Single turnovers of the EcoRI restriction endonuclease.

Limited proteolysis of the arom enzyme complex of Neurospora crassa by trypsin or subtilisin yielded a stable fragment of Mr 68000. This fragment, which was purified by two-dimensional polyacrylamide-gel electrophoresis, was shown by activity staining to contain the shikimate dehydrogenase active site, and by substrate labelling with 3-dehydroquinate and NaB3H4 to contain the 3-dehydroquinase active site. The fragment thus constitutes a bifunctional domain containing the two enzymic activities that are known, from genetic evidence, to be located adjacently at the C-terminal end of the pentafunctional arom polypeptide.     

Cation dependence of restriction endonuclease EcoRI activity

Restriction endonuclease EcoRI cleaves the DNA sequence 5'd(-G-A-A-T-T-C-) under optimum digestion conditions. A variation in pH and ionic strength can result in EcoRI activity when 5'd(-A-A-T-T-) is cut. A divalent cation, usually Mg2+, is required for enzyme activity, though Mn2+ can also be used. Eight different cations with ionic radius/charge ratios similar to Mg2+ were tested and Co2+ and Zn2+ were also found to act as cofactors for EcoRI. A comprehensive study has been made of the effect of NaCl and pH on the EcoRI/EcoRI transition in the presence of the above four cations. Generally, a decrease in NaCl and/or an increase in pH caused a decrease in enzyme specificity. The changeover depended on the cation. They may be placed in order of their ability to increase EcoRI specificity thus: Co2+ greater than Zn2+ greater than Mg2+ greater than Mn2+. The Km of EcoRI for ColE1 DNA, in the presence of Co2+, was found to be 0.4 nM, compared to 3 nM with Mg2+, whereas the turnover was only one double-stranded scission/min with Co2+ compared to eight/min with Mg2+. The implications of all these findings on the enzyme's mechanism are discussed.     

Purification and characterization of the restriction endonuclease RsrI, an isoschizomer of EcoRI

Rhodobacter sphaeroides strain 630 produces restriction enzyme RsrI which is an isoschizomer of EcoRI. We have purified this enzyme and initiated a comparison with the EcoRI endonuclease. The properties of RsrI are consistent with a reaction mechanism similar to that of EcoRI: the position of cleavage within the -GAATTC-site is identical, the MgCl2 optimum for the cleavage is identical, and the pH profile is similar. Methylation of the substrate sequence by the EcoRI methylase protects the site from cleavage by the RsrI endonuclease. RsrI cross-reacts strongly with anti-EcoRI serum indicating three-dimensional structural similarities. We have determined the sequence of 34 N terminal amino acids for RsrI and this sequence possesses significant similarity to the EcoRI N terminus.     

The essential carboxyl group in restriction endonuclease EcoRI

We have carried out studies on type II restriction endonuclease EcoRI, which cleaves the DNA sequence 5'd(-G-A-A-T-T-C-)3', as indicated. The active form of the enzyme consists of two subunits, each 31063 molecular weight. A water-soluble reagent, 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide metho-p-sulphonate, which reacts with carboxyl groups and also with tyrosine and cysteine residues, has been found to inactivate this enzyme. Results are presented which show the following. (1) This specific inactivation is not due to modification of tyrosine or cysteine residues. (2) There is one carboxyl group per subunit which, when modified with carbodiimide, inactivates the enzyme. (3) phi X174 DNA (which does not contain EcoRI sites) partially protects the enzyme from the carbodiimide; protection is unaffected by the additional presence of Mg2+, but significantly greater with Co2+ and phi X174 DNA.     

Restriction endonuclease RsrI from Rhodobacter sphaeroides, an isoschizomer of EcoRI: purification and properties.

We have purified RsrI endonuclease (R.RsrI), an isoschizomer of EcoRI, from Rhodobacter sphaeroides strain 630. The enzyme is homogeneous as judged by polyacrylamide gel electrophoresis and size-exclusion high-performance liquid chromatography. RsrI endonuclease is a dimer over the concentration range of 0.05 to 1.4 mg/ml. The reduced and denatured molecular weight of the enzyme is 30,000 Da. R.RsrI, like R.EcoRI, catalyzes the cleavage of duplex DNA and oligodeoxyribonucleotides between the first two residues of the sequence GAATTC. R.RsrI exhibits a KM of 14 nM and a kcat of 6.5 min-1 when reacting with pBR322 DNA at 25 degrees C. R.RsrI differs from R.EcoRI in its N-terminal amino acid sequence, susceptibility to inhibition by antibodies, sensitivity to N-ethylmaleimide, isoelectric point, state of aggregation at high concentrations, temperature lability, and conditions for optimal reaction. R.RsrI displays a reduction of specificity ("star activity") under conditions that also relax the specificity of R.EcoRI.     

Isolation and characterization of restriction endonuclease EclHKI from an Enterobacter cloacae strain

An Enterobacter cloacae strain, producing restriction enzyme EclHKI, was isolated from a decaying potato. The enzyme is an isoschizomer of Eam1105I, which recognizes and cleaves GACNNN!NNGTC. EclHKI was produced at high activity (4×10⁴ U/g wet cells) and was purified from contaminants which interfere with restriction digestion by passing the cell lysate through DEAE-Sephacel and heparin columns. Activity was optimal at 37°C in a medium salt buffer.H.-Y. Chan, Y.-C. Chan and P.-C. Shaw are with the Department of Biochemistry, The Chinese University of Hong Kong, Hong Kong: K.-M. Kam is with the Institute of Pathology, Sai Ying Pun Jockey Club Clinic, Hong Kong.     

A rapid procedure for purification of EcoRI endonuclease.

A convenient and rapid procedure has been developed to purify restriction endonuclease Eco RI. The method involves sonication of cells at low ionic strength, precipitation of the endonuclease with Polymin P (a polyethyleneimine), elution of the enzyme from the Polymin P precipitate, ammonium sulfate precipitation and chromatography on phosphocellulose. The purified restriction endonuclease is free of exonuclease and other endonucleases.     

Cleavage of single stranded oligonucleotides by EcoRI restriction endonuclease.

The 31mer 5'-TCA ACG CTA GAA TTC GGA TCC ATC GCT TGG T, the complementary 33mer 5'-CCA AGC GAT GGA TCC GAA TTC TAG CGT TGA GAT, the 40mer 5'-GGC CAG GAT GGT GAA GAA TTC GAT CCG GTA CGT AGC TAA G, and the complementary 42mer 5'-TAC TTA GCT ACG TAC CGG ATC GAA TTC TTC ACC ATC CTG GCC were synthesized and their reactivity towards EcoRI was studied. It was found that the 31mer and the 40mer were cleaved at a comparable rate to the 31mer-33mer hybrid and the 40mer-42mer hybrid, respectively. The rate of cleavage of the 33mer and the 42mer was an order of magnitude lower. To rule out possible intermolecular duplex formation, the 33mer was immobilized on cellulose by ligation and labeled with alpha 32P-dCTP using Klenow fragment of E. coli DNA polymerase. EcoRI cleaved this immobilized oligomer into specific fragments.     

Isolation and purification of restriction endonuclease BpeI from Bordetella pertussis

New restriction endonuclease has been isolated from Bordetella pertussis vaccine strain 305 and purified in 1 stage on Sepharose covalently bound with blue dextran. The isolated restrictase has been found capable of breaking down lambda-phag DNA into 7 fragments. According to its specificity, Bpe I is the isoschizomer of Hind III obtained from Haemophilus influenzae strain Rd.     

Construction of an overproducing strain,purification, and biochemical characterization of the 6His-Eco29kI restriction endonuclease

We constructed a strain of Escherichia coli overproducing 6His-tagged Eco29kI by placing the coding sequence under control of a strong bacteriophage T5 promoter. The yield of 6His-Eco29kI restriction endonuclease expression could be increased to about 20% of the total cellular protein, but inclusion bodies formed consisting of insoluble 6His-Eco29kI protein. We developed a fast and effective protocol for purification of the homogeneous enzyme from both soluble and insoluble fractions and established their identity by catalytic activity assay. The isolated enzymes were tested for recognition specificity and optimal reaction conditions as a function of NaCl and KCl concentrations, temperature, and pH compared with the native Eco29kI restriction endonuclease. The 6His-tagged enzyme retained the specificity of the native protein but had an altered optimum of its catalytic reaction.     

EcoRI restriction endonuclease map of the composite R plasmid NR1.

A physical map of the composite R plasmid NR1 has been constructed using specific cleavage of deoxyribonucleic acid (DNA) by the restriction endonuclease EcoR-. Digestion of composite NR1 DNA by EcoRI yields thirteen fragments. The six largest fragments (designated A to F) are from the resistance transfer factor component that harbors the tetracycline resistance genes (RTF-TC). The seven smallest fragments (designated G to M) are from the r-determinants component that harbors the chloramphenicol (CM), streptomycin-spectinomycin (SM/SP), and sulfonamide (SA) resistance genes. The largest fragment of several RTF-TC segregants of NR1 that have deleted the r-determinants component is 0.8 X 10(6) daltons larger than fragment A of composite NR1. Only a part of fragment H of the r-determinants component is amplified in transitioned NR1 DNA in Proteus mirabilis, which consists of multiple, tandem sequences of r-determinants attached to a single copy of the RTF-TC component. Both of these changes can be explained by the locations of the excision sites at the RTF-TC: r-determinants junctions that are involved in the dissociation and reassociation of the RTF-TC and r-determinants components. The thirteen fragments of composite NR1 DNA produced by EcoRI have been ordered using partial digestion techniques. The order of the fragments is: A-D-C-E-F-B-H-I-L-K-G-M-J. The approximate locations of the TC, CM, SM/SP, and SA resistance genes on the EcoRI map were determined by analyzing several deletion mutants of NR1.     

Studies on the cleavage of bacteriophage lambda DNA with EcoRI restriction endonuclease

The five EcoRI² restriction sites in bacteriophage lambda DNA have been mapped at 0.445, 0.543, 0.656, 0.810, and 0.931 fractional lengths from the left end of the DNA molecule. These positions were determined electron-microscopically by single-site cleavage of hydrogen-bonded circular λ DNA molecules and by cleavage of various DNA heteroduplexes between λ DNA and DNA from well defined λ mutants. The DNA lengths of the EcoRI fragments are in agreement with their electrophoretic mobility on agarose gels but are not in agreement with their mobilities on polyacrylamide gels. These positions are different from those previously published by Allet et al. (1973). Partial cleavage of pure λ DNA by addition of small amounts of EcoRI endonuclease does not lead to random cleavage between molecules. Also, the first site cleaved is not randomly distributed among the five sites within a molecule. The site nearest the right end is cleaved first about ten times more frequently than either of the two center sites.     

Polypeptide sequences involved in the cleavage of DNA by the restriction endonuclease EcoRI

We have prepared a variety of fragments of the restriction endonuclease EcoRI by partial or total CNBr or acid cleavage of the protein. These fragments were isolated by preparative polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. They were analyzed in a qualitative manner for phosphodiesterase activity. Antibodies against these fragments were elicited in rats and tested for binding to native EcoRI in an enzyme-linked immunoassay. We conclude from these experiments that the DNA binding site of EcoRI is located in the COOH-terminal half of the molecule, close to and probably comprising amino acid residues 137 to 157. This conclusion is reinforced by the observation that this sequence shows homology to the sequences of the recognition helix of other gene-regulatory proteins.     

The EcoRI restriction endonuclease, covalently closed DNA and ethidium bromide.

The reactions of the EcoRI restriction endonuclease on the covalently closed DNA of plasmid pMB9 were studied in the presence of ethidium bromide. At the concentrations of ethidium bromide tested, which covered the range over which the DNA is changed from negatively to positively supercoiled, the dye caused no alteration to the rate at which this enzyme cleaved the covalently closed DNA to yield the open-circle form, but the rate at which these open circles were cleaved to the linear product could be inhibited. The fluorescence change, caused by ethidium bromide binding with different stoichiometries to covalently closed and open-circle DNA, provided a direct and sensitive signal for monitoring the cleavage of DNA by this enzyme. This method was used for a steady-state kinetic analysis of the reaction catalysed by the EcoRI restriction enzyme. Reaction mechanisms where a complex between DNA and Mg2+ is the substrate for this enzyme were eliminated, and instead DNA and Mg2+ must bind to the enzyme in separate stages. The requisite controls for this fluorimetric assay in both steady-state and transient kinetics studies, and its application to other enzymes that alter the structure of covalently closed DNA, are described.     

Expression and purification of BmrI restriction endonuclease and its N-terminal cleavage domain variants

BmrI (ACTGGG N5/N4) is one of the few metal-independent restriction endonucleases (REases) found in bacteria. The BmrI restriction-modification system was cloned by the methylase selection method, inverse PCR, and PCR. BmrI REase shows significant amino acid sequence identity to BfiI and a putative endonuclease MspBNCORF3798 from the sequenced Mesorhizobium sp. BNC1 genome. The EDTA-resistant BmrI REase was successfully over-expressed in a pre-modified E. coli strain from pET21a or pBAC-expIQ vectors. The recombinant BmrI REase shows strong promiscuous activity (star activity) in NEB buffers 1, 4, and an EDTA buffer. Star activity was diminished in buffers with 100-150 mM NaCl and 10 mM MgCl(2). His-tagged BmrI192, the N-terminal cleavage domain of BmrI, was expressed in E. coli and purified from inclusion bodies. The refolded BmrI192 protein possesses non-specific endonuclease activity. BmrI192 variants with a single Ser to Cys substitution (S76C or S90C) and BmrI200 (T200C) with a single Cys at the C-terminal end were also constructed and purified. BmrI200 digests both single-strand (ss) and double-strand (ds) DNA and the nuclease activity on ss DNA is at least 5-fold higher than that on ds DNA. The Cys-containing BmrI192 and BmrI200 nuclease variants may be useful for coupling to other DNA binding elements such as synthetic zinc fingers, thio-containing locked nucleic acids (LNA) or peptide nucleic acids (PNA).     

EcoRI restriction endonuclease cleavage site map of bacteriophage P22DNA.

The F plasmid is able to co-transfer (mobilize) the small, chimeric R plasmid pBR322 during conjugation only at a very low frequency (Bolivar et al., 1977). Mobilization has been found here to be invariably (> 99%) associated with a structural alteration of pBR322. The alteration was shown, by restriction endonuclease analysis and electron microscopy, to be an insertion of the F attachment sequence λδ (2.8 to 8.5F). λδ is, therefore, an insertion sequence.