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1.  Related bifunctional restriction endonuclease-methyltransferase triplets: TspDTI, Tth111II/TthHB27I and TsoI with distinct specificities 
BMC Molecular Biology  2012;13:13.
We previously defined a family of restriction endonucleases (REases) from Thermus sp., which share common biochemical and biophysical features, such as the fusion of both the nuclease and methyltransferase (MTase) activities in a single polypeptide, cleavage at a distance from the recognition site, large molecular size, modulation of activity by S-adenosylmethionine (SAM), and incomplete cleavage of the substrate DNA. Members include related thermophilic REases with five distinct specificities: TspGWI, TaqII, Tth111II/TthHB27I, TspDTI and TsoI.
TspDTI, TsoI and isoschizomers Tth111II/TthHB27I recognize different, but related sequences: 5'-ATGAA-3', 5'-TARCCA-3' and 5'-CAARCA-3' respectively. Their amino acid sequences are similar, which is unusual among REases of different specificity. To gain insight into this group of REases, TspDTI, the prototype member of the Thermus sp. enzyme family, was cloned and characterized using a recently developed method for partially cleaving REases.
TspDTI, TsoI and isoschizomers Tth111II/TthHB27I are closely related bifunctional enzymes. They comprise a tandem arrangement of Type I-like domains, like other Type IIC enzymes (those with a fusion of a REase and MTase domains), e.g. TspGWI, TaqII and MmeI, but their sequences are only remotely similar to these previously characterized enzymes. The characterization of TspDTI, a prototype member of this group, extends our understanding of sequence-function relationships among multifunctional restriction-modification enzymes.
PMCID: PMC3384240  PMID: 22489904
2.  Three-stage biochemical selection: cloning of prototype class IIS/IIC/IIG restriction endonuclease-methyltransferase TsoI from the thermophile Thermus scotoductus 
BMC Molecular Biology  2013;14:17.
In continuing our research into the new family of bifunctional restriction endonucleases (REases), we describe the cloning of the tsoIRM gene. Currently, the family includes six thermostable enzymes: TaqII, Tth111II, TthHB27I, TspGWI, TspDTI, TsoI, isolated from various Thermus sp. and two thermolabile enzymes: RpaI and CchII, isolated from mesophilic bacteria Rhodopseudomonas palustris and Chlorobium chlorochromatii, respectively. The enzymes have several properties in common. They are large proteins (molecular size app. 120 kDa), coded by fused genes, with the REase and methyltransferase (MTase) in a single polypeptide, where both activities are affected by S-adenosylmethionine (SAM). They recognize similar asymmetric cognate sites and cleave at a distance of 11/9 nt from the recognition site. Thus far, we have cloned and characterised TaqII, Tth111II, TthHB27I, TspGWI and TspDTI.
TsoI REase, which originate from thermophilic Thermus scotoductus RFL4 (T. scotoductus), was cloned in Escherichia coli (E. coli) using two rounds of biochemical selection of the T. scotoductus genomic library for the TsoI methylation phenotype. DNA sequencing of restriction-resistant clones revealed the common open reading frame (ORF) of 3348 bp, coding for a large polypeptide of 1116 aminoacid (aa) residues, which exhibited a high level of similarity to Tth111II (50% identity, 60% similarity). The ORF was PCR-amplified, subcloned into a pET21 derivative under the control of a T7 promoter and was subjected to the third round of biochemical selection in order to isolate error-free clones. Induction experiments resulted in synthesis of an app. 125 kDa protein, exhibiting TsoI-specific DNA cleavage. Also, the wild-type (wt) protein was purified and reaction optima were determined.
Previously we identified and cloned the Thermus family RM genes using a specially developed method based on partial proteolysis of thermostable REases. In the case of TsoI the classic biochemical selection method was successful, probably because of the substantially lower optimal reaction temperature of TsoI (app. 10-15°C). That allowed for sufficient MTase activity in vivo in recombinant E. coli. Interestingly, TsoI originates from bacteria with a high optimum growth temperature of 67°C, which indicates that not all bacterial enzymes match an organism’s thermophilic nature, and yet remain functional cell components. Besides basic research advances, the cloning and characterisation of the new prototype REase from the Thermus sp. family enzymes is also of practical importance in gene manipulation technology, as it extends the range of available DNA cleavage specificities.
PMCID: PMC3751577  PMID: 23919831
3.  Modified ‘one amino acid-one codon’ engineering of high GC content TaqII-coding gene from thermophilic Thermus aquaticus results in radical expression increase 
An industrial approach to protein production demands maximization of cloned gene expression, balanced with the recombinant host’s viability. Expression of toxic genes from thermophiles poses particular difficulties due to high GC content, mRNA secondary structures, rare codon usage and impairing the host’s coding plasmid replication.
TaqII belongs to a family of bifunctional enzymes, which are a fusion of the restriction endonuclease (REase) and methyltransferase (MTase) activities in a single polypeptide. The family contains thermostable REases with distinct specificities: TspGWI, TaqII, Tth111II/TthHB27I, TspDTI and TsoI and a few enzymes found in mesophiles. While not being isoschizomers, the enzymes exhibit amino acid (aa) sequence homologies, having molecular sizes of ~120 kDa share common modular architecture, resemble Type-I enzymes, cleave DNA 11/9 nt from the recognition sites, their activity is affected by S-adenosylmethionine (SAM).
We describe the taqIIRM gene design, cloning and expression of the prototype TaqII. The enzyme amount in natural hosts is extremely low. To improve expression of the taqIIRM gene in Escherichia coli (E. coli), we designed and cloned a fully synthetic, low GC content, low mRNA secondary structure taqIIRM, codon-optimized gene under a bacteriophage lambda (λ) P R promoter. Codon usage based on a modified ‘one amino acid–one codon’ strategy, weighted towards low GC content codons, resulted in approximately 10-fold higher expression of the synthetic gene. 718 codons of total 1105 were changed, comprising 65% of the taqIIRM gene. The reason for we choose a less effective strategy rather than a resulting in high expression yields ‘codon randomization’ strategy, was intentional, sub-optimal TaqII in vivo production, in order to decrease the high ‘toxicity’ of the REase-MTase protein.
Recombinant wt and synthetic taqIIRM gene were cloned and expressed in E. coli. The modified ‘one amino acid–one codon’ method tuned for thermophile-coded genes was applied to obtain overexpression of the ‘toxic’ taqIIRM gene. The method appears suited for industrial production of thermostable ‘toxic’ enzymes in E. coli. This novel variant of the method biased toward increasing a gene’s AT content may provide economic benefits for industrial applications.
PMCID: PMC3893498  PMID: 24410856
4.  A new Thermus sp. class-IIS enzyme sub-family: isolation of a ‘twin’ endonuclease TspDTI with a novel specificity 5′-ATGAA(N11/9)-3′, related to TspGWI, TaqII and Tth111II 
Nucleic Acids Research  2003;31(14):e74.
The TspDTI restriction endonuclease, which shows a novel recognition specificity 5′-ATGAA(N11/9)-3′, was isolated from Thermus sp. DT. TspDTI appears to be a ‘twin’ of restriction endonuclease TspGWI from Thermus sp. GW, as we have previously reported. TspGWI was isolated from the same location as TspDTI, it recognizes a related sequence 5′-ACGGA(N11/9)-3′ and has conserved cleavage positions. Both enzymes resemble two other class-IIS endonucleases from Thermus sp.: TaqII and Tth111II. N-terminal amino acid sequences of TspGWI tryptic peptides exhibit 88.9–100% similarity to the TaqII sequence. All four enzymes were purified to homogeneity; their polypeptide sizes (114.5–122 kDa) make them the largest class-IIS restriction endonucleases known to date. The existence of a Thermus sp. sub-family of class-IIS restriction endonucleases of a common origin is herein proposed.
PMCID: PMC167652  PMID: 12853651
5.  Cloning and analysis of a bifunctional methyltransferase/restriction endonuclease TspGWI, the prototype of a Thermus sp. enzyme family 
BMC Molecular Biology  2009;10:52.
Restriction-modification systems are a diverse class of enzymes. They are classified into four major types: I, II, III and IV. We have previously proposed the existence of a Thermus sp. enzyme family, which belongs to type II restriction endonucleases (REases), however, it features also some characteristics of types I and III. Members include related thermophilic endonucleases: TspGWI, TaqII, TspDTI, and Tth111II.
Here we describe cloning, mutagenesis and analysis of the prototype TspGWI enzyme that recognises the 5'-ACGGA-3' site and cleaves 11/9 nt downstream. We cloned, expressed, and mutagenised the tspgwi gene and investigated the properties of its product, the bifunctional TspGWI restriction/modification enzyme. Since TspGWI does not cleave DNA completely, a cloning method was devised, based on amino acid sequencing of internal proteolytic fragments. The deduced amino acid sequence of the enzyme shares significant sequence similarity with another representative of the Thermus sp. family – TaqII. Interestingly, these enzymes recognise similar, yet different sequences in the DNA. Both enzymes cleave DNA at the same distance, but differ in their ability to cleave single sites and in the requirement of S-adenosylmethionine as an allosteric activator for cleavage. Both the restriction endonuclease (REase) and methyltransferase (MTase) activities of wild type (wt) TspGWI (either recombinant or isolated from Thermus sp.) are dependent on the presence of divalent cations.
TspGWI is a bifunctional protein comprising a tandem arrangement of Type I-like domains; particularly noticeable is the central HsdM-like module comprising a helical domain and a highly conserved S-adenosylmethionine-binding/catalytic MTase domain, containing DPAVGTG and NPPY motifs. TspGWI also possesses an N-terminal PD-(D/E)XK nuclease domain related to the corresponding domains in HsdR subunits, but lacks the ATP-dependent translocase module of the HsdR subunit and the additional domains that are involved in subunit-subunit interactions in Type I systems. The MTase and REase activities of TspGWI are autonomous and can be uncoupled. Structurally and functionally, the TspGWI protomer appears to be a streamlined 'half' of a Type I enzyme.
PMCID: PMC2700111  PMID: 19480701
6.  Cofactor analogue-induced chemical reactivation of endonuclease activity in a DNA cleavage/methylation deficient TspGWI N473A variant in the NPPY motif 
Molecular Biology Reports  2014;41:2313-2323.
We reported previously that TspGWI, a prototype enzyme of a new Thermus sp. family of restriction endonucleases-methyltransferases (REases-MTases), undergoes the novel phenomenon of sinefungin (SIN)-caused specificity transition. Here we investigated mutant TspGWI N473A, containing a single amino acid (aa) substitution in the NPPY motif of the MTase. Even though the aa substitution is located within the MTase polypeptide segment, DNA cleavage and modification are almost completely abolished, indicating that the REase and MTase are intertwined. Remarkably, the TspGWI N473A REase functionality can be completely reconstituted by the addition of SIN. We hypothesize that SIN binds specifically to the enzyme and restores the DNA cleavage-competent protein tertiary structure. This indicates the significant role of allosteric effectors in DNA cleavage in Thermus sp. enzymes. This is the first case of REase mutation suppression by an S-adenosylmethionine (SAM) cofactor analogue. Moreover, the TspGWI N473A clone strongly affects E. coli division control, acting as a ‘selfish gene’. The mutant lacks the competing MTase activity and therefore might be useful for applications in DNA manipulation. Here we present a case study of a novel strategy for REase activity/specificity alteration by a single aa substitution, based on the bioinformatic analysis of active motif locations, combining (a) aa sequence engineering (b) the alteration of protein enzymatic properties, and (c) the use of cofactor–analogue cleavage reconstitution and stimulation.
PMCID: PMC3968444  PMID: 24442320
Endonuclease-methyltransferase; Thermus sp. enzyme; Enzymatic reaction cofactor; Cofactor analogue; Sinefungin; S-adenosylmethione; Mutant activation; Specificity change
7.  TspGWI, a thermophilic class-IIS restriction endonuclease from Thermus sp., recognizes novel asymmetric sequence 5′-ACGGA(N11/9)-3′ 
Nucleic Acids Research  2002;30(7):e33.
A novel prototype class-IIS restriction endonuclease, TspGWI, was isolated from the thermophilic bacterium Thermus sp. GW. The recognition sequence and cleavage positions have been established: TspGWI recognizes the non-palindromic 5-bp sequence 5′-ACGGA-3′ and cleaves the DNA 11 and 9 nt downstream in the top and bottom strand, respectively. In addition, an accompanying endonuclease, TspGWII, an isoschizomer of Pst I, was found in Thermus sp. GW cells.
PMCID: PMC101857  PMID: 11917039
8.  A second type II restriction endonuclease from Thermus aquaticus with an unusual sequence specificity. 
Nucleic Acids Research  1984;12(14):5567-5581.
A type II restriction endonuclease activity free of TaqI was prepared from Thermus Aquaticus YT. The fraction contains two endonucleolytic components with apparently different specificities, however the major activity is sufficiently dominant to allow partial digestion analysis of the position of recognition sites. A precise determination of the location of cleavage sites in pBR322 DNA and a computer-aided search for regions of homology in the vicinity of the cut sites indicate that this enzyme recognizes the nonpalindromic sequences GACCGA or CACCCA. Other related sequences are not cleaved, in particular, GACCCA and CACCGA, indicating that the enzyme requires the identity of nucleotides in the first and fifth positions, a type of specificity that has not been previously reported. The position of cleavage is located outside of the site and is represented as: (Formula: see text).
PMCID: PMC320015  PMID: 6087291
9.  A new genomic tool, ultra-frequently cleaving TaqII/sinefungin endonuclease with a combined 2.9-bp recognition site, applied to the construction of horse DNA libraries 
BMC Genomics  2013;14:370.
Genomics and metagenomics are currently leading research areas, with DNA sequences accumulating at an exponential rate. Although enormous advances in DNA sequencing technologies are taking place, progress is frequently limited by factors such as genomic contig assembly and generation of representative libraries. A number of DNA fragmentation methods, such as hydrodynamic sharing, sonication or DNase I fragmentation, have various drawbacks, including DNA damage, poor fragmentation control, irreproducibility and non-overlapping DNA segment representation. Improvements in these limited DNA scission methods are consequently needed. An alternative method for obtaining higher quality DNA fragments involves partial digestion with restriction endonucleases (REases).
We have shown previously that class-IIS/IIC/IIG TspGWI REase, the prototype member of the Thermus sp. enzyme family, can be chemically relaxed by a cofactor analogue, allowing it to recognize very short DNA sequences of 3-bp combined frequency. Such frequently cleaving REases are extremely rare, with CviJI/CviJI*, SetI and FaiI the only other ones found in nature. Their unusual features make them very useful molecular tools for the development of representative DNA libraries.
We constructed a horse genomic library and a deletion derivative library of the butyrylcholinesterase cDNA coding region using a novel method, based on TaqII, Thermus sp. family bifunctional enzyme exhibiting cofactor analogue specificity relaxation. We used sinefungin (SIN) – an S-adenosylmethionine (SAM) analogue with reversed charge pattern, and dimethylsulfoxide (DMSO), to convert the 6-bp recognition site TaqII (5′-GACCGA-3′ [11/9]) into a theoretical 2.9-bp REase, with 70 shortened variants of the canonical recognition sequence detected. Because partial DNA cleavage is an inherent feature of the Thermus sp. enzyme family, this modified TaqII is uniquely suited to quasi-random library generation.
In the presence of SIN/DMSO, TaqII REase is transformed from cleaving every 4096 bp on average to cleaving every 58 bp. TaqII SIN/DMSO thus extends the palette of available REase prototype specificities. This phenomenon, employed under partial digestion conditions, was applied to quasi-random DNA fragmentation. Further applications include high sensitivity probe generation and metagenomic DNA amplification.
PMCID: PMC3681635  PMID: 23724933
10.  Tsp49I (ACGT/), a thermostable neoschizomer of the Type II restriction endonuclease MaeII (A/CGT), discovered in isolates of the genus Thermus from the Azores, Iceland and New Zealand. 
Nucleic Acids Research  1996;24(10):1799-1801.
One hundred and forty eight isolates of the genus Thermus, from neutral and alkaline hot water springs on four continents, have been screened for the presence of restriction endonuclease activity. An isolate (SM49) from the island of Sao Miguel, in the Azores, showed a high level of restriction endonuclease activity when a cell-free extract was incubated with lambda phage DNA at 65 degrees C. A Type II restriction endonuclease (Tsp49I) has been partially purified from this isolate and the recognition and cleavage site determined. Tsp49I recognizes the four base sequence ACGT, which is the same as the recognition sequence of the mesophilic Type II restriction endonuclease MaeII. However, unlike MaeII, which cleaves DNA between the first and second bass of the recognition sequence (A/CGT), Tsp49I hydrolyses the phosphodiester bond in both strands of the substrate after the last base of the recognition sequence 5'-ACGT/-3', producing four base 3'-OH overhangs (sticky ends). The enzyme has a pH optimum of 9.0, requires 2 mM MgCl2 for maximum activity and retains full enzyme activity following incubation for 10 min at temperatures up to 8O degrees C. Two further examples of the same restriction endonuclease specificity as Tsp491 were detected in Thermus isolates from Iceland (TspIDSI) and New Zealand (TspWAM8AI). The three MaeII neoschizomers, Tsp49I, TspIDSI and TspWAM8AI, exhibit similar pH optima, heat stabilities and MgCl2 requirements, but differ in their requirements for NaCl and KCl.
PMCID: PMC145888  PMID: 8657557
11.  Taq52 I, a novel and thermostable type II restriction endonuclease from the genus Thermus, recognising the pentanucleotide sequence GC(A or T)GC and cleaving DNA between the first and second bases of the recognition sequence: G decreased or reduced C(A or T)GC. 
Nucleic Acids Research  1995;23(22):4573-4575.
127 isolates of the genus Thermus, from neutral and alkaline hot water springs on four continents, have been screened for the presence of restriction endonuclease activity. An isolate (YS52) from Yellowstone National Park, USA, showed a high level of restriction endonuclease activity when a cell free extract was incubated with lambda phage DNA at 65 degrees C. A Type II restriction endonuclease (Taq52 I) has been partially purified from this isolate and the recognition and cleavage site determined. Taq52 I has a novel interrupted palindromic tetranucleotide recognition sequence GCWGC, where W can be either adenine (A) or thymine (T). It hydrolyses the phosphodiester bond in both strands of the substrate between the first and second bases of the recognition sequence: 5'G decreased or reduced CWGC3', producing three-base 5'-OH overhangs (sticky ends). The enzyme has a pH optimum of 7.0, requires 8 mM MgCl2 for maximum activity and is thermally stable, retaining full enzyme activity following incubation at 79 degrees C for 10 min. Taq52 I not only represents a new addition to the Type II restriction endonucleases, but also increases the small list of thermostable restriction endonucleases.
PMCID: PMC307427  PMID: 8524644
12.  Purification and characterization of two new modification methylases: MClaI from Caryophanon latum L and MTaqI from Thermus aquaticus YTI. 
Nucleic Acids Research  1981;9(24):6795-6804.
A method for detecting Type II modification methylases and determining their methylation site by assaying the ability of methylated DNA to be cleaved by heterologous restriction enzymes is described and applied to the isolation of the restriction modification methylases from Thermus thermophilus HB8, Thermus aquaticus YTI and Caryophanon latum L. M.TaqI is shown to have a methylation specificity identical to M.ThI (TCGmeA). M.ClaI methylates at adenine and protects a subset of TthI sites indicating that it methylates the sequence ATCGmeAT. Methylation by M.ThI also protects against cleavage by SalI, XhoI and at some HindII, AccI and MboI sites.
PMCID: PMC327642  PMID: 6278447
13.  Directional cloning of DNA fragments using deoxyinosine-containing oligonucleotides and endonuclease V 
BMC Biotechnology  2013;13:81.
DNA fragments carrying internal recognition sites for the restriction endonucleases intended for cloning into a target plasmid pose a challenge for conventional cloning.
A method for directional insertion of DNA fragments into plasmid vectors has been developed. The target sequence is amplified from a template DNA sample by PCR using two oligonucleotides each containing a single deoxyinosine base at the third position from the 5′ end. Treatment of such PCR products with endonuclease V generates 3′ protruding ends suitable for ligation with vector fragments created by conventional restriction endonuclease reactions.
The developed approach generates terminal cohesive ends without the use of Type II restriction endonucleases, and is thus independent from the DNA sequence. Due to PCR amplification, minimal amounts of template DNA are required. Using the robust Taq enzyme or a proofreading Pfu DNA polymerase mutant, the method is applicable to a broad range of insert sequences. Appropriate primer design enables direct incorporation of terminal DNA sequence modifications such as tag addition, insertions, deletions and mutations into the cloning strategy. Further, the restriction sites of the target plasmid can be either retained or removed.
PMCID: PMC3856533  PMID: 24090222
Cohesive ends; DNA cleavage; Genetic vectors; Modified primers; Molecular methods; Polymerase chain reaction; Recombinant Escherichia coli; Restriction enzymes
14.  Type II restriction endonucleases cleave single-stranded DNAs in general. 
Nucleic Acids Research  1985;13(16):5747-5760.
Restriction endonucleases (13 out of 18 species used for the test) were certified to cleave single-stranded(ss)DNA. Such enzymes as AvaII, HaeII, DdeI, AluI, Sau3AI, AccII,TthHB8I and HapII were newly reported to cleave ssDNA. A model to account for the cleavage of ssDNA by restriction enzymes was proposed with supportive data. The essential part of the model was that restriction enzymes preferentially cleave transiently formed secondary structures (called canonical structures) in ssDNA composed of two recognition sequences with two fold rotational symmetry. This means that a restriction enzyme can cleave ssDNAs in general so far as the DNAs have the sequences of restriction sites for the enzyme, and that the rate of cleavage depends on the stabilities of canonical structures.
PMCID: PMC321909  PMID: 2994012
15.  Characterization of cleavage intermediate and star sites of RM.Tth111II 
Scientific Reports  2014;4:3838.
Tth111II is a thermostable Type IIGS restriction enzyme that recognizes DNA sites CAARCA (R = A or G) and cleaves downstream at N11/N9. Here, the tth111IIRM gene was cloned and expressed in E. coli, and Tth111II was purified. The purified enzyme contains internally-bound S-adenosylmethionine (SAM). When the internal SAM was removed, the endonuclease activity was stimulated by adding SAM or its analog sinefungin. The cleavage intermediate is mostly top-strand nicked DNA on a single-site plasmid. Addition of duplex oligos with a cognate site stimulates cleavage activity of the one-site substrate. Tth111II cleaves a two-site plasmid DNA with equal efficiency regardless of site orientation. We propose the top-strand nicking is carried out by a Tth111II monomer and bottom-strand cleavage is carried out by a transient dimer. Tth111II methylates cleavage product-like duplex oligos CAAACAN9, but the modification rate is estimated to be much slower than the top-strand nicking rate. We cloned and sequenced a number of Tth111II star sites which are 1-bp different from the cognate sites. A biochemical pathway is proposed for the restriction and methylation activities of Tth111II.
PMCID: PMC3899748  PMID: 24452415
16.  The Role of the Methyltransferase Domain of Bifunctional Restriction Enzyme RM.BpuSI in Cleavage Activity 
PLoS ONE  2013;8(11):e80967.
Restriction enzyme (REase) RM.BpuSI can be described as a Type IIS/C/G REase for its cleavage site outside of the recognition sequence (Type IIS), bifunctional polypeptide possessing both methyltransferase (MTase) and endonuclease activities (Type IIC) and endonuclease activity stimulated by S-adenosyl-L-methionine (SAM) (Type IIG). The stimulatory effect of SAM on cleavage activity presents a major paradox: a co-factor of the MTase activity that renders the substrate unsusceptible to cleavage enhances the cleavage activity. Here we show that the RM.BpuSI MTase activity modifies both cleavage substrate and product only when they are unmethylated. The MTase activity is, however, much lower than that of M1.BpuSI and is thought not to be the major MTase for host DNA protection. SAM and sinefungin (SIN) increase the Vmax of the RM.BpuSI cleavage activity with a proportional change in Km, suggesting the presence of an energetically more favorable pathway is taken. We further showed that RM.BpuSI undergoes substantial conformational changes in the presence of Ca2+, SIN, cleavage substrate and/or product. Distinct conformers are inferred as the pre-cleavage/cleavage state (in the presence of Ca2+, substrate or both) and MTase state (in the presence of SIN and substrate, SIN and product or product alone). Interestingly, RM.BpuSI adopts a unique conformation when only SIN is present. This SIN-bound state is inferred as a branch point for cleavage and MTase activity and an intermediate to an energetically favorable pathway for cleavage, probably through increasing the binding affinity of the substrate to the enzyme under cleavage conditions. Mutation of a SAM-binding residue resulted in altered conformational changes in the presence of substrate or Ca2+ and eliminated cleavage activity. The present study underscores the role of the MTase domain as facilitator of efficient cleavage activity for RM.BpuSI.
PMCID: PMC3817140  PMID: 24224063
17.  A spite specific endonuclease from thermus thermophilus 111, Tth111I. 
Nucleic Acids Research  1980;8(1):43-56.
A site specific endonuclease with novel specificity has been isolated from Thermus thermophilus strain 111 and named Tth111I. Tth111I cleaves lambda DNA into three fragments of 23.5, 25.7 and 50.8% of the total length, and ColE1 DNA into two fragments of nearly equal length. The sequences around Tth111I cleavage sites of ColE1 and lambda DNA were determined by the Maxam and Gilbert method and the two dimensional mapping method. The results suggest that Tth111I recognizes the DNA sequence (formula: see text) and cleaves the site as indicated by the arrows. Assuming that the first T.A pair in the sequence can be replaced for any base pair, the Tth111I recognition sequence has the symmetry with the two-fold axis as most type II restriction endonucleases do.
PMCID: PMC327241  PMID: 6243779
18.  SURVEY AND SUMMARY: Diversity of Type II restriction endonucleases that require two DNA recognition sites 
Nucleic Acids Research  2003;31(21):6079-6084.
Orthodox Type IIP restriction endonucleases, which are commonly used in molecular biological work, recognize a single palindromic DNA recognition sequence and cleave within or near this sequence. Several new studies have reported on structural and biochemical peculiarities of restriction endonucleases that differ from the orthodox in that they require two copies of a particular DNA recognition sequence to cleave the DNA. These two sites requiring restriction endonucleases belong to different subtypes of Type II restriction endonucleases, namely Types IIE, IIF and IIS. We compare enzymes of these three types with regard to their DNA recognition and cleavage properties. The simultaneous recognition of two identical DNA sites by these restriction endonucleases ensures that single unmethylated recognition sites do not lead to chromosomal DNA cleavage, and might reflect evolutionary connections to other DNA processing proteins that specifically function with two sites.
PMCID: PMC275478  PMID: 14576294
19.  A second site specific endonuclease from Thermus thermophilus 111, Tth111II. 
Nucleic Acids Research  1980;8(15):3275-3285.
A second site specific endonuclease with novel specificity has been purified from Thermus thermophilus strain 111 and named Tth111II. The enzyme is active at temperature up to 80 degrees C and requires Mg2+ or Mn2+ for endonuclease activity. Tth111II cleaves phi X174RFDNA into 11 fragments and lambda NA into more than 25 fragments. From the 5'-terminal sequences of TthlllII fragments of phi X174RFDNA determined by the two dimensional homochromatography and the survey on nucleotide sequence of phi X174RFDNA, it was concluded that Tth111II recognizes the DNA sequence (see former index) and cleaves the sites as indicated by the arrows.
PMCID: PMC324152  PMID: 6255411
20.  Characterization and crystal structure of the type IIG restriction endonuclease RM.BpuSI 
Nucleic Acids Research  2011;39(18):8223-8236.
A type IIG restriction endonuclease, RM.BpuSI from Bacillus pumilus, has been characterized and its X-ray crystal structure determined at 2.35Å resolution. The enzyme is comprised of an array of 5-folded domains that couple the enzyme's N-terminal endonuclease domain to its C-terminal target recognition and methylation activities. The REase domain contains a PD-x15-ExK motif, is closely superimposable against the FokI endonuclease domain, and coordinates a single metal ion. A helical bundle domain connects the endonuclease and methyltransferase (MTase) domains. The MTase domain is similar to the N6-adenine MTase M.TaqI, while the target recognition domain (TRD or specificity domain) resembles a truncated S subunit of Type I R–M system. A final structural domain, that may form additional DNA contacts, interrupts the TRD. DNA binding and cleavage must involve large movements of the endonuclease and TRD domains, that are probably tightly coordinated and coupled to target site methylation status.
PMCID: PMC3185434  PMID: 21724614
21.  Simple repeated sequences in human satellite DNA. 
Nucleic Acids Research  1982;10(2):547-563.
In an extensive analysis, using a range of restriction endonucleases, HinfI and TaqI were found to differentiate satellites I, II and III & IV. Satellite I is resistant to digestion by TaqI, but is cleaved by HinfI to yield three major fragments of approximate size 770, 850 and 950bp, associated in a single length of DNA. The 770bp fragment contains recognition sites for a number of other enzymes, whereas the 850 and 950bp fragments are "silent" by restriction enzyme analysis. Satellite II is digested by HinfI into a large number of very small (10-80bp) fragments, many of which also contain TaqI sites. A proportion of the HinfI sites in satellite II have the sequence 5'GA(GC)TC. The HinfI digestion products of satellites III and IV form a complete ladder, stretching from 15bp or less to more than 250bp, with adjacent multimers separated by an increment of 5bp. The ladder fragments do not contain TaqI sites and all HinfI sites have the sequence 5'GA(AT)TC. Three fragments from the HinfI ladder of satellite III have been sequenced, and all consist of a tandemly repeated 5bp sequence, 5'TTCCA, with a non-repeated, G+C rich sequence, 9bp in length, at the 3' end.
PMCID: PMC326157  PMID: 6278420
22.  Cloning, sequencing and expression of the Taq I restriction-modification system. 
Nucleic Acids Research  1987;15(23):9781-9796.
The Taq I modification and restriction genes (recognition sequence TCGA) have been cloned in E. coli and their DNA sequences have been determined. Both proteins were characterized and the N-terminal sequence of the endonuclease was determined. The genes have the same transcriptional orientation with the methylase gene 5' to the endonuclease gene. The methylase gene is 1089 bp in length (363 amino acids, 40,576 daltons); the endonuclease gene is 702 bp in length (234 amino acids, 27,523 daltons); they are separated by 132 bp. Both methylase and endonuclease activity can be detected in cell extracts. The clones fully modify the vector and chromosomal DNA but they fail to restrict infecting phage. Clones carrying only the restriction gene are viable even in the absence of modification. The restriction gene contains 7 Taq I sites; the modification gene contains none. This asymmetric distribution of sites could be important in the regulation of the expression of the endonuclease gene.
PMCID: PMC306531  PMID: 2827113
23.  alpha-Putrescinylthymine and the sensitivity of bacteriophage phi W-14 DNA to restriction endonucleases. 
Nucleic Acids Research  1985;13(7):2559-2568.
The modified base alpha-putrescinylthymine (putT) in phi W-14 DNA blocks cleavage of the DNA by 17 of 32 Type II restriction endonucleases. The enzymes cleaving the DNA do so to widely varying extents. The frequencies of cleavage of three altered forms of the DNA show that putT blocks recognition sites either when it occurs within the site or when it is in a sequence flanking the site. The blocking is dependent on both charge and steric factors. The charge effects can be greater than the steric effects for some of the enzymes tested. All the enzymes cleaving phi W-14 DNA release discrete fragments, showing that the distribution of putT is ordered. The cleavage frequencies for different enzymes suggest that the sequence CAputTG occurs frequently in the DNA. Only TaqI of the enzymes tested appeared not to be blocked by putT, but it was slowed down. TaqI generated fragments are joinable by T4 DNA ligase.
PMCID: PMC341175  PMID: 2987859
24.  Restriction endonucleases HindII and TaqI cleave DNA with mismatched nucleotides within their recognition sequences. 
Nucleic Acids Research  1986;14(5):1943-1949.
Restriction endonucleases HindII and TaqI, but not SalI, were found to efficiently cleave synthetic hexadecanucleotide duplexes which contained either an A/C or a G/T mismatch within their respective restriction sites. Double-stranded M13 DNAs with identical mismatches were also cleaved under the assay conditions. These results suggest that the distortion of the DNA duplex, caused by these purine/pyrimidine mismatches is not sufficiently large so as to interfere with the recognition and the subsequent cleavage of the DNA by these two enzymes. HindII and SalI, but not TaqI, were furthermore shown to hydrolyze the two strands of the duplex with different rates. The differences between the mode of recognition of their respective restriction sites by these three enzymes are discussed.
PMCID: PMC339633  PMID: 3008080
25.  Creation of a type IIS restriction endonuclease with a long recognition sequence 
Nucleic Acids Research  2009;37(9):3061-3073.
Type IIS restriction endonucleases cleave DNA outside their recognition sequences, and are therefore particularly useful in the assembly of DNA from smaller fragments. A limitation of type IIS restriction endonucleases in assembly of long DNA sequences is the relative abundance of their target sites. To facilitate ligation-based assembly of extremely long pieces of DNA, we have engineered a new type IIS restriction endonuclease that combines the specificity of the homing endonuclease I-SceI with the type IIS cleavage pattern of FokI. We linked a non-cleaving mutant of I-SceI, which conveys to the chimeric enzyme its specificity for an 18-bp DNA sequence, to the catalytic domain of FokI, which cuts DNA at a defined site outside the target site. Whereas previously described chimeric endonucleases do not produce type IIS-like precise DNA overhangs suitable for ligation, our chimeric endonuclease cleaves double-stranded DNA exactly 2 and 6 nt from the target site to generate homogeneous, 5′, four-base overhangs, which can be ligated with 90% fidelity. We anticipate that these enzymes will be particularly useful in manipulation of DNA fragments larger than a thousand bases, which are very likely to contain target sites for all natural type IIS restriction endonucleases.
PMCID: PMC2685105  PMID: 19304757

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