Type II Restriction Enzymes Research Articles

The Recognition Domain of the BpuJI Restriction Endonuclease in Complex with Cognate DNA at 1.3-Å Resolution

Type IIS restriction endonucleases recognize asymmetric DNA sequences and cleave both DNA strands at fixed positions downstream of the recognition site. The restriction endonuclease BpuJI recognizes the asymmetric sequence 5′-CCCGT; however, it cuts at multiple sites in the vicinity of the target sequence. BpuJI consists of two physically separate domains, with catalytic and dimerization functions in the C-terminal domain and DNA recognition functions in the N-terminal domain. Here we report the crystal structure of the BpuJI recognition domain bound to cognate DNA at 1.3-Å resolution. This region folds into two winged-helix subdomains, D1 and D2, interspaced by the DL subdomain. The D1 and D2 subdomains of BpuJI share structural similarity with the similar subdomains of the FokI DNA-binding domain; however, their orientations in protein–DNA complexes are different. Recognition of the 5′-CCCGT target sequence is achieved by BpuJI through the major groove contacts of amino acid residues located on both the helix–turn–helix motifs and the N-terminal arm. The role of these interactions in DNA recognition is also corroborated by mutational analysis.

Identification of a Single HNH Active Site in Type IIS Restriction Endonuclease Eco31I

Type IIS restriction endonuclease Eco31I is a “short-distance cutter”, which cleaves DNA strands close to its recognition sequence, 5′-GGTCTC(1/5). Previously, it has been proposed that related endonucleases recognizing a common sequence core GTCTC possess two active sites for cleavage of both strands in the DNA substrate. Here, we present bioinformatic identification and experimental evidence for a single nuclease active site. We identified a short region of homology between Eco31I and HNH nucleases, constructed a three-dimensional model of the putative catalytic domain and validated our predictions by random and site-specific mutagenesis. The restriction mechanism of Eco31I is suggested by analogy to the mechanisms of phage T4 endonuclease VII and homing endonuclease I- PpoI. We propose that residues D311 and N334 coordinate the cofactor. H312 acts as a general base-activating water molecule for the nucleophilic attack. K337 together with R340 and D345 are located in close proximity to the active center and are essential for correct folding of catalytic motif, while D345 together with R264 and D273 could be directly involved in DNA binding. We also predict that the Eco31I catalytic domain contains a putative Zn-binding site, which is essential for its structural integrity. Our results suggest that the HNH-like active site is involved in the cleavage of both strands in the DNA substrate. On the other hand, analysis of site-specific mutants in the region, previously suggested to harbor the second active site, revealed its irrelevance to the nuclease activity. Thus, our data argue against the earlier prediction and indicate the presence of a single conserved active site in type IIS restriction endonucleases that recognize common sequence core GTCTC.

An EM View of the FokI Synaptic Complex by Single Particle Analysis

FokI is a type IIS restriction endonuclease that recognizes the 5′-GGATG-3′ sequence and cleaves non-specifically at 9 and 13 base-pairs away on the top and bottom strands, respectively, to produce a 5′ overhang. FokI is a bipartite endonuclease with separate recognition and cleavage domains. Because of its bipartite nature, FokI has received considerable interest in generating chimeric nucleases for use in biotechnology, and recently as possible therapeutic agents in gene therapy by initiating homologous gene recombination and repair. Here we show, using single-particle electron microscopic studies, that the FokI active complex prefers a single conformation in which the subunits are arranged in a doughnut shape complex with protein–protein and possibly protein–DNA interactions stabilizing the cleavage complex. Our electron microscopy (EM) model provides new insights into the activation mechanism of FokI and how non-specific cleavage is avoided.

REPSA: General combinatorial approach for identifying preferred ligand–DNA binding sequences

Most DNA-binding ligands, ranging from protein transcription factors to small molecule antineoplastic agents, recognize duplex DNA with some degree of sequence specificity. Determining this binding specificity is important for biochemists, molecular biologists, and medicinal chemists. Some information can be obtained through the study of defined DNA sequences, but a full picture of a ligand’s binding specificity can only be obtained through combinatorial means, whereby vast libraries of sequences are screened. Several combinatorial methods have been developed for the study of ligand–DNA interactions, all of which require the physical separation of ligand-bound DNA from uncomplexed DNA before amplification by PCR. Here, we describe the novel combinatorial method Restriction Endonuclease Protection Selection and Amplification (REPSA). REPSA selects for ligand-bound DNAs through their inhibition of an enzymatic process—cleavage by a type IIS restriction endonuclease—which inactivates templates for subsequent PCR amplification. We have used REPSA to identify the preferred binding sites of oligonucleotides, proteins, and small molecules on duplex DNA. Unlike conventional combinatorial methods, REPSA is amenable to the study of mixtures of native ligands with relatively unknown identities and properties. Thus, REPSA is a powerful, versatile, general method for the combinatorial determination of ligand-binding specificity and a functional means of ligand discovery.

Restriction endonuclease BpuJI specific for the 5′-CCCGT sequence is related to the archaeal Holliday junction resolvase family

Type IIS restriction endonucleases (REases) recognize asymmetric DNA sequences and cleave both DNA strands at fixed positions downstream of the recognition site. REase BpuJI recognizes the asymmetric sequence 5′-CCCGT, however it cuts at multiple sites in the vicinity of the target sequence. We show that BpuJI is a dimer, which has two DNA binding surfaces and displays optimal catalytic activity when bound to two recognition sites. BpuJI is cleaved by chymotrypsin into an N-terminal domain (NTD), which lacks catalytic activity but binds specifically to the recognition sequence as a monomer, and a C-terminal domain (CTD), which forms a dimer with non-specific nuclease activity. Fold recognition approach reveals that the CTD of BpuJI is structurally related to archaeal Holliday junction resolvases (AHJR). We demonstrate that the isolated catalytic CTD of BpuJI possesses end-directed nuclease activity and preferentially cuts 3 nt from the 3′-terminus of blunt-ended DNA. The nuclease activity of the CTD is repressed in the apo-enzyme and becomes activated upon specific DNA binding by the NTDs. This leads to a complicated pattern of specific DNA cleavage in the vicinity of the target site. Bioinformatics analysis identifies the AHJR-like domain in the putative Type III enzymes and functionally uncharacterized proteins.

USER fusion: a rapid and efficient method for simultaneous fusion and cloning of multiple PCR products

We present a method that allows simultaneous fusion and cloning of multiple PCR products in a rapid and efficient manner. The procedure is based on the use of PCR primers that contain a single deoxyuridine residue near their 5′ end. Treatment of the PCR products with a commercial deoxyuridine-excision reagent generates long 3′ overhangs designed to specifically complement each other. The combination of this principle with the improved USER cloning technique provides a simple, fast and very efficient method to simultaneously fuse and clone multiple PCR fragments into a vector of interest. Around 90% positive clones were obtained when three different PCR products were fused and cloned into a USER-compatible vector in a simple procedure that, apart from the single PCR amplification step and the bacterial transformation, took approximately one hour. We expect this method to replace overlapping PCR and the use of type IIS restriction enzymes in many of their applications.

Development of an automated SNP analysis method using a paramagnetic beads handling robot

Biological and medical importance of the single nucleotide polymorphism (SNP) has led to development of a wide variety of methods for SNP typing. Aiming for establishing highly reliable and fully automated SNP typing, we have developed the adapter ligation method in combination with the paramagnetic beads handling technology, Magtration(R). The method utilizes sequence specific ligation between the fluorescently labeled adapter and the sample DNAs at the cohesive end produced by a type IIS restriction enzyme. Evaluation of the method using human genomic DNA showed clear discrimination of the three genotypes without ambiguity using the same reaction condition for any SNPs examined. The operations following PCR amplification were automatically performed by the Magtration(R)-based robot that we have previously developed. Multiplex typing of two SNPs in a single reaction by using four fluorescent dyes was successfully preformed at the almost same sensitivity and reliability as the single typing. These results demonstrate that the automated paramagnetic beads handling technology, Magtration(R), is highly adaptable to the automated SNP analysis and that our method best fits to an automated in-house SNP typing for laboratory and medical uses.

Differential dependence on DNA ligase of type II restriction enzymes: A practical way toward ligase-free DNA automaton

DNA computing study is a new paradigm in computer science and biological computing fields. As one of DNA computing approaches, DNA automaton is composed of the hardware, input DNA molecule and state transition molecules. By now restriction enzymes are key hardware for DNA computing automaton. It has been found that DNA computing efficiency may be independent on DNA ligases when type IIS restriction enzymes like FokI are used as hardware. In this study, we compared FokI with four other distinct enzymes HgaI, BsmFI, BbsI, and BseMII, and found their differential independence on T4 DNA ligase when performing automaton reactions. Since DNA automaton is a potential powerful tool to tackle gene relationship in genomic network scale, the feasible ligase-free DNA automaton may set an initial base to develop functional DNA automata for various DNA technology development and implications in genetics study in the near future.

Total synthesis of multi-kilobase DNA sequences from oligonucleotides

A method for synthesizing DNA from 40-mer oligonucleotides, which we used to generate a 32-kb DNA fragment, is explained. DNA sequences are synthesized as approximately 500 bp fragments (synthons) in a two-step PCR reaction and cloned using ligation-independent cloning (LIC). Synthons are then assembled into longer full-length sequences in a stepwise manner. By initially synthesizing smaller fragments (synthons), the number of clones sequenced is low compared with synthesizing complete multi-kilobase DNA sequences in a single step. LIC eliminates the need for purification of fragments before cloning, making the process amenable to high-throughput operation and automation. Type IIs restriction enzymes allow seamless assembly of synthons without placing restrictions on the sequence being synthesized. Synthetic fragments are assembled in pairs to generate the final construct using vectors that allow selection of desired clones with two unique antibiotic resistance markers, and this eliminates the need for purification of fragments after digestion with restriction endonucleases.

Domain organization and metal ion requirement of the Type IIS restriction endonuclease MnlI

A two-domain structure of the Type IIS restriction endonuclease MnlI has been identified by limited proteolysis. An N-terminal domain of the enzyme mediates the sequence-specific interaction with DNA, whereas a monomeric C-terminal domain resembles bacterial colicin nucleases in its requirement for alkaline earth as well as transition metal ions for double- and single-stranded DNA cleavage activities. The results indicate that the fusion of the non-specific HNH-type nuclease to the DNA binding domain had transformed MnlI into a Mg 2+-, Ni 2+-, Co 2+-, Mn 2+-, Zn 2+-, Ca 2+-dependent sequence-specific enzyme. Nevertheless, MnlI retains a residual single-stranded DNA cleavage activity controlled by its C-terminal colicin-like nuclease domain.

Use of Single-Point Genome Signature Tags as a Universal Tagging Method for Microbial Genome Surveys

We developed single-point genome signature tags (SP-GSTs), a generally applicable, high-throughput sequencing-based method that targets specific genes to generate identifier tags from well-defined points in a genome. The technique yields identifier tags that can distinguish between closely related bacterial strains and allow for the identification of microbial community members. SP-GSTs are determined by three parameters: (i) the primer designed to recognize a conserved gene sequence, (ii) the anchoring enzyme recognition sequence, and (iii) the type IIS restriction enzyme which defines the tag length. We evaluated the SP-GST method in silico for bacterial identification using the genes rpoC, uvrB, and recA and the 16S rRNA gene. The best distinguishing tags were obtained with the restriction enzyme Csp6I upstream of the 16S rRNA gene, which discriminated all organisms in our data set to at least the genus level and most organisms to the species level. The method was successfully used to generate Csp6I-based tags upstream of the 16S rRNA gene and allowed us to discriminate between closely related strains of Bacillus cereus and Bacillus anthracis. This concept was further used successfully to identify the individual members of a defined microbial community.

787. Engineered Fok I Heterodimers for Enhanced Zinc Finger Nuclease Specificity

Zinc finger nucleases have been shown to be effective tools for high-frequency genome editing with potential therapeutic applications (Urnov et al., Nature 435:646-651). Such nucleases are created by fusing the cleavage domain of the type IIs restriction enzyme Fok I to engineered zinc finger proteins. One limitation of this architecture is that the natural Fok I cleavage domain must dimerize in order to cleave DNA; therefore two different zinc finger protein-Fok I fusions are required to introduce a double-stranded break at an asymmetric target site.

Mva1269I: A Monomeric Type IIS Restriction Endonuclease from Micrococcus Varians with Two EcoRI- and FokI-like Catalytic Domains

Type II restriction endonuclease Mva1269I recognizes an asymmetric DNA sequence 5'-GAATGCN / -3'/5'-NG / CATTC-3' and cuts top and bottom DNA strands at positions, indicated by the "/" symbol. Most restriction endonucleases require dimerization to cleave both strands of DNA. We found that Mva1269I is a monomer both in solution and upon binding of cognate DNA. Protein fold-recognition analysis revealed that Mva1269I comprises two "PD-(D/E)XK" domains. The N-terminal domain is related to the 5'-GAATTC-3'-specific restriction endonuclease EcoRI, whereas the C-terminal one resembles the nonspecific nuclease domain of restriction endonuclease FokI. Inactivation of the C-terminal catalytic site transformed Mva1269I into a very active bottom strand-nicking enzyme, whereas mutants in the N-terminal domain nicked the top strand, but only at elevated enzyme concentrations. We found that the cleavage of the bottom strand is a prerequisite for the cleavage of the top strand. We suggest that Mva1269I evolved the ability to recognize and to cleave its asymmetrical target by a fusion of an EcoRI-like domain, which incises the bottom strand within the target, and a FokI-like domain that completes the cleavage within the nonspecific region outside the target sequence. Our results have implications for the molecular evolution of restriction endonucleases, as well as for perspectives of engineering new restriction and nicking enzymes with asymmetric target sites.

Whole-Genome Validation of High-Information-Content Fingerprinting

Fluorescent-based high-information-content fingerprinting (HICF) techniques have recently been developed for physical mapping. These techniques make use of automated capillary DNA sequencing instruments to enable both high-resolution and high-throughput fingerprinting. In this article, we report the construction of a whole-genome HICF FPC map for maize (Zea mays subsp. mays cv B73), using a variant of HICF in which a type IIS restriction enzyme is used to generate the fluorescently labeled fragments. The HICF maize map was constructed from the same three maize bacterial artificial chromosome libraries as previously used for the whole-genome agarose FPC map, providing a unique opportunity for direct comparison of the agarose and HICF methods; as a result, it was found that HICF has substantially greater sensitivity in forming contigs. An improved assembly procedure is also described that uses automatic end-merging of contigs to reduce the effects of contamination and repetitive bands. Several new features in FPC v7.2 are presented, including shared-memory multiprocessing, which allows dramatically faster assemblies, and automatic end-merging, which permits more accurate assemblies. It is further shown that sequenced clones may be digested in silico and located accurately on the HICF assembly, despite size deviations that prevent the precise prediction of experimental fingerprints. Finally, repetitive bands are isolated, and their effect on the assembly is studied.

DNA Sequencing by Indexer Walking

There is a need for DNA sequencing methods that are faster, more accurate, and less expensive than existing techniques. Here we present a new method for DNA analysis by means of indexer walking. For DNA sequencing by indexer walking, we ligated double-stranded synthetic oligonucleotides (indexers) to DNA fragments that were produced by type IIS restriction endonucleases, which generate nonidentical 4-nucleotide 5' overhangs. The subsequent amplification (30 thermal cycles) of indexed DNA provided a template for automated DNA sequencing with fluorescent dideoxy terminators. The data gathered in the first sequencing reaction permitted further movement into the unknown nucleotide sequence by digestion of analyzed DNA with selected type IIS restriction endonuclease followed by ligation of the next indexer. A library of presynthesized indexers consisting of 256 oligonucleotides was used for bidirectional analysis of DNA molecules and provided universal primers for sequencing. The proposed protocol was successfully applied to sequencing of cryptic plasmids isolated from pathogenic strains of Escherichia coli. The overall error rate for base-calling was 0.5%, with a mean read length of 550 nucleotides. Approximately 1000 nucleotides of high-quality sequence could be obtained per day from a single clone. Indexer walking can be used as a low-cost procedure for nucleotide sequence determination of DNA molecules, such as natural plasmids, cDNA clones, and longer DNA fragments. It can also serve as an alternative method for gap filling at the final stage of genome sequencing projects.

Random Gene Dissection: A Tool for the Investigation of Protein Structural Organization

To investigate the domain structure of proteins and the function of individual domains, proteins are usually subjected to limited proteolysis, followed by isolation of protein fragments and determination of their functions. We have developed an approach we call random gene dissection (RGD) for the identification of functional protein domains and their interdomain regions as well as their in vivo complementing fragments. The approach was tested on a two-domain protein, the type IIS restriction endonuclease BfiI. The collection of BfiI insertional mutants was screened for those that are endonucleolytically active and thus induce the SOS DNA repair response. Sixteen isolated mutants of the wild-type specificity contained insertions that were dispersed in a relatively large region of the target recognition domain. They split the gene into two complementing parts that separately were unable to induce the SOS DNA repair response. In contrast, all 19 mutants of relaxed specificity contained the cassette inserted into a very narrow interdomain region that connects BfiI domains responsible for DNA recognition and for cleavage. As expected, only the N-terminal fragment of BfiI was required to induce SOS response. Our results demonstrate that RGD can be used as a general method to identify complementing fragments and functional domains in enzymes.

Population Genotyping of Hepatitis C Virus by Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry Analysis of Short DNA Fragments

Identifying hepatitis C virus (HCV) genotypes has become increasingly important for determining clinical course and the outcome of antiviral therapy. Here we describe the development of restriction fragment mass polymorphism (RFMP) analysis, a novel matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) assay suitable for high-throughput, sensitive, specific genotyping of multiple HCV species. The assay is based on PCR amplification and mass measurement of oligonucleotides containing genotype-specific motifs in the 5' untranslated region, into which a type IIS restriction endonuclease recognition was introduced by PCR amplification. Enzymatic cleavage of the products led to excision of multiple oligonucleotide fragments representing variable regions whose masses were determined by MALDI-TOF MS. The RFMP assay identified viral genotypes present at concentrations as low as 0.5% and reliably determined their relative abundance. When sera from 318 patients were analyzed, the RFMP assay exhibited 100% concordance with results obtained by clonal sequencing and identified mixed-genotype infections in 22% of the samples, in addition to several subtype variants. The RFMP assay has practical advantages over existing methods, including better quantitative detection of mixed populations and detection of genotype variants without need for population-based cloning, enabling reliable viral genotyping in laboratories and efficient study of the relationship between viral genotypes and clinical outcome.

MnlI—The member of H-N-H subtype of Type IIS restriction endonucleases

The Type IIS restriction endonuclease MnlI recognizes the non-palindromic nucleotide sequence 5′-CCTC(N)7/6↓ and cleaves DNA strands as indicated by the arrow. The genes encoding MnlI restriction–modification system were cloned and sequenced. It comprises N6-methyladenine and C5-methylcytosine methyltransferases and the restriction endonuclease. Biochemical studies revealed that MnlI restriction endonuclease cleaves double- and single-stranded DNA, and that it prefers different metal ions for hydrolysis of these substrates. Mg 2+ ions were shown to be required for the specific cleavage of double-stranded DNA, whereas Ni 2+ and some other transition metal ions were preferred for nonspecific cleavage of single-stranded DNA. The C-terminal part of MnlI restriction endonuclease revealed an intriguing similarity with the H-N-H type nucleolytic domain of bacterial toxins, Colicin E7 and Colicin E9. Alanine replacements in the conserved sequence motif 306Rx 3ExHHx 14Nx 8H greatly reduced specific activity of MnlI, and some mutations even completely inactivated the enzyme. However, none of these mutations had effect on MnlI binding to the specific DNA, and on its oligomerisation state as well. We interpret the presented experimental evidence as a suggestion that the motif 306Rx 3ExHHx 14Nx 8H represents the active site of MnlI. Consequentially, MnlI seems to be the member of Type IIS with the active site of the H-N-H type.

A rapid and efficient PCR-based mutagenesis method applicable to cell physiology study

PCR-based mutagenesis is a cornerstone of molecular biology and protein engineering studies. Herein we describe a rapid and highly efficient mutagenesis method using type IIs restriction enzymes. A template gene is amplified into two separate PCR fragments using two pairs of anchor and mutagenic primers. Mutated sequences are located near the recognition site of a type IIs restriction enzyme. After digestion of two fragments with a type IIs enzyme, exposed cohesive ends that are complementary to each other are then ligated together to generate a mutated gene. We applied this method to introduce multiple site-directed mutations in EGFP and Bcl-2 family genes and observed perfect mutagenesis efficiency at the desired sites. This efficient and cost-effective mutagenesis method can be applied to a wide variety of structural and functional studies in cell physiology.

The isolation of strand-specific nicking endonucleases from a randomized SapI expression library

The Type IIS restriction endonuclease SapI recognizes the DNA sequence 5'-GCTCTTC-3' (top strand by convention) and cleaves downstream (N1/N4) indicating top- and bottom-strand spacing, respectively. The asymmetric nature of DNA recognition presented the possibility that one, if not two, nicking variants might be created from SapI. To explore this possibility, two parallel selection procedures were designed to isolate either top-strand nicking or bottom-strand nicking variants from a randomly mutated SapI expression library. These procedures take advantage of a SapI substrate site designed into the expression plasmid, which allows for in vitro selection of plasmid clones possessing a site-specific and strand-specific nick. A procedure designed to isolate bottom-strand nicking enzymes yielded Nb.SapI-1 containing a critical R420I substitution near the end of the protein. The top-strand procedure yielded several SapI variants with a distinct preference for top-strand cleavage. Mutations present within the selected clones were segregated to confirm a top-strand nicking phenotype for single variants Q240R, E250K, G271R or K273R. The nature of the amino acid substitutions found in the selected variants provides evidence that SapI may possess two active sites per monomer. This work presents a framework for establishing the mechanism of SapI DNA cleavage.