Type II Restriction Enzymes Research Articles

BfiI, a restriction endonuclease from Bacillus firmus S8120, which recognizes the novel non-palindromic sequence 5'-ACTGGG(N)5/4-3'.

A new type IIS restriction endonuclease Bfi I hasbeen partially purified from Bacillus firmus S8120. Bfi I recognizes the non-palindromic hexanucleotide sequence 5'-ACTGGG(N)5/4-3' and makes a staggered cut at the fifth base pair downstream of the recognition sequence on the upper strand, producing a single base 3' protruding end.

Construction of a Z-DNA-specific restriction endonuclease.

Novel restriction enzymes can be created by fusing the nuclease domain of FokI endonuclease with defined DNA binding domains. Recently, we have characterized a domain (Z alpha) from the N-terminal region of human double-stranded RNA adenosine deaminase (hADAR1), which binds the Z-conformation with high specificity. Here we report creation of a conformation-specific endonuclease, Z alpha nuclease, which is a chimera of Z alpha and FokI nuclease. Purified Z alpha nuclease cleaves negatively supercoiled plasmids only when they contain a Z-DNA forming insert, such as (dC-dG)13. The precise location of the cleavage sites was determined by primer extension. Cutting has been mapped to the edge of the B-Z junction, suggesting that Z alpha nuclease binds within the Z-DNA insert, but cleaves in the nearby B-DNA, by using a mechanism similar to type IIs restriction enzymes. These data show that Z alpha binds Z-DNA in an environment similar to that in a cell. Z alpha nuclease, a structure-specific restriction enzyme, may be a useful tool for further study of the biological role of Z-DNA.

Recognition and cleavage of DNA by type-II restriction endonucleases.

Restriction endonucleases are enzymes which recognize short DNA sequences and cleave the DNA in both strands. Depending on the enzymological properties different types are distinguished. Type II restriction endonucleases are homodimers which recognize short palindromic sequences 4-8 bp in length and, in the presence of Mg2+, cleave the DNA within or next to the recognition site. They are capable of non-specific binding to DNA and make use of linear diffusion to locate their target site. Binding and recognition of the specific site involves contacts to the bases of the recognition sequence and the phosphodiester backbone over approximately 10-12 bp. In general, recognition is highly redundant which explains the extreme specificity of these enzymes. Specific binding is accompanied by conformational changes over both the protein and the DNA. This mutual induced fit leads to the activation of the catalytic centers. The precise mechanism of cleavage has not yet been established for any restriction endonuclease. Currently two models are discussed: the substrate-assisted catalysis mechanism and the two-metal-ion mechanism. Structural similarities identified between EcoRI, EcoRV, BamHI, PvuII and Cfr10I suggest that many type II restriction endonucleases are not only functionally but also evolutionarily related.

Crystallization and preliminary X-ray analysis of restriction endonuclease FokI bound to DNA

FokI is a type IIs restriction endonuclease which recognizes an asymmetric DNA sequence and cleaves DNA a short distance away from the sequence. The enzyme is bipartite in nature with its DNA recognition and cleavage functions located on distinct domains. We report here cocrystals of the complete FokI enzyme (579 amino acids) bound to a 20-bp DNA fragment containing its recognition sequence. The complex is amongst the largest protein-DNA complexes to be crystallized, and required macroseeding techniques for optimal crystal growth. The cocrystals diffract to at least 2.8 Å in resolution and belong to space group P2 1 with unit cell dimensions of a=67.9 Å, b=119.8 Å, c=69.1 Å, β=96.6°. Using specific amino acid analysis we show that asymmetric unit contains a single FokI molecule bound to the 20-bp DNA fragment. This paper reports the first cocrystals of a type IIs restriction endonuclease. © 1997 Federation of European Biochemical Societies.

NOMAD: a versatile strategy for in vitro DNA manipulation applied to promoter analysis and vector design.

Molecular analysis of complex modular structures, such as promoter regions or multi-domain proteins, often requires the creation of families of experimental DNA constructs having altered composition, order, or spacing of individual modules. Generally, creation of every individual construct of such a family uses a specific combination of restriction sites. However, convenient sites are not always available and the alternatives, such as chemical resynthesis of the experimental constructs or engineering of different restriction sites onto the ends of DNA fragments, are costly and time consuming. A general cloning strategy (nucleic acid ordered assembly with directionality, NOMAD; WWW resource locator http:@Lmb1.bios.uic.edu/NOMAD/NOMAD.htm l) is proposed that overcomes these limitations. Use of NOMAD ensures that the production of experimental constructs is no longer the rate-limiting step in applications that require combinatorial rearrangement of DNA fragments. NOMAD manipulates DNA fragments in the form of "modules" having a standardized cohesive end structure. Specially designed "assembly vectors" allow for sequential and directional insertion of any number of modules in an arbitrary predetermined order, using the ability of type IIS restriction enzymes to cut DNA outside of their recognition sequences. Studies of regulatory regions in DNA, such as promoters, replication origins, and RNA processing signals, construction of chimeric proteins, and creation of new cloning vehicles, are among the applications that will benefit from using NOMAD.

Type IIS restriction enzyme footprinting I. Measurement of a triple helix dissociation constant with Eco57I at 25 degrees C.

A method is described to measure triple helix dissociation constants by inhibiting the cleavage of a plasmid constructed to contain a target sequence for the triplex forming oligonucleotide (TFO) dT20 by the type IIS restriction enzyme Eco57I. The method relies upon the TFO's ability to block the cleavage reaction by occupying the enzymes cleavage site but not its specific binding sequence. Using this protocol, the dissociation constant for dT20 bound to its target was 0.16 +/- 0.01 microM at 25 degrees C. The accuracy of this experiment was demonstrated by measuring the Kd of an affinity cleavage TFO using Eco57I and Quantitative Affinity Cleavage Titration. Type IIS restriction endonuclease footprinting should be useful for the qualitative and quantitative investigation of ligand-DNA interactions.

Molecular Phylogeny of the Neotoma floridana Species Group

A phylogeny of the Neotoma floridana species group of woodrats was inferred using mi-tochondrial-DNA (mtDNA) restriction sites identified by 17 type-II restriction endonucleas-es. Wagner, Dollo, and generalized parsimony analyses revealed a sharp dichotomy among populations of Neotoma albigula found east and west of the Rio Grande. Additional polymorphism among samples of N. albigula from northern Mexico also was detected. Data from mtDNA suggest paraphyly exists within N. albigula and that at least two cryptic species are present within the taxon. Phylogenetic and phenetic analyses of the mtDNA data reveal a sister-group relationship between Neotoma micropus and N. albigula , whereas N. floridana and Neotoma magister form sister taxa to this group. These results clarify discrepancies reported for this species group based on morphologic, allozymic, and karyotype data. Additionally, Neotoma phenax, Neotoma cinerea, Neotoma mexicana , and Neotoma stephensi were included to provide an intrageneric perspective on relationships within the N. floridana species group. N. mexicana and N. stephensi formed a clade associated with the N. floridana species-group clade with N. phenax and N. cinerea appearing as sole members of branches basal to the remaining Neotoma .

Sequence specificity of triplex DNA formation: Analysis by a combinatorial approach, restriction endonuclease protection selection and amplification.

We have devised a combinatorial method, restriction endonuclease protection selection and amplification (REPSA), to identify consensus ligand binding sequences in DNA. In this technique, cleavage by a type IIS restriction endonuclease (an enzyme that cleaves DNA at a site distal from its recognition sequence) is prevented by a bound ligand while unbound DNA is cleaved. Since the selection step of REPSA is performed in solution under mild conditions, this approach is amenable to the investigation of ligand-DNA complexes that are either insufficiently stable or not readily separable by other methods. Here we report the use of REPSA to identify the consensus duplex DNA sequence recognized by a G/T-rich oligodeoxyribonucleotide under conditions favoring purine-motif triple-helix formation. Analysis of 47 sequences indicated that recognition between 13 bases on the oligonucleotide 3' end and the duplex DNA was sufficient for triplex formation and indicated the possible existence of a new base triplet, G.AT. This information should help identify appropriate target sequences for purine-motif triplex formation and demonstrates the power of REPSA for investigating ligand-DNA interactions.

Characterisation of a complex restriction/modification system detected in a Bifidobacterium longum strain

Two type-II restriction endonucleases, BloI and BloII, have been detected in a Bifidobacterium longum strain. BloI is influenced by dam methylation: it cleaves dam − but not dam + DNA. It shows a temperature and pH optimum of 45°C and pH 7.5. Restriction analysis and cloning experiments showed that the recognition sequence is RGATCY and that the enzyme cuts 5′ to the guanine residue. It is an isoschizomer of commercial enzymes, Bst YI and XhoII. The second activity is not inhibited by dam methylation. It has a temperature optimum between 25°C and 30°C and shows a broad pH optimum between 4.5 and 7.0. The activity is thermolabile and can be heat-killed by a 5 min incubation at 60°C. Cloning and sequencing experiments revealed that its recognition sequence is CTGCAG and that it cuts 5′ to the second guanine residue in the sequence. This enzyme is the first described isoschizomer of PstI.

Creating seamless junctions independent of restriction sites in PCR cloning

A method is described for the efficient cloning of any given DNA sequence into any desired location without the limitation of naturally occurring restriction sites. The technique employs the polymerase chain reaction (PCR) combined with the capacity of the type-IIS restriction endonuclease (ENase) Eam1104I to cut outside its recognition sequence. Primers that contain the Eam1104I recognition site (5′-CTCTTC) are used to amplify the DNA fragments being manipulated. Because the ENase is inhibited by site-specific methylation in the recognition sequence, all internal Eam1104I sites present in the DNA can be protected by performing the PCR amplification in the presence of 5-methyl-deoxycytosine ( m5dCTP). The primer-encoded Eam1104I sites are not affected by the modified nucleotides (nt) since the newly synthesized strand does not contain any cytosine residues in the recognition sequence. In addition, the ENase's ability to cleave several bases downstream from its recognition site allows the removal of superfluous, terminal sequences from the amplified DNA fragments, resulting in 5′ overhangs that are defined by the nt present within the cleavage site. Thus, the elimination of extraneous nt and the generation of unique, non-palindromic sticky ends permits the formation of seamless junctions in a directional fashion during the subsequent ligation event.

Two different isoschizomers of the type-II restriction endonuclease Taq I (T/CGA) within the same Thermus isolate: Tsp32 I, an enzyme with similar heat stability properties to the prototype enzyme Taq I, and Tsp32 II, a hyperthermostable isoschizomer of Taq I

We have recently screened 112 separate isolates of the genus Thermus, collected from neutral and alkaline hot water springs on four continents, for the presence of the Type-II restriction endonuclease Taq I (T/CGA). One particular isolate from the Azores (strain 32) was found to contain high levels of a restriction endonuclease with the same recognition and cleavage site as Taq I. Initial studies revealed that the partially purified enzyme from strain 32 was considerably more resistant to heat inactivation than the prototype enzyme Taq I, being able to withstand temperatures at least 10 degrees C higher than Taq I, before showing evidence of heat inactivation. Subsequently it became clear that the partially purified extract from strain 32 contains two separate enzymes, both of which are isoschizomers of Taq I. One of the enzymes, Tsp32 I, has similar thermal stability characteristics to Taq I, whereas the second Taq I isoschizomer, Tsp32 II, found in the same Thermus isolate as Tsp32 I, is considerably more thermostable than Taq I, retaining full enzyme activity up to a temperature of 85 degrees C. Tsp32 I and Tsp32 II were further distinguished by virtue of their different requirements for magnesium ions.

Evidence for an evolutionary relationship among type-II restriction endonucleases

Type-II restriction-modification (R-M) systems comprise two enzymes, a DNA methyltransferase (MTase) and a restriction endonuclease (ENase), each of which specifically interact with the same 4–8-bp sequence. All type-II MTases share several amino acid (aa) sequence motifs, which makes an evolutionary relatedness among these enzymes probable. The type-II ENases, in contrast, except for some homologous isoschizomers, do not share significant aa sequence similarity. Therefore, ENases in general have been considered unrelated. Here we show that in addition to the analysis of the genotype (aa sequence), a comparison of the phenotype (recognition sequence) of these enzymes can provide independent information regarding evolutionary relationships, and thereby, help to analyze the significance of weak aa sequence similarities. Multistep Monte-Carlo analyses were employed to demonstrate that the recognition sequences of those ENases, which were found to be related by a progressive multiple aa sequence alignment, are more similar to each other than would be expected by chance. This analysis supports the notion that not only type-II MTases, but also type-II ENases did not arise independently in evolution, but rather evolved from one or a few primordial DNA-modifying and DNA-cleaving enzymes, respectively.

SgfI, a new type-II restriction endonuclease that recognizes the octanucleotide sequence 5′-GCGAT/CGC-3′

A new restriction endonuclease (ENase), SgfI, has been isolated from the bacterium Streptomyces sp. SgfI recognizes the 8-bp palindrome 5′-GCGATCGC-3′ and cleaves double-stranded DNA after the T in this sequence, producing a two-base 3′ overhang compatible with PvuI termini. SgfI is a rare-cutting ENase and should be useful for megabase mapping experiments.

Sequence similarity among type-II restriction endonucleases, related by their recognized 6-bp target and tetranucleotide-overhang cleavage

The type-II restriction endonucleases (ENases) EcoRI (recognition sequence G ↓AATTC), RsrI (G ↓AATTC), Xcyl (C ↓CCGGG), Cfr91 (C ↓CCGGG) and MunI (C ↓AATTG), all cleave hexanucleotide palindromic sequences, leaving tetranucleotide 5′-overhangs. Two regions of similarity that appear in the same order and relative position were identified among the amino-acid sequences of ENases. These regions map to the structural elements of EcoRI involved in the building of the catalytic site and in interactions with the central nucleotides of the recognized sequence. We propose that these ENases might all share a similar structural organization of the active site and structural motifs involved in interactions with specific DNA recognition sequences.

Restriction endonucleases functionally interacting with two DNA sites

Simultaneous interaction with two recognition sites was found to be a precondition for DNA cleavage by certain type-II and type-III restriction endonucleases. Nevertheless, the molecular mechanisms of the protein-DNA interaction are different between members of both classes of enzymes.

Determination of methylation specificity of DsaV methyltransferase by a simple biochemical method.

We have developed a simple new method that can identify the base methylated by a sequence-specific DNA methyltransferase and have used it to identify the cytosine that is methylated by DsaV methyltransferase (M. DsaV) within its recognition sequence 5'-CCNGG. The method utilizes the fact that exonuclease III of E. coli does not degrade DNA ends with 3' overhangs and cannot hydrolyze a phosphorothioate linkage. DNA duplexes containing phosphorothioate linkages at specific positions were methylated with M. DsaV in the presence of [methyl-3H] S-adenosylmethionine and were subjected to exonuclease III digestion. The pattern of [methyl-3H] dCMP release from the duplexes was consistent with the methylation of the internal cytosine in CCNGG, but not of the outer cytosine. To establish the accuracy of this method, we confirmed the known specificity of EcoRII methyltransferase by the method. We also confirmed the specificity of M. DsaV using an established biochemical method that involves the use of a type IIS restriction enzyme. Methylation of CCWGG (W = A or T) sequences at the internal cytosines is native to E. coli and is not restricted by the modified cytosine restriction (Mcr) systems. Surprisingly, the gene for M. DsaV was significantly restricted by the McrBC system. We interpret this to mean that M. DsaV may occasionally methylate at sequences other than CCNGG or may occasionally methylate the outer cytosine in its recognition sequence.

Construction of a Translation Enhancer-Containing Vector for Gene Expression in a Prokaryotic Two-Cistron System

Abstract A prokaryotic two-cistron system for gene expression, based on the pGEM1 plasmid and containing a translation enhancer in the coding part of the first cistron, has been constructed. The gene to be expressed can be inserted into the vector by means of a PCR-mediated approach using type IIS restriction endonucleases (SDL method). Efficiency of the system is exemplified by the expression of a synthetic gene encoding human interleukin lα.

LlaAI and LlaBI, two type-II restriction endonucleases from Lactococcus lactis subsp. cremoris W9 and W56 recognizing, respectively, 5'-/GATC-3' and 5'-C/TRYAG-3'

Two type-II restriction endonucleases have been purified from Lactococcus lactis subsp. cremoris W9 and W56, the strains isolated from a mixed Cheddar starter. Their characterization showed that LlaAI was an isoschizomer of MboI from Morexella bovis with the cleaving sequence, 5'-/GATC-3', being sensitive to methylation of the adenine residue; LlaBI was an isoschizomer to SfcI from Streptococcus faecium with the cleaving sequence, 5'-C/TRYAG-3'. Both LlaAI and LlaBI restriction-modification (R-M) systems are encoded by the plasmids, respectively, pFW094 and pJW563, protecting the harboring strain against phage attack.

Single amino acid substitutions uncouple the DNA binding and strand scission activities of Fok I endonuclease.

Single alanine substitution mutations at Asp-450 or Asp-467 of the type IIS restriction enzyme Fok I have no effect on the ability of the enzyme to bind strongly and selectively to its recognition site but completely eliminate its ability to cleave either strand of substrate DNA. Since wild-type Fok I shows no kinetic preference or required order of strand cleavage, these results indicate that Fok I, which evidently functions as a monomer, uses a single catalytic center to cleave both strands of DNA. In this respect, Fok I may resemble other monomeric enzymes that cleave double-stranded DNA.

Ligation-mediated PCR of restriction fragments from large DNA molecules.

A general method is described for PCR amplification of single restriction fragments from large DNA molecules. The method involves sequence-specific ligation of synthetic oligonucleotides to ambiguous 4-base 5' overhangs produced by type IIS restriction endonucleases. Such "adapter-tags" provide one target for primer annealing in subsequent PCR reactions. The second target for primer annealing is provided by a universal "bubble-tag" ligated to blunt ends produced with another endonuclease. The key advantage of this approach is that specific fragments can be isolated without any prior knowledge of the nucleotide sequence of the target. Using bacteriophage lambda DNA as a test system, unique PCR products could be generated consistently. Conditions of temperature, ionic strength, and substrate concentration in the adapter-tag ligations--which affect sequence specificity--were found to have a major influence on the purity of PCR-generated fragments. In principle, the method permits the amplification of virtually any sequence from purified cosmid or YAC DNA using a library of only 240 adapter-tags.