribonucleases de Sítio Específico do Tipo III/metabolismo , Adenosina Trifosfatases/metabolismo , Clivagem do DNA , Hidrólise , Cinética , Ressonância de Plasmônio de Superfície
5.
Br J Biomed Sci ; 71(2): 66-72, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24974681

RESUMO

Two examples of Campylobacter upsaliensis RM3195 and JV21 strains are shown to carry putative type III restriction (res)-modification (mod) enzyme gene clusters, following genome sequence analyses. It is suggested that the cluster is composed of at least three structural genes, res, internal methylase gene and mod, in the strains, based on the nucleotide sequence information. A ribosome binding site, a putative promoter consisting of a consensus sequence at the -10-like structure and a semiconserved T-rich region and a putative intrinsic p-independent transcriptional terminator were identified for the gene cluster in the two strains. Using two primer pairs, f-/r-res and f-/r-mod, 34 of 41 C. upsaliensis isolates generated two expected amplicons of the res and mod gene segments, and using another primer pair, the same number of isolates also generated an amplicon of the res and mod gene segments cluster, including the third internal methylase gene. Thus, C. upsaliensis isolates frequently carried putative type III R-M gene clusters, encoding the three enzymes. Interestingly, two possible overlaps were identified within the three tandem structural genes. In addition, the type III R-M gene cluster loci appear to be very similar among the C. upsaliensis isolates and very different from other thermophilic campylobacters.


Assuntos
Campylobacter upsaliensis/enzimologia , Desoxirribonucleases de Sítio Específico do Tipo III/química , Sequência de Aminoácidos , Animais , Sequência de Bases , Campylobacter upsaliensis/genética , Campylobacter upsaliensis/isolamento & purificação , Clonagem Molecular , Desoxirribonucleases de Sítio Específico do Tipo III/genética , Desoxirribonucleases de Sítio Específico do Tipo III/isolamento & purificação , Dados de Sequência Molecular
6.
Mikrobiol Z ; 76(2): 59-66, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25000732

RESUMO

A set of lysogenic strains of phytopathogenic bacteria Erwinia "horticola" and Erwinia amylovora associated with woody plants was obtained using bacteriophage P1 Cmc1ts100. The phenotype conversion from Cm(S) to Cm(R) was shown to be connected with introducing of authentic prophage DNA of 94.8 kb as a single-copy plasmid into the cells. Prophage state is unstable: P1 plasmid is spontaneously lost with high frequency by the cells. In lysogenic cells the prophage genes of type III restriction-modification complex EcoP1I are actively expressed. The system formed by E. "horticola" 450 and 60 as well as their lysogenic derivatives and specific bacteriophages provides an opportunity to divide the latter into three groups according to the level of restriction in the course of their interaction with the enzyme EcoP1I. The difference in phage responses to the endonuclease presence in a lysogenized host presumably correlates with the number of enzyme recognition sequences and the adsorption sites availability. After the prophage plasmid DNA curing the characteristic value of phage sensitivity of cells is changed. The lysogenic strains obtained in this work allow for the exploration of EcoP1I restriction-modification gene complex interaction with polyvalent phages able to grow not only on E. coli, but also on such phytopathogens as E. "horticola" and E. amylovora.


Assuntos
Bacteriófago P1/genética , Erwinia amylovora/virologia , Erwinia/virologia , Genes Virais , Lisogenia/genética , Interações Microbianas/genética , DNA Viral , Desoxirribonucleases de Sítio Específico do Tipo III/genética , Desoxirribonucleases de Sítio Específico do Tipo III/metabolismo , Genótipo , Metiltransferases/genética , Metiltransferases/metabolismo , Fenótipo , Plantas/microbiologia , Plasmídeos , Prófagos/genética
7.
Nucleic Acids Res ; 42(8): 5139-50, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24510100

RESUMO

Fundamental aspects of the biochemistry of Type III restriction endonucleases remain unresolved despite being characterized by numerous research groups in the past decades. One such feature is the subunit stoichiometry of these hetero-oligomeric enzyme complexes, which has important implications for the reaction mechanism. In this study, we present a series of results obtained by native mass spectrometry and size exclusion chromatography with multi-angle light scattering consistent with a 1:2 ratio of Res to Mod subunits in the EcoP15I, EcoPI and PstII complexes as the main holoenzyme species and a 1:1 stoichiometry of specific DNA (sDNA) binding by EcoP15I and EcoPI. Our data are also consistent with a model where ATP hydrolysis activated by recognition site binding leads to release of the enzyme from the site, dissociation from the substrate via a free DNA end and cleavage of the DNA. These results are discussed critically in the light of the published literature, aiming to resolve controversies and discuss consequences in terms of the reaction mechanism.


Assuntos
Metilases de Modificação do DNA/metabolismo , DNA/metabolismo , Desoxirribonucleases de Sítio Específico do Tipo III/química , Desoxirribonucleases de Sítio Específico do Tipo III/metabolismo , Trifosfato de Adenosina/metabolismo , Metilases de Modificação do DNA/química , Holoenzimas/metabolismo , Multimerização Proteica , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , DNA Metiltransferases Sítio Específica (Adenina-Específica)/metabolismo
8.
Nucleic Acids Res ; 42(6): 3908-18, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24423871

RESUMO

In this study, we examined the intracellular whereabouts of Mrr, a cryptic type IV restriction endonuclease of Escherichia coli K12, in response to different conditions. In absence of stimuli triggering its activity, Mrr was found to be strongly associated with the nucleoid as a number of discrete foci, suggesting the presence of Mrr hotspots on the chromosome. Previously established elicitors of Mrr activity, such as exposure to high (hydrostatic) pressure (HP) or expression of the HhaII methyltransferase, both caused nucleoid condensation and an unexpected coalescence of Mrr foci. However, although the resulting Mrr/nucleoid complex was stable when triggered with HhaII, it tended to be only short-lived when elicited with HP. Moreover, HP-mediated activation of Mrr typically led to cellular blebbing, suggesting a link between chromosome and cellular integrity. Interestingly, Mrr variants could be isolated that were specifically compromised in either HhaII- or HP-dependent activation, underscoring a mechanistic difference in the way both triggers activate Mrr. In general, our results reveal that Mrr can take part in complex spatial distributions on the nucleoid and can be engaged in distinct modes of activity.


Assuntos
Enzimas de Restrição do DNA/análise , Enzimas de Restrição do DNA/metabolismo , Proteínas de Escherichia coli/análise , Proteínas de Escherichia coli/metabolismo , Enzimas de Restrição do DNA/genética , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Desoxirribonucleases de Sítio Específico do Tipo III , Proteínas de Escherichia coli/genética , Pressão Hidrostática , Mutação
9.
Nucleic Acids Res ; 42(1): 45-55, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23863841

RESUMO

Restriction endonucleases interact with DNA at specific sites leading to cleavage of DNA. Bacterial DNA is protected from restriction endonuclease cleavage by modifying the DNA using a DNA methyltransferase. Based on their molecular structure, sequence recognition, cleavage position and cofactor requirements, restriction-modification (R-M) systems are classified into four groups. Type III R-M enzymes need to interact with two separate unmethylated DNA sequences in inversely repeated head-to-head orientations for efficient cleavage to occur at a defined location (25-27 bp downstream of one of the recognition sites). Like the Type I R-M enzymes, Type III R-M enzymes possess a sequence-specific ATPase activity for DNA cleavage. ATP hydrolysis is required for the long-distance communication between the sites before cleavage. Different models, based on 1D diffusion and/or 3D-DNA looping, exist to explain how the long-distance interaction between the two recognition sites takes place. Type III R-M systems are found in most sequenced bacteria. Genome sequencing of many pathogenic bacteria also shows the presence of a number of phase-variable Type III R-M systems, which play a role in virulence. A growing number of these enzymes are being subjected to biochemical and genetic studies, which, when combined with ongoing structural analyses, promise to provide details for mechanisms of DNA recognition and catalysis.


Assuntos
Desoxirribonucleases de Sítio Específico do Tipo III/metabolismo , Colífagos/enzimologia , Clivagem do DNA , Metilases de Modificação do DNA/genética , Desoxirribonucleases de Sítio Específico do Tipo III/química , Desoxirribonucleases de Sítio Específico do Tipo III/genética , Desoxirribonucleases de Sítio Específico do Tipo III/história , História do Século XX , História do Século XXI
10.
Nucleic Acids Res ; 42(1): 3-19, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24141096

RESUMO

In the early 1950's, 'host-controlled variation in bacterial viruses' was reported as a non-hereditary phenomenon: one cycle of viral growth on certain bacterial hosts affected the ability of progeny virus to grow on other hosts by either restricting or enlarging their host range. Unlike mutation, this change was reversible, and one cycle of growth in the previous host returned the virus to its original form. These simple observations heralded the discovery of the endonuclease and methyltransferase activities of what are now termed Type I, II, III and IV DNA restriction-modification systems. The Type II restriction enzymes (e.g. EcoRI) gave rise to recombinant DNA technology that has transformed molecular biology and medicine. This review traces the discovery of restriction enzymes and their continuing impact on molecular biology and medicine.


Assuntos
Enzimas de Restrição do DNA/história , Metilases de Modificação do DNA/história , Desoxirribonucleases de Sítio Específico do Tipo I/história , Desoxirribonucleases de Sítio Específico do Tipo II/história , Desoxirribonucleases de Sítio Específico do Tipo III/história , História do Século XX
11.
Science ; 340(6130): 353-6, 2013 Apr 19.
Artigo em Inglês | MEDLINE | ID: mdl-23599494

RESUMO

Helicases are ubiquitous adenosine triphosphatases (ATPases) with widespread roles in genome metabolism. Here, we report a previously undescribed functionality for ATPases with helicase-like domains; namely, that ATP hydrolysis can trigger ATP-independent long-range protein diffusion on DNA in one dimension (1D). Specifically, using single-molecule fluorescence microscopy we show that the Type III restriction enzyme EcoP15I uses its ATPase to switch into a distinct structural state that diffuses on DNA over long distances and long times. The switching occurs only upon binding to the target site and requires hydrolysis of ~30 ATPs. We define the mechanism for these enzymes and show how ATPase activity is involved in DNA target site verification and 1D signaling, roles that are common in DNA metabolism: for example, in nucleotide excision and mismatch repair.


Assuntos
Trifosfato de Adenosina/metabolismo , Clivagem do DNA , DNA Helicases/metabolismo , DNA/metabolismo , Desoxirribonucleases de Sítio Específico do Tipo III/metabolismo , DNA/química , DNA Helicases/química , Desoxirribonucleases de Sítio Específico do Tipo III/química , Hidrólise , Microscopia de Fluorescência/métodos , Conformação de Ácido Nucleico , Estrutura Terciária de Proteína
12.
Biochimie ; 95(4): 817-23, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23220200

RESUMO

For efficient DNA hydrolysis, Type III restriction endonuclease EcoP15I interacts with two inversely oriented recognition sites in an ATP-dependent process. EcoP15I consists of two methylation (Mod) subunits and a single restriction (Res) subunit yielding a multifunctional enzyme complex able to methylate or to hydrolyse DNA. Comprehensive sequence alignments, limited proteolysis and mass spectroscopy suggested that the Res subunit is a fusion of a motor or translocase (Tr) domain of superfamily II helicases and an endonuclease domain with a catalytic PD…EXK motif. In the Tr domain, seven predicted helicase motifs (I, Ia, II-VI), a recently discovered Q-tip motif and three additional regions (IIIa, IVa, Va) conserved among Type III restriction enzymes have been identified that are predicted to be involved in DNA binding and ATP hydrolysis. Because DNA unwinding activity for EcoP15I (as for bona fide helicases) has never been found and EcoP15I ATPase rates are only low, the functional importance of the helicase motifs and regions was questionable and has never been probed systematically. Therefore, we mutated all helicase motifs and conserved regions predicted in Type III restriction enzyme EcoP15I and examined the functional consequences on EcoP15I enzyme activity and the structural integrity of the variants by CD spectroscopy. The resulting eleven enzyme variants all, except variant IVa, are properly folded showing the same secondary structure distribution as the wild-type enzyme. Classical helicase motifs I-VI are important for ATP and DNA cleavage by EcoP15I and mutations therein led to complete loss of ATPase and cleavage activity. Among the catalytically inactive enzyme variants three preserved the ability to bind ATP. In contrast, newly assigned motifs Q-tip, Ia and Va are not essential for EcoP15I activity and the corresponding enzyme variants were still catalytically active. DNA binding was only marginally reduced (2-7 fold) in all enzyme variants tested.


Assuntos
Sequência Conservada , DNA Helicases/química , Desoxirribonucleases de Sítio Específico do Tipo III/química , Desoxirribonucleases de Sítio Específico do Tipo III/metabolismo , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Mutação , Motivos de Aminoácidos , Sequência de Aminoácidos , Desoxirribonucleases de Sítio Específico do Tipo III/genética , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese , Proteínas Mutantes/genética , Estrutura Terciária de Proteína
13.
Nucleic Acids Res ; 40(14): 6752-64, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22523084

RESUMO

DNA cleavage by the Type III Restriction-Modification (RM) enzymes requires the binding of a pair of RM enzymes at two distant, inversely orientated recognition sequences followed by helicase-catalysed ATP hydrolysis and long-range communication. Here we addressed the dissociation from DNA of these enzymes at two stages: during long-range communication and following DNA cleavage. First, we demonstrated that a communicating species can be trapped in a DNA domain without a recognition site, with a non-specific DNA association lifetime of ∼ 200 s. If free DNA ends were present the lifetime became too short to measure, confirming that ends accelerate dissociation. Secondly, we observed that Type III RM enzymes can dissociate upon DNA cleavage and go on to cleave further DNA molecules (they can 'turnover', albeit inefficiently). The relationship between the observed cleavage rate and enzyme concentration indicated independent binding of each site and a requirement for simultaneous interaction of at least two enzymes per DNA to achieve cleavage. In light of various mechanisms for helicase-driven motion on DNA, we suggest these results are most consistent with a thermally driven random 1D search model (i.e. 'DNA sliding').


Assuntos
Clivagem do DNA , Desoxirribonucleases de Sítio Específico do Tipo III/metabolismo , DNA/metabolismo , DNA Helicases/metabolismo , Ensaios Enzimáticos/métodos , Cinética , Movimento (Física)
14.
J Bacteriol ; 194(1): 49-60, 2012 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-22037402

RESUMO

The genomes of two Bacillus cereus strains (ATCC 10987 and ATCC 14579) have been sequenced. Here, we report the specificities of type II/III restriction (R) and modification (M) enzymes. Found in the ATCC 10987 strain, BceSI is a restriction endonuclease (REase) with the recognition and cut site CGAAG 24-25/27-28. BceSII is an isoschizomer of AvaII (G/GWCC). BceSIII cleaves at ACGGC 12/14. The BceSIII C terminus resembles the catalytic domains of AlwI, MlyI, and Nt.BstNBI. BceSIV is composed of two subunits and cleaves on both sides of GCWGC. BceSIV activity is strongly stimulated by the addition of cofactor ATP or GTP. The large subunit (R1) of BceSIV contains conserved motifs of NTPases and DNA helicases. The R1 subunit has no endonuclease activity by itself; it strongly stimulates REase activity when in complex with the R2 subunit. BceSIV was demonstrated to hydrolyze GTP and ATP in vitro. BceSIV is similar to CglI (GCSGC), and homologs of R1 are found in 11 sequenced bacterial genomes, where they are paired with specificity subunits. In addition, homologs of the BceSIV R1-R2 fusion are found in many sequenced microbial genomes. An orphan methylase, M.BceSV, was found to modify GCNGC, GGCC, CCGG, GGNNCC, and GCGC sites. A ParB-methylase fusion protein appears to nick DNA nonspecifically. The ATCC 14579 genome encodes an active enzyme Bce14579I (GCWGC). BceSIV and Bce14579I belong to the phospholipase D (PLD) family of endonucleases that are widely distributed among Bacteria and Archaea. A survey of type II and III restriction-modification (R-M) system genes is presented from sequenced B. cereus, Bacillus anthracis, and Bacillus thuringiensis strains.


Assuntos
Bacillus cereus/metabolismo , Proteínas de Bactérias/metabolismo , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Desoxirribonucleases de Sítio Específico do Tipo III/metabolismo , Regulação Bacteriana da Expressão Gênica/fisiologia , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Bacillus cereus/classificação , Bacillus cereus/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sequência de Bases , Clonagem Molecular , DNA Bacteriano/metabolismo , Desoxirribonucleases de Sítio Específico do Tipo II/genética , Desoxirribonucleases de Sítio Específico do Tipo III/genética , Regulação Enzimológica da Expressão Gênica , Genoma Bacteriano , Guanosina Trifosfato/metabolismo , Dados de Sequência Molecular , Subunidades Proteicas
15.
Nucleic Acids Res ; 40(8): 3610-22, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22199260

RESUMO

For efficient DNA cleavage, the Type III restriction endonuclease EcoP15I communicates with two inversely oriented recognition sites in an ATP-dependent process. EcoP15I consists of methylation (Mod) and restriction (Res) subunits forming a multifunctional enzyme complex able to methylate or to cleave DNA. In this study, we determined by different analytical methods that EcoP15I contains a single Res subunit in a Mod(2)Res stoichiometry. The Res subunit comprises a translocase (Tr) domain carrying functional motifs of superfamily 2 helicases and an endonuclease domain with a PD..D/EXK motif. We show that the isolated Tr domain retains ATP-hydrolyzing activity and binds single- and double-stranded DNA in a sequence-independent manner. To localize the regions of DNA binding, we screened peptide arrays representing the entire Res sequence for their ability to interact with DNA. We discovered four DNA-binding regions in the Tr domain and two DNA-binding regions in the endonuclease domain. Modelling of the Tr domain shows that these multiple DNA-binding regions are located on the surface, free to interact with DNA. Interestingly, the positions of the DNA-binding regions are conserved among other Type III restriction endonucleases.


Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Desoxirribonucleases de Sítio Específico do Tipo III/química , Desoxirribonucleases de Sítio Específico do Tipo III/metabolismo , Adenosina Trifosfatases/química , Trifosfato de Adenosina/metabolismo , Cromatografia em Gel , Clonagem Molecular , DNA/metabolismo , Desoxirribonucleases de Sítio Específico do Tipo III/genética , Hidrólise , Estrutura Terciária de Proteína , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , DNA Metiltransferases Sítio Específica (Adenina-Específica)/química , DNA Metiltransferases Sítio Específica (Adenina-Específica)/metabolismo
16.
Nucleic Acids Res ; 39(18): 8042-51, 2011 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-21724613

RESUMO

DNA cleavage by the Type III Restriction-Modification enzymes requires communication in 1D between two distant indirectly-repeated recognitions sites, yet results in non-specific dsDNA cleavage close to only one of the two sites. To test a recently proposed ATP-triggered DNA sliding model, we addressed why one site is selected over another during cleavage. We examined the relative cleavage of a pair of identical sites on DNA substrates with different distances to a free or protein blocked end, and on a DNA substrate using different relative concentrations of protein. Under these conditions a bias can be induced in the cleavage of one site over the other. Monte-Carlo simulations based on the sliding model reproduce the experimentally observed behaviour. This suggests that cleavage site selection simply reflects the dynamics of the preceding stochastic enzyme events that are consistent with bidirectional motion in 1D and DNA cleavage following head-on protein collision.


Assuntos
Clivagem do DNA , Desoxirribonucleases de Sítio Específico do Tipo III/metabolismo , DNA/metabolismo , Modelos Biológicos , Método de Monte Carlo , Movimento (Física)
17.
Toxicol Sci ; 123(1): 103-12, 2011 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-21659616

RESUMO

Methyleugenol (MEG), a constituent of human food, induces malignant tumors in multiple tissues of rats and mice. Although MEG forms DNA adducts and induces unscheduled DNA synthesis in rat liver, it is negative in many in vitro genetic toxicity assays. In the present study, we evaluated MEG-induced DNA damage in the rat using (1) the alkaline Comet assay, (2) the oxidative Comet assay, and (3) expression profiling of genes associated with DNA damage pathways. Male F344 rats received single oral doses of 400 or 1000 mg/kg body weight (bw) MEG and DNA damage was assessed by the Comet assay in liver, bladder, bone marrow, kidney, and lung 3 h and 24 h later. MEG failed to produce any increase in DNA damage. In addition, rats were given a single oral dose of 2000 mg/kg bw MEG, and Comet assays were performed with liver, bone marrow, and bladder 1, 3, 6, and 8 h later. With one exception (bone marrow at 8 h), no DNA damage was detected. Enzyme-modified Comet assays were conducted in parallel with standard Comet assays in liver. Whereas no MEG-induced DNA damage was detected following formamidopyrimidine DNA glycosylase digestion, digestion with endonuclease III resulted in increases in DNA damage at the 6- and 8-h sampling times. Gene expression analysis on the livers from MEG-exposed rats showed significant reduction in genes associated with DNA repair. The results indicate that MEG induces DNA damage in rat liver and that oxidative DNA damages may be partly responsible for the genotoxicity of MEG in rodents.


Assuntos
Ensaio Cometa , Dano ao DNA , DNA/efeitos dos fármacos , Eugenol/análogos & derivados , Regulação da Expressão Gênica/efeitos dos fármacos , Mutagênicos/toxicidade , Animais , Células da Medula Óssea/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , Reparo do DNA/genética , Desoxirribonucleases de Sítio Específico do Tipo III/genética , Desoxirribonucleases de Sítio Específico do Tipo III/metabolismo , Eugenol/classificação , Eugenol/toxicidade , Perfilação da Expressão Gênica , Fígado/efeitos dos fármacos , Masculino , Mutagênicos/classificação , Oxirredução , Ratos , Ratos Endogâmicos F344 , Bexiga Urinária/efeitos dos fármacos
18.
Nucleic Acids Res ; 39(14): 5991-6001, 2011 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-21504983

RESUMO

The Mrr protein of Escherichia coli is a laterally acquired Type IV restriction endonuclease with specificity for methylated DNA. While Mrr nuclease activity can be elicited by high-pressure stress in E. coli MG1655, its (over)expression per se does not confer any obvious toxicity. In this study, however, we discovered that Mrr of E. coli MG1655 causes distinct genotoxicity when expressed in Salmonella typhimurium LT2. Genetic screening enabled us to contribute this toxicity entirely to the presence of the endogenous Type III restriction modification system (StyLTI) of S. typhimurium LT2. The StyLTI system consists of the Mod DNA methyltransferase and the Res restriction endonuclease, and we revealed that expression of the LT2 mod gene was sufficient to trigger Mrr activity in E. coli MG1655. Moreover, we could demonstrate that horizontal acquisition of the MG1655 mrr locus can drive the loss of endogenous Mod functionality present in S. typhimurium LT2 and E. coli ED1a, and observed a strong anti-correlation between close homologues of MG1655 mrr and LT2 mod in the genome database. This apparent evolutionary antagonism is further discussed in the light of a possible role for Mrr as defense mechanism against the establishment of epigenetic regulation by foreign DNA methyltransferases.


Assuntos
Enzimas de Restrição do DNA/metabolismo , Desoxirribonucleases de Sítio Específico do Tipo III/metabolismo , Proteínas de Escherichia coli/metabolismo , Evolução Molecular , Metilases de Modificação do DNA/metabolismo , Enzimas de Restrição do DNA/genética , Desoxirribonucleases de Sítio Específico do Tipo III/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Salmonella typhimurium/enzimologia , Salmonella typhimurium/metabolismo
19.
Biochem Soc Trans ; 39(2): 589-94, 2011 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21428945

RESUMO

To cleave DNA, the Type III RM (restriction-modification) enzymes must communicate the relative orientation of two recognition sequences, which may be separated by many thousands of base pairs. This long-range interaction requires ATP hydrolysis by a helicase domain, and both active (DNA translocation) and passive (DNA sliding) modes of motion along DNA have been proposed. Potential roles for ATP binding and hydrolysis by the helicase domains are discussed, with a focus on bipartite ATPases that act as molecular switches.


Assuntos
Trifosfato de Adenosina/farmacologia , DNA/metabolismo , Desoxirribonucleases de Sítio Específico do Tipo III/metabolismo , Desoxirribonucleases de Sítio Específico do Tipo III/fisiologia , Movimento/fisiologia , Animais , Sítios de Ligação/efeitos dos fármacos , Sítios de Ligação/genética , DNA/química , Humanos , Modelos Biológicos , Conformação de Ácido Nucleico/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/fisiologia , Transporte Proteico , Especificidade por Substrato
20.
Nucleic Acids Res ; 39(11): 4525-31, 2011 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-21310716

RESUMO

Much insight into the interactions of DNA and enzymes has been obtained using a number of single-molecule techniques. However, recent results generated using two of these techniques-atomic force microscopy (AFM) and magnetic tweezers (MT)-have produced apparently contradictory results when applied to the action of the ATP-dependent type III restriction endonucleases on DNA. The AFM images show extensive looping of the DNA brought about by the existence of multiple DNA binding sites on each enzyme and enzyme dimerisation. The MT experiments show no evidence for looping being a requirement for DNA cleavage, but instead support a diffusive sliding of the enzyme on the DNA until an enzyme-enzyme collision occurs, leading to cleavage. Not only do these two methods appear to disagree, but also the models derived from them have difficulty explaining some ensemble biochemical results on DNA cleavage. In this 'Survey and Summary', we describe several different models put forward for the action of type III restriction enzymes and their inadequacies. We also attempt to reconcile the different models and indicate areas for further experimentation to elucidate the mechanism of these enzymes.


Assuntos
DNA/metabolismo , Desoxirribonucleases de Sítio Específico do Tipo III/metabolismo , Modelos Biológicos , DNA/química , Microscopia de Força Atômica , Transporte Proteico
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