Purification and properties of the P15 specific restriction endonuclease from Escherichia coli.

The specific restriction endonuclease of the Escherichia coli plasmid, P15, has been purified to apparent homogeneity by a procedure that includes DNA-cellulose chromatography as well as a new endonuclease assay. Sedimentation on glycerol gradients showed two peaks of activity with values of 11.3 S and 15.7 S. The highly purified enzyme requires ATP and Mg2+ for activity and is stimulated by S-adenosylmethionine. A methylase activity is observed in the course of the endonucleolytic reaction which protects some of the DNA sites from cleavage.     

Purification, properties and specificity of the restriction endonuclease from Bacillus stearothermophilus.

The restriction endonuclease BstI was purified from 70kg of Bacillus stearothermophilus. The final product is at least 97% pure as judged by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis; this major protein species co-migrates with the enzyme activity on native polyacrylamide-gel electrophoresis and isoelectric focusing. Pure restriction endonuclease BstI has a subunit mol.wt. of 26,000 and is probably a loosely associated dimer. The enzyme shows maximum activity at pH values between 7 and 9.5, and in the presence of 0.5-2mM-Mg2+. NaCl inhibits the restriction enzyme activity. Restriction endonuclease BstI cleaves DNA in a position identical with that cleaved by endonuclease BamHI (for Bacillus amyloliquefaciens), i.e.: (formula: see text). In the presence of high concentrations of enzyme, DNA cleavage occurs at secondary sites. This side-specificity is enhanced by the addition of glycerol. Preliminary studies indicate that these sites are of the type: (formula: see text).     

The EcoDXX1 restriction and modification system of Escherichia coli ET7. Purification, subunit structure and properties of the restriction endonuclease

The Escherichia coli plasmid pDXX1 codes for a new restriction-modification system. The specific restriction endonuclease coded by this system has been purified by a procedure that includes phosphocellulose and heparin-agarose chromatography. Sedimentation on glycerol gradients showed one peak of activity with a value of about 12 S. The highly purified enzyme require ATP and Mg2+ for activity as well as S-adenosylmethionine, although some S-adenosylmethionine molecules are probably bound to the enzyme. The enzyme does not cleave lambda DNA at well-defined sites and has a strong non-modified DNA-dependent ATPase activity. The enzyme has also methylase activity acting against non-modified DNA.     

The bacteriophage P1 restriction endonuclease

The bacteriophage P1 restriction endonuclease has been purified from Escherichia coli lysogenic for P1. This restriction endonuclease P has a sedimentation coefficient of 9.3 S. Unlike the E. coli K restriction endonuclease, endonuclease P does not require S-adenosylmethionine for breakage of DNA. S-adenosylmethionine does, however, stimulate the rate of double-strand breakage of DNA by endonuclease P. Hydrolysis of ATP by endonuclease P could not be detected under conditions in which the K restriction endonuclease massively degrades ATP.The enzyme makes a limited number of double-strand breaks in unmodified or heterologously modified λ DNA. In the presence of S-adenosylmethionine, it does not cut every DNA molecule to the same extent. Incubation of λ DNA with excess amounts of enzyme in the presence of S-adenosylmethionine results in less breakage of the DNA than with smaller amounts of enzyme. This effect is not seen in the absence of S-adenosylmethionine. The maximum amount of cutting in the absence of S-adenosylmethionine appears to be greater than the maximum amount of cutting in its presence. This is most likely due to the modification methylase activity of P1 restriction endonuclease.     

The EcoA restriction and modification system of Escherichia coli 15T-: enzyme structure and DNA recognition sequence

The EcoA restriction enzyme from Escherichia coli 15T- has been isolated. It proves to be an unusual enzyme, clearly related functionally to the classical type I restriction enzymes. The basic enzyme is a two subunit modification methylase. Another protein species can be purified which by itself has no enzymatic activities but which converts the modification methylase to an ATP and S-adenosylmethionine-dependent restriction endonuclease. The DNA recognition sequence of EcoA has an overall structure that is very similar to previously determined type I sequences. It is: 5'-GAGNNNNNNNGTCA-3' 3'-CTCNNNNNNNCAGT-5' where N can be any nucleotide. Modification methylates the adenosyl residue in the specific trinucleotide and the adenosyl residue in the lower strand of the specific tetranucleotide.     

Restriction and modification in B. subtilis. Purification and general properties of a restriction endonuclease from strain R.

Summary All Bacillus subtilis R-type strains showing the phenomena of restriction and modification contain an endonuclease that inactivates in vitro the biological activity of a variety of DNAs lacking R-specific modification, such as transfecting SPP1, SPO2 and 105 DNA, and transforming B. subtilis 168-type DNA. The corresponding DNAs carrying R-specific modification are resistant to the enzyme. The enzyme has been purified approximately 400-fold and is essentially free from contaminating double strand-directed unspecific exo-or endonuclease activity. Only Mg²⁺ is required as cofactor. The substrate DNAs are cleaved at specific sites. The double-stranded fragments produced from SPP1 DNA (molecular weight 2.5×10⁷) have an average molecular weight of about 3×10⁵.     

限制性内切酶EcoR的高效表达与纯化

EcoR Ⅱ was the first restriction endonuclease ever found requiring the cooperative interaction with the least two DNA sites for digestion activity.To study the specific activity, Eco R Ⅱ was purified from hyperexpression engineering bacteria in which the specific expression products increased to about 20% of total cellular protein.By using chromatography on DEAE cellulose column,phosphocellulose column and FPLC of Resource Q,the enzyme was purified from soluble protein fraction.The inclusion bodies were solved and renatured,and the enzyme was purified from this part of protein with higher specific activity by using FPLC of Resource Q.Detection showed that the enzyme was purified to homogeneity and was free of detectable contamination by other DNase(exo and endo).     

Mapping of New Escherichia coli K and 15 Restriction Sites on Specific Fragments of Bacteriophage φX174 DNA

We have isolated several new phiX174 mutants which contain sites sensitive to restriction by Escherichia coli. One contains an E. coli 15 restriction site and three are double mutants containing an E. coli K site as well as the E. coli 15 site. The replicative form (RF) DNA of one of the mutants containing a K site has been shown to be restricted in spheroplasts of a K-12 strain. The infectivity of this RF, but not wild-type RF, has also been shown to be inactivated by an E. coli K extract and by purified K restriction enzyme in vitro. The product of the RF treated with purified K restriction enzyme in vitro is a full length linear molecule. The mutant sites have also been localized to specific regions of the phiX174 genome by a fragment mapping technique, making use of specific fragments of phiX174 RF DNA obtained by digestion with a specific endonuclease.     

Antirestriction activity of the IncI1 transmissive plasmid R64 ArdA protein

The transmissive plasmid IncI1 R64 contains the ardA gene encoding the ArdA antirestriction protein. The R64 ardA gene locating in the leading region of plasmid R64 has been cloned and their sequence has been determined. Antirestriction proteins belonging to the Ard family are specific inhibitors of type I restriction-modification enzymes. The IncI1 ColIb-P9 and R64 are closely related plasmids, and the latter specifies an ArdA homologue that is predicted to be 97.6% (162 residues from 166) identical at the amino acid sequence level with the ColIb = P9 equivalent. However, the R64 ArdA selectively inhibits the restriction activity of EcoKi enzyme leaving significant levels of modification activity under conditions in which restriction was almost completely prevented. The ColIb-P9 ArdA inhibits restriction endonuclease and methyltransferase activities simultaneously. It is hypothesized that the ArdA protein forms two complexes with the type I restriction-modification enzyme (R2M2S): (1) with a specific region in the S subunit involved in contact with the sK site in DNA; and (2) with nonspecific region in the R subunit involved in DNA translocation and degradation by restriction endonuclease. The association of the ColIb-P9 ArdA with the specific region inhibits restriction endonuclease and methyltransferase activities simultaneously, whereas the association of the R64 ArdA with a nonspecific region inhibits only restriction endonuclease activity of the R2M2S enzyme.     

Recognition sequence of the dam methylase of Escherichia coli K12 and mode of cleavage of Dpn I endonuclease.

The recognition sequence for the dam methylase of Escherichia coli K12 has been determined directly by use of in vivo methylated ColE1 DNA or DNA methylated in vitro with purified enzyme. The methylase recognizes the symmetric tetranucleotide d(pG-A-T-C) and introduces two methyl groups per site in duplex DNA with the product of methylation being 6-methylaminopurine. This work has also demonstrated that Dpn I restriction endonuclease cleaves on the 3' side of the modified adenine within the methylated sequence to yield DNA fragments possessing fully base-paired termini. All sequences in ColE1 DNA methylated by the dam enzyme are subject to double strand cleavage by Dpn I endonuclease. Therefore, this restriction enzyme can be employed for mapping the location of sequences possessing the dam modification.     

Characterization of human enzymes specific for damaged DNA: resolution of endonuclease for irradiated DNA from an apparent N-glycosidase active on alkylated DNA.

An endonuclease partially purified from human lymphoblasts, and active against ultraviolet-irradiated DNA, was found to act additionally on DNA damaged by either x-radiation or methylmethanesulfonate. To determine if these activities were truly endonucleolytic, the reaction products were analyzed under conditions that prevented conversion of apurinic or apyrimidinic sites to single-strand breaks. With either ultraviolet- or x-irradiated DNA, strand breakage remained maximal, hence confirming the endonucleolytic character of the enzyme. By contrast, with DNA alkylated with methylmethanesulfonate, strand breakage was sharply reduced. Additional experiments indicated that the activity for alkylated DNA induces strand breaks only in concert with a purified endonuclease specific for apurinic sites, suggesting that it is an N-glycosidase that depurinates alkylated bases. This enzyme was separated from the endonuclease specific for irradiated DNA, by chromatography on DNA-agarose.     

The DNA translocation and ATPase activities of restriction-deficient mutants of Eco KI.

Eco KI, a type I restriction enzyme, specifies DNA methyltransferase, ATPase, endonuclease and DNA translocation activities. One subunit (HsdR) of the oligomeric enzyme contributes to those activities essential for restriction. These activities involve ATP-dependent DNA translocation and DNA cleavage. Mutations that change amino acids within recognisable motifs in HsdR impair restriction. We have used an in vivo assay to monitor the effect of these mutations on DNA translocation. The assay follows the Eco KI-dependent entry of phage T7 DNA from the phage particle into the host cell. Earlier experiments have shown that mutations within the seven motifs characteristic of the DEAD-box family of proteins that comprise known or putative helicases severely impair the ATPase activity of purified enzymes. We find that the mutations abolish DNA translocation in vivo. This provides evidence that these motifs are relevant to the coupling of ATP hydrolysis to DNA translocation. Mutations that identify an endonuclease motif similar to that found at the active site of type II restriction enzymes and other nucleases have been shown to abolish DNA nicking activity. When conservative changes are made at these residues, the enzymes lack nuclease activity but retain the ability to hydrolyse ATP and to translocate DNA at wild-type levels. It has been speculated that nicking may be necessary to resolve the topological problems associated with DNA translocation by type I restriction and modification systems. Our experiments show that loss of the nicking activity associated with the endonuclease motif of Eco KI has no effect on ATPase activity in vitro or DNA translocation of the T7 genome in vivo.     

Host specificity of DNA in Haemophilus influenzae: DNA restriction enzyme from H. influenzae Rf232.

A restriction endonuclease has been partially purified from Haemophilus influenzae Rf232 containing the genetically determined system of restriction and modification of DNA. The enzyme requires ATP for the degradation of transfecting phage DNA.     

The HsdR subunit of R.EcoR124II: cloning and over-expression of the gene and unexpected properties of the subunit.

Type I restriction endonucleases are composed of three subunits, HsdR, HsdM and HsdS. The HsdR subunit is absolutely required for restriction activity; while an independent methylase is composed of HsdM and HsdS subunits. DNA cleavage is associated with a powerful ATPase activity during which DNA is translocated by the enzyme prior to cleavage. The presence of a Walker type I ATP-binding site within the HsdR subunit suggested that the subunit may be capable of independent enzymatic activity. Therefore, we have, for the first time, cloned and over-expressed the hsdRgene of the type IC restriction endonuclease EcoR124II. The purified HsdR subunit was found to be a soluble monomeric protein capable of DNA- and Mg2+-dependent ATP hydrolysis. The subunit was found to have a weak nuclease activity both in vivo and in vitro, and to bind plasmid DNA; although was not capable of binding a DNA oligoduplex. We were also able to reconstitute the fully active endonuclease from purified M. EcoR124I and HsdR. This is the first clear demonstration that the HsdR subunit of a type I restriction endonuclease is capable of independent enzyme activity, and suggests a mechanism for the evolution of the endonuclease from the independent methylase.     

Mechanism of action of Micrococcus luteus gamma-endonuclease

Micrococcus luteus extracts contain gamma-endonuclease, a Mg2+-independent endonuclease that cleaves gamma-irradiated DNA. This enzyme has been purified approximately 1000-fold, and the purified enzyme was used to study its substrate specificity and mechanism of action. gamma-Endonuclease cleaves DNA containing either thymine glycols, urea residues, or apurinic sites but not undamaged DNA or DNA containing reduced apurinic sites. The enzyme has both N-glycosylase activity that releases thymine glycol residues from OsO4-treated DNA and an associated apurinic endonuclease activity. The location and nature of the cleavage site produced has been determined with DNA sequencing techniques. gamma-Endonuclease cleaves DNA containing thymine glycols or apurinic sites immediately 3' to the damaged or missing base. Cleavage results in a 5'-phosphate terminus and a 3' baseless sugar residue. Cleavage sites can be converted to primers for DNA polymerase I by subsequent treatment with Escherichia coli exonuclease III. The mechanism of action of gamma-endonuclease and its substrate specificity are very similar to those identified for E. coli endonuclease III.     

A new restriction endonuclease from Acetobacter pasteurianus.

A restriction endonuclease, ApaI, has been partially purified from Acetobacter pasteurianus. This enzyme cleaves bacteriophage lambda DNA and Simian virus 40 DNA at one site, adenovirus-2 DNA at more than nine sites, but it does not cleave phi X174 DNA nor plasmid pBR322 DNA. This enzyme recognizes the sequence (formula; see text) and cuts at the sites indicated by the arrows.     

Subunit structure of a yeast site-specific endodeoxyribonuclease, endo SceI. A study using monoclonal antibodies

Endo SceI is a eucaryotic site-specific endoDNase of 120 kDa that causes double-stranded scission at well-defined sites, but is distinguishable from procaryotic restriction endonucleases by its mode of sequence recognition and lack of related specific DNA modification. In purified preparations of endoSceI, only two polypeptide species of 75 kDa (75-kDa peptide) and 50 kDa (50-kDa peptide) are detected in apparently equal amounts. We prepared mouse monoclonal IgGs that bound specifically to the 75-kDa peptide (but not the 50-kDa peptide) without inhibiting the endoSceI activity. Immunoprecipitation experiments with these IgGs revealed that the 75-kDa peptide and the 50-kDa peptide are physically associated with each other and with the endonucleolytic activity. Full endoSceI activity was recovered by mixing the purified 75-kDa peptide and the partially purified 50-kDa peptide, each of which exhibited little or no endonuclease activity alone. These observations indicate that endoSceI consists of two non-identical subunits of 75 kDa and 50 kDa, and that both subunits are required for full enzyme activity.     

Characterization of a restriction enzyme from Escherichia coli K carrying a mutation in the modification subunit.

The restriction enzyme from a restriction and modification-deficient strain of Escherichia coli K mutated in the modification gene (hsdM) has been purified using an in vitro complementation assay with a mutant restriction enzyme from a strain lacking only restriction. The restriction enzyme from the hsdM mutant lacks all of the activities that are associated with the wild type enzyme: binding of unmodified DNA to filters, cleavage, or methylation of unmodified DNA and ATP hydrolysis. It is shown that the enzyme from this hsdM mutant cannot bind S-adenosylmethionine, an allosteric effector in the restriction reaction. In the absence of enzyme activation by S-adenosylmethionine, no binding to unmodified DNA takes place. A comparison with other mutant restriction enzymes allows us to outline the biochemical role of the subunits of the E. coli K restriction endonuclease.     

Nmel, a restriction endonuclease from Neisseria meningitidis

A restriction endonuclease, Nmel, present in Neisseria meningitidis was partially purified by passing through a blue 2-cross linked agarose column; no contaminating nucleases remained detectable. This enzyme cleaved phage lambda, adenovirus type 2 and phi x 174 DNA but did not cleave SV40 DNA. It had an absolute requirement for Mg2+ for its activity and was inhibited by high concentrations of NaCl or MgCl2. Nmel activity was completely abolished after 1 h of incubation at 65 degrees C. S-adenosyl-L-methionine and ATP had no effect on its activity suggesting that Nmel is a type II restriction endonuclease enzyme. It is the first report of a restriction enzyme present in N. meningitidis.