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.     

Genetic structure of the IncN plasmid N3

N3, a plasmid of incompatibility group N (IncN) was mapped by cleavage with restriction endonucleases. The restriction fragments were cloned into vector plasmids. All of the genes unique to IncN plasmids such as specific replication machinery, a restriction-modification system, and repair functions were located on a large portion which had no cleavage sites for many of the site-specific six base-identifying restriction endonucleases tested.     

Properties of the plasmid pFT15/10-1 isolated from the vaccine strain of Francisella tularensis

Plasmid, designated pFT15/10-1, was isolated from Francisella tularensis vaccine strain 15/10. The plasmid is presented by the homogeneous 5.02 +/- 0.054 Md monomeric circular DNA molecules in electron microscopic preparations. Plasmid size is 7-7.3 kb as defined by electrophoresis in agarose gel. The restriction analysis has revealed that plasmid pFT15/10-1 possesses a single specific cleavage site for restriction endonuclease EcoRI, two sites for restriction endonucleases BamHI, BgIII, HincII, HindIII, PstI, three sites for BglI and SalI, some for AluI, TagI, MvaI, CfrI. Plasmid is not digested by restriction endonucleases SmaI, XmaI, KpnI, MluI. Restriction map of the plasmid was constructed for most frequently used restriction endonucleases.     

Biospecific fractionation matrices for sequence specific endonucleases

Fractionation of several type II specific restriction endonucleases was achieved by separation on two novel biospecific matrices. The matrices are pyran, a copolymer of divinyl ether of maleic anhydride, and Cibacron Blue F3GA, a blue dye commonly used for the calibration of molecular sieves. Both compounds are insolubilized by coupling to sepharose through a cyanogen bromide linkage and in their soluble form inhibit the restriction endonucleases which we have tested. These affinity matrices can be used to obtain restriction endonucleases from crude extracts after removal of nucleic acids. They have also proven to have a high capacity when used as subsequent steps in enzyme purification. Their additional advantage is the rapid development time and reusability of columns packed with the two matrices.     

Chromosome banding in Amphibia

Fixed metaphase chromosomes of several species of Amphibia were treated with various restriction endonucleases and subsequently stained with Giemsa. Metaphases of man and chicken were examined in parallel under the same experimental conditions for comparison. The restriction enzymes always induce subsets of the C-banding patterns present in the amphibian karyotypes. The heterochromatic regions can be either resistant or sensitive to the restriction enzyme. The modified C-banding patterns revealed by different restriction endonucleases in the karyotype of the same species can be either extremely dissimilar or almost completely congruent. Correspondingly, the action of the same restriction enzyme on the karyotypes of different species may vary greatly. There is only rarely a correlation between the type of C-banding patterns produced by different restriction endonucleases and their specific base pair recognition sequences. In contrast to mammalian and avian chromosomes, restriction enzymes induce no multiple G-banding patterns in amphibian chromosomes. This is attributed to the difference in organization of the DNA in the genomes of poikilothermic vertebrates. The possible mechanisms of restriction endonuclease banding and the various uses of this technique for amphibian chromosomes are discussed.     

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.     

Use of the antibiotics actinomycin D and distamycin A to limit the action of restriction endonucleases and to map DNA

It is shown that distamycin A and actinomycin D protect the recognition sites of certain restriction endonucleases from the attack by these nucleases due to specific interaction of these antibiotics with double-stranded DNA. Distamycin A protects A-T containing sites and actinomycin G-C rich sites. Among Hind II recognition sites which have alternative structure (GTPyPuAC) distamycin A protects only Hpa I similar sites (GTTAAC). It is shown with several restriction endonucleases that antibiotic action depends on the nucleotide sequences in the recognition sites and in their closest environment. Proper concentrations of antibiotic give rise to larger fragments. Use of both distamycin A and actinomycin D allows to obtain a set of overlapping fragments. The data obtained with various DNAs and restriction endonucleases allow to conclude that these antibiotics may be useful for DNA mapping and for preparation of large functional fragments of DNA.     

Efficient solubilization and partial purification of sea urchin histone genes as chromatin

Soluble chromatin fragments are rapidly and efficiently produced when nuclei are digested with restriction endonucleases in buffers containing very low concentrations of magnesium. Under these conditions, the sequence specificity of the restriction endonucleases is maintained, resulting in release of specific genes as fragments with discrete molecular weights that can be fractionated by size on glycerol gradients. Gradient fractions can be chosen to be significantly enriched in specific genes and their associated proteins. For instance, we can achieve a 16-fold enrichment of the chromatin containing the early histone genes of sea urchin. The enrichments produced by these methods are useful as a first step in techniques to purify specific genes as chromatin. Glycerol gradient analyses can also be used to test whether putative gene-specific proteins are actually bound to the same sequences in vivo.     

Organization of chicken DNA sequences homologous to the transforming gene of avian myeloblastosis virus. II. Isolation and characterization of lambda proto-amv DNA recombinant clones from a library of leukemic chicken DNA.

Several lambda proto-amv recombinants isolated from a lambda Charon 4A library of leukemic chicken DNA were analyzed by using various restriction endonucleases and hybridization with specific probes representing different regions of the transforming gene of avian myeloblastosis virus. The position of 30 sites for 11 different restriction endonucleases was established in the proto-amv region of chicken DNA. Identical restriction endonuclease maps were obtained for the normal and leukemic DNAs in the proto-amv domain, which covers 8 to 9 kilobases of DNA. The cellular genetic elements homologous to the cellular sequence (amv) inserted into the avian myeloblastosis virus genome are contained within six major proto-amv segments which are interrupted by at least five large DNA regions lacking homology with amv.     

Diversity of type II restriction endonucleases that require two DNA recognition sites

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.     

The effect of sequence specific DNA methylation on restriction endonuclease cleavage.

Sequence specific DNA methylation sometimes results in the protection of some or all of a restriction endonucleases' cleavage sites. This is usually, but not always, the result of methylation of one or both strands of DNA at the site characteristic of the corresponding "cognate" modification methylase. The known effects of sequence specific methylation on restriction endonucleases are compiled.     

WebFARM: web server for finite automated restriction mapping

Restriction endonucleases are indispensable tools in molecular biology and biotechnology. Type II restriction endonucleases are part of restriction modification systems. DNA fragment extraction and restriction mapping are the basis for several biotechnological activities. WebFARM is a server application for identifying restriction endonuclease recognition sites and to give information regarding restriction mapping for given nucleotide sequences. WebFARM analyses given nucleotide sequence and identify restriction site for selected restriction endonucleases. It will also provide frequency of restriction for each restriction endonuclease. AVAILABILITY: http://webfarm.bioinfoindia.org/     

Restriction endonucleases from Bifidobacteria

The sitespecific restriction endonucleases were found in four strains among the twelve strains of anaerobic bacteria of generum Bifidobacterium. Two of the restriction endonucleases studied, BadI from B. adolescentis LVA1 and BbfI from B. bifidum LVA3, are isoshizomers of XhoI and recognize the nucleotide sequence CTCGAG. The restriction endonucleases Bbf7411I from B. bifidum 7411 and Bla7920I from B. lactentis 7920 recognize and hydrolize the nucleotide sequence TCCGGA having the specifity analogous to the one of restriction endonuclease CauB3I. Like CauB3I, these restriction endonucleases are unable to hydrolyize DNA if the adenine residues in the recognition site are methylated.     

Detection of cryptic bands by AluI in eukaryotic chromosomes

Selective digestion of fixed chromatin with the restriction endonuclease AluI (which cuts the sequence AG CT) uncovers a specific and repeatable pattern of bands within the euchromatin of two species of grasshoppers and of the L929 mouse cell line, which are not detectable by means of other banding techniques such as C-bands, specific fluorochromes, or other restriction endonucleases. It is tentatively suggested that this chromatin represents a special class of repetitive DNA embedded in the euchromatin, not containing the AluI restriction site to the same extent as in euchromatin and not associated with C-banded heterochromatic material.     

Sequences hybridizing to mRNA, oligo(dT) and dsRNA from pre-mRNA are contiguous in the cloned mouse DNA fragments.

Fragments from the DNA of mouse embryos produced by restriction endonucleases HindIII were cloned in pBR322 plasmid and examined for the ability to hybridize in situ with [32P] labeled cDNA synthesized from the polysomal poly(A)+mRNA template. Several of the selected clones were examined for the presence of specific sequences inside the cloned mouse DNA fragments by the blotting procedure of southern [1]. The data obtained indicate that the majority of the cloned mouse DNA fragments contained sequences hybridizing with cDNA, oligo(dT) and double-stranded regions from pre-mRNA. The results of hybridization experiments and double digestion with HindIII+HaeIII endonucleases provide evidence that these sequences could be contiguous in the given restriction DNA fragments.     

REF Select: expert system software for selecting restriction endonucleases for restriction endonuclease fingerprinting

REF Select, expert system software, has been developed to assist in the selection of optimal restriction endonucleases for restriction endonuclease fingerprinting (REF), a method for rapid and sensitive mutation screening of long DNA segments (1-2 kb). The REF method typically involves six separate digestions with up to two restriction endnonucleases used in each digestion. If done manually, performing a comprehensive review of the large number of possible sets of restriction endonucleases that could be used (over 10(19) in the example presented here) and making an optimal choice is not feasible. Furthermore, the typical nonoptimal manual selection takes approximately 8 h by someone experienced with REF. REF Select enables a comprehensive review of the possible sets and a consistent, objective and fast selection of an optimal set by using a two-step strategy: the selection of sets that meet specific constraints, which is followed by a ranking of those sets by an optimality score. Based on our experience with REF, we chose default selection and ranking parameters to help the user get started quickly. These parameters form a knowledge base that can be customized and then saved by the user. In conclusion, REF Select facilitates the general application of REF by serving as an expert system for the selection of optimal restriction endonucleases. We demonstrated REF Select using an example segment from the human p53 gene.     

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.     

GATC-specific restriction and modification systems in treponemes

AIMS: To investigate the presence of GATC-specific modification and restriction activities in rumen isolates of Treponema sp. METHODS: The presence of N6-methyladenine within GATC (Dam) sequences was analysed using isoschizomeric restriction endonucleases having different sensitivities to the methylation of the target sequence. A fast screening method was used for testing of site-specific endonuclease activities directly in crude cell extracts. Three out of six rumen isolates of Treponema sp. showed restriction activities. Restriction endonucleases were further purified by Heparin-Sepharose chromatography. Using PCR and specific primers, no sequence homologous to the T. pallidum dam gene was found. CONCLUSIONS: Three rumen treponemal strains were documented to possess MboI isoschizomeric restriction-modification systems. SIGNIFICANCE: This is the first report on restriction activity in rumen treponemes.