Characterization of two intein homing endonucleases encoded in the DNA polymerase gene of Pyrococcus kodakaraensis strain KOD1.

Two intein endonucleases, denoted PI- Pko I and PI- Pko II, in the DNA polymerase gene of the hyperthermophilic archaeon Pyrococcus kodakaraensis KOD1 were expressed in Escherichia coli and the recombinant endonucleases were characterized. Both endonucleases were thermostable and cleaved their intein-less DNA sequences leaving four base 3'-hydroxyl overhangs. PI-Pko I exhibited 22 times higher specific activity than PI-Pko II and the activity of PI-Pko II was enhanced at higher potassium ion concentrations (1 M). Recognition sequences were also determined using synthetic oligonucleotides inserted into plasmid pUC19. It was shown that DNA sequences of 19 and 16 bp are needed for cleavage by PI-Pko I and PI-Pko II, respectively. PI-Pko II could cleave the downstream junction region between intein-encoding and mature DNA polymerase regions and cleavage by PI-Pko II could be detected even when chromosomal DNA of P.kodakaraensis KOD1 was used as substrate. Therefore, it is suggested that these endonucleases are switching endonucleases whose function lies in the rearrangement of chromosomal DNA.     

Microarray-based method to evaluate the accuracy of restriction endonucleases HpaII and MspI

A double-strand DNA (ds DNA) microarray was fabricated to analyze the structural perturbations caused by methylation and the different base mismatches in the interaction of the restriction endonucleases HpaII and MspI with DNA. First, a series of synthesized oligonucleotides were arrayed on the aldehyde-coated glass slides. Second, these oligonucleotides were hybridized with target sequences to obtain ds DNA microarray, which includes several types of double strands with the fully methylated, semi-methylated, and unmethylated canonical recognition sequences, semi-methylated and unmethylated base mismatches within the recognition sequences. The cleavage experiments were carried out under normal buffer conditions. The results indicated that MspI could partially cleave methylated and semi-methylated canonical recognition sequences. In contrast, HpaII could not cleave methylated and semi-methylated canonical recognition sequences. HpaII and MspI could both cleave the unmethylated canonical recognition sequence. However, HpaII could partially cleave the sequence containing one GG mismatch and not cleave other base mismatches in the corresponding recognition site. In contrast, MspI could not recognize the base mismatches within the recognition sequence. A good reproducibility was observed in several parallel experiments. The experiment indicates that the microarray technology has great potentials in high-throughput identifying important interactions between protein and DNA.     

Massively parallel characterization of restriction endonucleases

Restriction endonucleases are highly specific in recognizing the particular DNA sequence they act on. However, their activity is affected by sequence context, enzyme concentration and buffer composition. Changes in these factors may lead to either ineffective cleavage at the cognate restriction site or relaxed specificity allowing cleavage of degenerate ‘star’ sites. Additionally, uncharacterized restriction endonucleases and engineered variants present novel activities. Traditionally, restriction endonuclease activity is assayed on simple substrates such as plasmids and synthesized oligonucleotides. We present and use high-throughput Illumina sequencing-based strategies to assay the sequence specificity and flanking sequence preference of restriction endonucleases. The techniques use fragmented DNA from sequenced genomes to quantify restriction endonuclease cleavage on a complex genomic DNA substrate in a single reaction. By mapping millions of restriction site–flanking reads back to the Escherichia coli and Drosophila melanogaster genomes we were able to quantitatively characterize the cognate and star site activity of EcoRI and MfeI and demonstrate genome-wide decreases in star activity with engineered high-fidelity variants EcoRI-HF and MfeI-HF, as well as quantify the influence on MfeI cleavage conferred by flanking nucleotides. The methods presented are readily applicable to all type II restriction endonucleases that cleave both strands of double-stranded DNA.     

Nicking endonucleases

Nicking endonucleases are a new type of enzymes. Like restriction endonucleases, they recognize short specific DNA sequence and cleave DNA at a fixed position relatively to the recognition sequence. However, unlike restriction endonucleases, nicking endonucleases cleave only one predetermined DNA strand. Until recently, nicking endonucleases were suggested to be naturally mutated restriction endonucleases which had lost their ability to dimerize and as a result the ability to cleave the second strand. We have shown that nicking endonucleases are one of the subunits of heterodimeric restriction endonucleases. Mechanisms used by various restriction endonucleases for double-stranded cleavage, designing of artificial nicking endonucleases on the basis of restriction endonucleases, and application of nicking endonucleases in molecular biology are reviewed.     

Evolutionary relationship between different subgroups of restriction endonucleases

The type II restriction endonuclease SsoII shows sequence similarity with 10 other restriction endonucleases, among them the type IIE restriction endonuclease EcoRII, which requires binding to an effector site for efficient DNA cleavage, and the type IIF restriction endonuclease NgoMIV, which is active as a homotetramer and cleaves DNA with two recognition sites in a concerted reaction. We show here that SsoII is an orthodox type II enzyme, which is active as a homodimer and does not require activation by binding to an effector site. Nevertheless, it shares with EcoRII and NgoMIV a very similar DNA-binding site and catalytic center as shown here by a mutational analysis, indicative of an evolutionary relationship between these three enzymes. We suggest that a similar relationship exists between other orthodox type II, type IIE, and type IIF restriction endonucleases. This may explain why similarities may be more pronounced between members of different subtypes of restriction enzymes than among the members of a given subtype.     

Restriction endonucleases and their applications

Restriction endonucleases are deoxyribonucleases which cleave double-stranded DNA into fragments. With only one exception, all restriction endonucleases recognize short, non-methylated DNA sequences. Restriction endonucleases can be divided into two groups based on the position of the cleavage site relative to the recognition sequence. Class I restriction endonucleases cleave double-stranded DNA at positions outside the recognition sequence and generate fragments of random size. The cleavage sites of Class II restriction endonucleases are located, in most cases, within the recognition sequence. Most of the Class II restriction endonucleases recognize 4, 5, or 6 base pair palindromes and generate fragments with either flush ends or staggered ends. DNA fragments with staggered ends contain 3, 4, or 5 nucleotide single-stranded tails called ‘sticky ends’. DNA fragments produced by Class II restriction endonuclease cleavage can be separated on gels according to their molecular weight. The fragments can be isolated from the gel and used for sequence analysis to elucidate genetic information stored in DNA. Further, an isolated fragment can be inserted into a small extrachromosomal DNA, e.g. plasmid, phage or viral DNA, and its replication and expression can be studied in clones of prokaryotic or eukaryotic cells. Restriction endonucleases and cloning technology are powerful modern tools for attacking genetic problems in medicine, agriculture and industrial microbiology.     

Best minimally modified antisense oligonucleotides according to cell nuclease activity.

Minimally modified oligonucleotides belong to the second-generation antisense class. They are phosphodiester oligonucleotides with a minimum of phosphorothioate linkages in order to be protected against serum and cellular exonucleases and endonucleases. They activate RNase H, have weak interactions with proteins, and have thus a better antisense efficiency. Two of them have been designed from an all-phosphorothioate antisense oligonucleotide directed against mdrl-expressing cells. They are protected against serum and cellular enzymatic degradation by the self-forming hairpin d(GCGAAGC) at their 3'-end and by judiciously located phosphorothioate residues, depending on the cellular composition in exonucleases or endonucleases. Besides their already demonstrated ability to cleave pyrimidine sites, endonucleases show some specificity for CpG sites. Their activity is hindered if specific sites are involved in secondary structure as hairpin.     

Cleavage and protection of locked nucleic acid-modified DNA by restriction endonucleases

Locked nucleic acid (LNA) is one of the most prominent nucleic acid analogues reported so far. We herein for the first time report cleavage by restriction endonuclease of LNA-modified DNA oligonucleotides. The experiments revealed that RsaI is an efficient enzyme capable of recognizing and cleaving LNA-modified DNA oligonucleotides. Furthermore, introduction of LNA nucleotides protects against cleavage by the restriction endonucleases PvuII, PstI, SacI, KpnI and EcoRI.     

Construction of a multi RE module: exploitation of mechanochemistry of restriction endonucleases.

We describe the construction of a multi-immobilized restriction endonuclease module (Multi RE module). We demonstrate that the applied mechanical stress enables modulation of enzyme activity and modulation of recognition site selectivity (in oligonucleotides of approximately 200 bp) of immobilized restriction endonucleases. The central module which is consisted of different strips of immobilized restriction endonucleases allows limited digestion of a large DNA sample in a controlled manner as a function of applied mechanical stress on strips. The stress-activity relationship and the effect of repeated cycles of stress and relaxation on the immobilized strips are presented here. © 1999 John Wiley & Sons, Inc.     

Type IIE and IIF restriction endonucleases interacting with two recognition sites in DNA

Recently, it was revealed that restriction endonucleases widely used in genetic engineering and molecular biology are diverse not only in DNA sequence specificities but also in mechanisms of their interaction with DNA. In the review type IIE and IIF restriction endonucleases which require the simultaneous interaction with two copies of their recognition sequence for effective hydrolysis of DNA are considered. Crystal structures of these enzymes and their complexes with DNA as well as stepwise interaction with DNA, mechanisms of catalysis and enzyme-mediated DNA looping are discussed. A novel type of DNA-protein recognition was found for type IIE endonucleases when two copies of the same DNA sequence specifically interact with two different amino acid sequences and two structural motifs located in one polypeptide chain.     

Activity of single-stranded DNA endonucleases in mung bean is associated with cell division

A single-strand-specific endonuclease from mung bean sprouts is widely usedin molecular biology. However, the biological role of this enzyme is unknown. We studied the spatial and temporal activity of single-stranded DNA endonucleases in mung bean seedling by following enzyme activity that linearizes supercoiled plasmid DNA, a characteristic of this type of enzyme. The formation of a linear molecule from supercoiled DNA was found to occur in two distinguishable steps. The first, which involves introducing a nick into the supercoiled DNA and relaxing it, is very rapid and complete within a few seconds. The second step of cleaving the opposite strand to generate a unit-length linear duplex DNA is a relatively slow process. Analysis of the DNA cleavage sites showed the nuclease preferentially cuts supercoiled DNA at an AT-rich region. Varying levels of nuclease activity could be detected in different tissues of the mung bean seedling. The highest activity was in the root tip and was correlated with histone H1 kinase activity. This implies a link between nuclease activity and cell division. Induction of cell division in mung bean hypocotyls with auxin promoted formation of root primordia and considerably increased the activity of single-stranded DNA endonucleases. The nuclease activity and histone H1 kinase activity were reduced in mung bean cuttings treated with hydroxyurea, but not in cuttings treated with oryzalin. The potential function of single-stranded DNA endonucleases is discussed.     

Relaxed circular SV40 DNA as cleavage intermediate of two restriction endonucleases.

We have determined the mode of cleavage of superhelical SV40 DNA (Form I) by restriction endonucleases EcoRI and HpaII at 37 degrees C. By analysis with agarose gel electrophoresis and direct examination with dark field electron microscopy, we found that a large amount of the single-nicked circular DNA (Form II) was produced before the linear SV40 DNA (Form III) appeared. Thus, both restriction enzymes cleave only one strand of the superhelical DNA first. The second cleavage on the complementary strand occurred after a lag period. The first order rate constant for the second cleavage by EcoRI endonuclease was determined and a kinetic reaction scheme for both enzymes is proposed.     

Current approaches to the search for new restriction endonucleases

The main methodological approaches to the search of new restriction endonucleases are reviewed. These methods include obtaining acellular extracts by ultrasonic desintegration of microbial cells, osmotic shock effects, the effects of organic solvents, mechanical disruption of bacterial cells, biphase division after Albertson and others. The resolving power of any method discussed depends mainly on the level of restriction endonuclease activity, the presence of nonspecific endonucleases in the biomass, the presence of exonucleases and the taxonomy of the used microorganisms.     

Immobilized oligonucleotides as affinity sorbents for restriction endonucleases

Affinity chromatography of IIS type restriction endonucleases is proposed. It is shown that endonucleases HgaI, FokI, and SfaNI have affinity to the matrix with immobilized oligonucleotides which contain the endonuclease's recognition sites resistant to the hydrolysis.     

Analysis of brain chromatin subunit composition using different endonucleases]

A comparison of the processes of chromatin digestion in brain and liver nuclei by Ca, Mg-dependent and staphylococcal endonucleases demonstrates a similarity of the subunit composition of chromatin from both tissues and reveals the same type of linked DNA regions. However, a formation of low molecular weight DNP fragments during hydrolysis and the DNA spectra of soluble and insoluble DNP fragments suggest that brain chromatin contains these fragments alongside with the regions, which are specific for this particular tissue, predominate in it and are resistant to staphylococcal and, particularly, to Ca, Mg-dependent endonucleases. This is paralleled with a non-histone protein enrichment of different brain chromatin fractions and an expansion of the electrophoretic monomer band towards the fragment with a greater molecular weight. It may be assumed that brain nucleosomes are characterized by a higher size heterogeneity of linked DNA, part of which are mostly covered by non-histone proteins, and/or are characterized by a greater set variety.     

Use of site-specific DNA endonucleases in genome-wide studies of human DNA

During the last decades, site-specific DNA endonucleases have served as a key instrument to study primary structure of DNA and genetic engineering. Here, we describe examples of these enzyme uses in genome-wide analysis of human DNA including restriction endonucleases involvement during sample preparation for sequencing using NGS devices, as well as visualization of cleavage of DNA repeats by endonucleases. The first studies on application of DNA endonucleases in the rapidly developing area of epigenetic analysis of genomes, which is facilitated by the recent discovery of a new class of enzymes, 5-methylcytosinedependent site-specific DNA endonucleases, are of special interest.     

Substrate specificity and properties of methyl-directed site-specific DNA endonucleases

Methyl-directed site-specific DNA endonucleases (MD endonucleases) are a small group of enzymes that specifically cleave only methylated DNA. The group includes N6-methyladenine- and 5-methylcytosine-directed enzymes. Although poorly understood, MD endonucleases are of interest for both basic research and application in biotechnology and epigenomics. The review for the first time summarizes the properties of MD endonucleases and considers their role in the bacterial cell and their possible uses in biotechnology and epigenomics.     

Functional characterization of isoschizomeric His-Cys box homing endonucleases from Naegleria.

Several species within the amoeboflagellate genus Naegleria harbor an optional ORF containing group I introns in their nuclear small subunit ribosomal DNA. The different ORFs encode homing endonucleases with 65 to 95% identity at the amino-acid level. I-NjaI, I-NanI and I-NitI, from introns in Naegleria jamiesoni, N. andersoni and N. italica, respectively, were analyzed in more detail and found to be isoschizomeric endonucleases that recognize and cleave an approximal 19-bp partially symmetrical sequence, creating a pentanucleotide 3' overhang upon cleavage. The optimal conditions for cleavage activity with respect to temperature, pH, salt and divalent metal ions were investigated. The optimal cleavage temperature for all three endonucleases was found to be 37 degrees C and the activity was dependent on the concentration of NaCl with an optimum at 200 mM. Divalent metal ions, primarily Mg2+, are essential for Naegleria endonuclease activity. Whereas both Mn2+ and Ca2+ could substitute for Mg2+, but with a slower cleavage rate, Zn2+ was unable to support cleavage. Interestingly, the pH dependence of DNA cleavage was found to vary significantly between the I-NitI and I-NjaI/I-NanI endonucleases with optimal pH values at 6.5 and 9, respectively. Site-directed mutagenesis of conserved I-NjaI residues strongly supports the hypothesis that Naegleria homing endonucleases share a similar zinc-binding structure and active site with the His-Cys box homing endonuclease I-PpoI.     

An activity gel assay for the detection of DNA helicases and nucleases from cell-free extracts.

An activity gel assay was developed for the detection of DNA helicases in crude extracts. The assay was based on the ability of DNA helicases to unwind radioactive fragments from single-stranded M13 circles that were immobilized in an SDS polyacrylamide gel. The displaced radioactive strands were detected by blotting them to a filter and visualizing the resulting bands by autoradiography. Experiments with purified proteins demonstrated that DNA helicases, endonucleases and exonucleases could produce activity bands. A one-dimensional gel assay was sufficiently sensitive to allow detection of DNA helicase I, DNA helicase II, DNA helicase IV, the RecQ helicase as well as 3 unidentified putative DNA helicases in crude extracts of Escherichia coli. Exonuclease and endonuclease activities from crude extracts could be distinguished from DNA helicase activities by their ATP-independence and from each other by their band morphologies.