The kinetic mechanism of EcoRI endonuclease.

Steady-state parameters governing cleavage of pBR322 DNA by EcoRI endonuclease are highly sensitive to ionic environment, with K(m) and k(cat) increasing 1,000-fold and 15-fold, respectively, when ionic strength is increased from 0.059 to 0.23 M. By contrast, pre-steady-state analysis has shown that recognition, as well as first and second strand cleavage events that occur once the enzyme has arrived at the EcoRI site, are essentially insensitive to ionic strength, and has demonstrated that the rate-limiting step for endonuclease turnover occurs after double-strand cleavage under all conditions tested. Furthermore, processive cleavage of a pBR322 variant bearing two closely spaced EcoRI sites is governed by the same turnover number as hydrolysis of parental pBR322, which contains only a single EcoRI sequence, ruling out slow release of the enzyme from the cleaved site or a slow conformational change subsequent to double-strand cleavage. We attribute the effects of ionic strength on steady-state parameters to nonspecific endonuclease.DNA interactions, reflecting facilitated diffusion processes, that occur prior to EcoRI sequence recognition and subsequent to DNA cleavage.     

Substrate specificity of restriction endonuclease VspI

The recognition sequence and cleavage point of restriction endonuclease VspI have been determined as 5'-AT decreases TAAT. This enzyme is not isoschizomer of any known restriction endonucleases. DNA pBR322 contains a single VspI recognition sequence in position 3539. Therefore this enzyme may be used for cloning DNA in the VspI site in AmpR-gene of pBR322.     

Alteration of the specificity of PvuII restriction endonuclease.

The restriction endonuclease PvuII which cleaves the sequence CAGCTG, at the position indicated by the arrow, was found to decrease its substrate specificity in the presence of organic solvents. Thirty-three sites, that we have named PvuII sites, were identified on the nucleotide sequence of pBR322 DNA. The new recognition sequences cleaved in pBR322 DNA, at the positions indicated by the arrows, were shown to be AAGCTG, GAGCTG, CNGCTG, CANCTG, CAGNTG, CAGCNG, CAGCTC and CAGCTT. (TAGCTG and the complementary sequence CAGCTA are not present in pBR322 DNA). From these recognition sequences, we deduced that PvuII activity recognizes and cleaves degenerate sequences which differ from the standard PvuII sequence CAGCTG at only one of the recognition site. Any substitution can occur at any one of the six positions in the hexanucleotide sequence. The optimum incubation medium for PvuII activity was found to be: 10-50 mM Tris-HCl, pH 8.5, 12-15 mM MgCl2, 50 mM NaCl, 10% ethanol + 10% dimethylsulfoxide (DMSO).     

Isolation and characterization of endonuclease J: a sequence-specific endonuclease cleaving immunoglobulin genes.

An endonuclease activity which cleaves close to the recombination sites of the immunoglobulin JK segments was found in extracts of chicken bursa of Fabricius and characterized after partial purification. The enzyme preparation also cleaved a VK segment at its 3' end. A similar activity was found in mouse liver, mouse myelomas and Hela cells. The enzyme designated as endonuclease J introduces double-stranded cleavages preferentially at sequences containing G clusters of pBR322 as well as the JK segments. However, not all the G clusters were cleaved by endonuclease J, suggesting that the enzyme recognizes additional sequences. Deletion of the conserved nonamer (GGTTTTTGT) located immediately 5' to the JK4 segment drastically reduced the cleavage activity of its immediate downstream G cluster. Although biological function of endonuclease J is not clear at this stage, the possibilities of its involvement in the immunoglobulin gene recombination and general recombination were discussed.     

Characterization of a site-specific restriction endonuclease from Rhodopseudomonas sphaeroides.

A type II restriction endonuclease, RshI, has been partially purified from photoheterotrophically grown Rhodopseudomonas sphaeroides strain 2.4.1. The enzyme preparation, after a single DE-52 column fractionation, is free of 5' exonuclease and phosphatase activities but contains a trace of 3' exonuclease activity. Based upon deoxyribonucleic acid (DNA) sequencing data in the vicinity of the enzyme-promoted cleavage of pBR322 DNA, we have concluded that RshI probably recognizes the palinodromic hexanucleotide sequence 5'-CGATCG-3' and cleaves between the T and C. lambda cI857 DNA contains three RshI sites, two of which lie in the replaceable region. The plasmid pBR322, which carries resistances to ampicillin and tetracycline, contains a single RshI site in the ampicillin resistance determinant. Insertion of DNA into the RshI site of pBR322 results in loss of ampicillin resistance but retention of tetracycline resistance, thereby providing a convenient screening procedure for recombinant plasmids.     

A novel endonuclease specified by bacteriophage lambda. Purification and properties of the enzyme

A DNA endonuclease whose expression is under the control of the b region of bacteriophage lambda has been partially purified from an induced lambda lysogen. In a reaction that requires single-stranded DNA, ATP, and Mg2+, the lambda-induced endonuclease makes one double strand break in pBR322 and other covalently closed circular DNA molecules, converting these substrates into unit-length linear forms. The double strand break in pBR322 DNA occurs at one of several preferred sites. Linear DNA appears not to be a good substrate for the enzyme.     

SwaI, a unique restriction endonuclease from Staphylococcus warneri, which recognizes 5'-ATTTAAAT-3'.

A novel class-II restriction endonuclease designated SwaI was purified from Staphylococcus warneri. This enzyme cleaves adenovirus 2 DNA, SV40 DNA and M13mp7 at one site each, but does not cleave lambda, PhiX174, pBR322 or pBR328 DNA. SwaI recognizes the octanucleotide sequence 5'-ATTTAAAT-3', cleaving in the center of the recognition sequence creating blunt ended DNA fragments. SwaI was used to digest chromosomal DNA from various microorganisms and human cells.     

NaeI endonuclease binding to pBR322 DNA induces looping.

Previous work has demonstrated the existence of both resistant and cleavable NaeI sites. Cleavable sites introduced on exogenous DNA can act in trans to increase the catalysis of NaeI endonuclease cleavage at resistant sites without affecting the apparent binding affinity of the enzyme for the resistant site [Conrad, M., & Topal, M. D. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 9707-9711]. This activation suggests allosteric regulation of NaeI cleavage by distant cis- and trans-acting sites in DNAs containing both resistant and cleavable sites. Plasmid pBR322 contains four NaeI sites, at least one of which is resistant to cleavage. Electron microscopy is used here to demonstrate that NaeI endonuclease simultaneously binds to multiple recognition sites in pBR322 DNA to form loops with NaeI protein bound at the loop's base. The maximum number of loops formed with a common base suggests four binding sites per enzyme molecule. Looping was inhibited by addition of enzyme-saturating amounts of double-stranded oligonucleotide containing an NaeI site, whereas another double-strand oligonucleotide without the NaeI site had no effect. The number of loops seen was not above background when double-stranded M13 DNA, which contains only a single NaeI recognition site, was used as substrate.     

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.     

Thermodynamic parameters governing interaction of EcoRI endonuclease with specific and nonspecific DNA sequences

Equilibrium binding of EcoRI endonuclease to DNA has been analyzed by nitrocellulose filter and preferential DNA cleavage methods. Association constants for pBR322 and a 34-base pair molecule containing the EcoRI site of this plasmid in a central position were determined to be 1.9 X 10(11) M-1 and 1.0 X 10(11) M-1 at 37 degrees C, respectively, with the stoichiometry of binding being 0.8 +/- 0.1 mol of endonuclease dimer per mol of DNA. In contrast, the affinity of the enzyme for a pBR322 derivative from which the EcoRI site has been deleted is 3.2 X 10(9) M-1 as judged by competitive binding experiments. If it is assumed that each base pair can define the beginning of a nonspecific binding site, this value corresponds to an affinity for nonspecific sites of 7.4 X 10(5) M-1. Furthermore, the affinity of the endonuclease for the EcoRI-methylated sequence is at least three orders of magnitude less than that for the unmodified recognition site. The dependence on temperature and ionic strength of the equilibrium constant governing specific interactions has also been examined. The temperature dependence of the reaction indicates that entropy increase accounts for 70% of the free energy of specific binding at 37 degrees C. Affinity of the endonuclease for the EcoRI site is highly dependent on NaCl concentration. Analysis of this dependence according to the theory of Record and colleagues (Record, T. M., Jr., Lohman, T. M., and deHaseth, P. (1976) J. Mol. Biol. 107, 145-158) has implicated 8 ion pairs in the stability of specific complexes, a value identical with the number of phosphate contacts determined by ethylation interference analysis (Lu, A. L., Jack, W. E., and Modrich, P. (1981) J. Biol. Chem. 256, 13200-13206). Extrapolation to 1 M NaCl suggests that nonelectrostatic interactions account for 40% of the free energy change associated with specific complex formation.     

A set of synthetic oligodeoxyribonucleotide primers for DNA sequencing in the plasmid vector pBR322

Seven oligonucleotide primers complementary to the plasmid vector pBR322 at positions adjacent to five of the unique restriction endonuclease cleavage sites (EcoRI, HindIII, BamHI, SalI and PstI) have been chemically synthesized. The polarity of the primers is such that any DNA inserted at one or a combination of two of the above restriction sites may be sequenced by the chain termination method using one of the synthetic DNA primers. One of the primers for sequencing inserts at the PstI site of pBR322 is also complementary to the M13 phage vector designated bla6. This set of universal primers is useful for rapid sequence determination of DNA cloned into pBR322 or M13bla6.     

Ability of high hydrostatic pressure treated plasmids and cells of Escherichia coli to genetically transform

The exposure of plasmid pUC18 and pBR322 DNA to high hydrostatic pressure increased the ability of plasmids to transform competent Escherichia coli cells. For pUC18 plasmid, a pressure of 400 MPa, and for pBR322, a pressure of 200 MPa was found to provide the highest transformation efficiency. The DNA duplexes of the two plasmids were found to be the most stable for melting conditions at these pressures. At pressures higher than these, both the stability of the duplex DNA and the transformation efficiency were affected. The stabilizing effect of high hydrostatic pressure on the hydrogen bond may be responsible for the observed increase in transformation efficiency of the pressure-exposed plasmid DNA. The possibility of pressure-induced changes in the structure and conformation of DNA was studied using various techniques. In agarose gel electrophoresis, pressure-treated plasmids (pUC18 at 400 MPa and pBR322 at 200 MPa) consistently showed visibly distinct higher mobility compared to untreated plasmids. Pressure-treated pUC18 as well as pBR322 DNA showed significant reduction in ethidium bromide binding as is evident from the reduced intensity of fluorescence of the dye bound pressure-treated DNA. Spectroscopic studies using circular dichroism and Fourier transform infrared (FTIR) spectroscopy also showed significant differences in the absorption profiles of pressure-treated plasmids as compared to an untreated control. These studies revealed that the pressure-induced changes in the conformation of these DNAs may be responsible for the observed increase in the transformation ability of the plasmids. On the other hand, the exposure of competent cells of E. coli to a high hydrostatic pressure of 50 MPa not only reduced their colony-forming ability but also drastically reduced their ability to take up plasmid DNA.     

Purification and Some Properties of Plant Endonuclease from Scallion Bulbs

A plant endonuclease with 3′-nucleotidase activity was purified from scallion bulbs to homogeneity as judged by SDS-PAGE. The molecular weight of the enzyme was estimated to be 38,000 by SDS-PAGE and 40,000 by Sephadex G-100 gel filtration. The enzyme rapidly hydrolyzed yeast RNA and denatured calf thymus DNA to acid-soluble substances, and hydrolyzed the plasmid pBR322 to yield small DNA fragments at low enzyme concentrations. These four activities were eliminated by treatments with EDTA and tetraethylenepentamine. The enzyme had maximum activity at pH 8.5-9.0 for 3′-AMP, 3′-GMP, and 3′-UMP, at pH 6.5 for 3′-CMP and yeast RNA, and at pH 6.0 for denatured calf thymus DNA and pBR322. During digestion of yeast RNA by the enzyme at pH 6.5, 5′-GMP was released most rapidly, followed by 5′-UMP, 5′-AMP, and 5′-CMP. These properties were different from those of endonucleases isolated from other sources such as mung bean sprouts and wheat seedlings.     

Cloning and cleavage site mapping of DNA from bovine herpesvirus 1 (Cooper strain).

Sequences representative of most of the bovine herpesvirus 1 (Cooper strain) DNa were cloned in the plasmid vector pBR322 at the HindIII site. EcoRI, HpaI, and BamHI restriction endonuclease sites were mapped in each of the cloned fragments, and this information was used to construct a restriction endonuclease cleavage site map of the entire viral genome for the four enzymes.     

Escherichia coli Fpg protein and UvrABC endonuclease repair DNA damage induced by methylene blue plus visible light in vivo and in vitro.

pBR322 plasmid DNA was treated with methylene blue plus visible light (MB-light) and tested for transformation efficiency in Escherichia coli mutants defective in either formamidopyrimidine-DNA glycosylase (Fpg protein) and/or UvrABC endonuclease. The survival of pBR322 DNA treated with MB-light was not significantly reduced when transformed into either fpg-1 or uvrA single mutants compared with that in the wild-type strain. In contrast, the survival of MB-light-treated pBR322 DNA was greatly reduced in the fpg-1 uvrA double mutant. The synergistic effect of these two mutations was not observed in transformation experiments using pBR322 DNA treated with methyl methanesulfonate, UV light at 254 nm, or ionizing radiation. In vitro experiments showed that MB-light-treated pBR322 DNA is a substrate for the Fpg protein and UvrABC endonuclease. The number of sites sensitive to cleavage by either Fpg protein or UvrABC endonuclease was 10-fold greater than the number of apurinic-apyrimidinic sites indicated as Nfo protein (endonuclease IR)-sensitive sites. Seven Fpg protein-sensitive sites per PBR322 molecule were required to produce a lethal hit when transformed into the uvrA fpg-1 mutant. These results suggest that MB-light induces DNA base modifications which are lethal and that these modifications are repaired by Fpg protein and UvrABC endonuclease in vivo and in vitro. Therefore, one of the physiological functions of Fpg protein might be to repair DNA base damage induced by photosensitizers and light.     

Molecular cloning of cDNA for rat glycine methyltransferase

Using a highly purified preparation of glycine methyltransferase mRNA, double-stranded cDNA was synthesized and inserted into the PstI site of pBR322. The resulting recombinant DNA was used to transform E. coli X 1776 by conventional methods. Among tetracycline-resistant transformants, a number of colonies were found to contain cDNA sequence for glycine methyltransferase as examined by hybrid-selected translation. A restriction endonuclease cleavage map was constructed covering about 720 base pairs. With the cDNA as the probe, the content of the glycine methyltransferase mRNA was quantitated in various rat tissues and was found to be proportional to the specific enzyme activity.     

A second type II restriction endonuclease from Thermus aquaticus with an unusual sequence specificity.

A type II restriction endonuclease activity free of TaqI was prepared from Thermus Aquaticus YT. The fraction contains two endonucleolytic components with apparently different specificities, however the major activity is sufficiently dominant to allow partial digestion analysis of the position of recognition sites. A precise determination of the location of cleavage sites in pBR322 DNA and a computer-aided search for regions of homology in the vicinity of the cut sites indicate that this enzyme recognizes the nonpalindromic sequences GACCGA or CACCCA. Other related sequences are not cleaved, in particular, GACCCA and CACCGA, indicating that the enzyme requires the identity of nucleotides in the first and fifth positions, a type of specificity that has not been previously reported. The position of cleavage is located outside of the site and is represented as: (Formula: see text).     

Relaxation of recognition sequence of specific endonuclease HindIII.

Under the standard reaction conditions, the restriction endonuclease HindIII cleaves double-stranded DNA, within the recognition sequence--A/AGCTT--at the position indicated by the arrow. In the presence of dimethyl sulfoxide the substrate specificity of this enzyme is reduced and cleavages occur at additional sites. We have determined the secondary sites in pBR322 DNA recognized by HindIII endonuclease under relaxed conditions and found that it cleaves the hexanucleotides: G/AGCTT, A/GGCTT, A/TGCTT, A/ATCTT, A/AGCCT, A/AGCAT, A/AGCGT, A/AGCTC, at the positions indicated by the arrows, producing fragments with cohesive ends.     

pBR322 contains glucocorticoid regulatory element DNA consensus sequences

A computer search of the pBR322 DNA sequence identified five sites matching reported glucocorticoid regulatory element (GRE) DNA consensus sequences and three related sites. A pBR322 DNA fragment containing one GRE site was shown to bind immobilized HeLa S3 cell glucocorticoid receptor and to compete for receptor binding in a competitive binding assay. Conversely, a pBR322 DNA fragment devoid of GRE sites showed barely detectable interaction with glucocorticoid receptor in either of these assays. These results demonstrate the importance of GRE consensus sequences in glucocorticoid receptor interactions with DNA, and further identify a cause for high background binding observed when pBR322 DNA is used as a negative control in studies of glucocorticoid receptor-DNA interactions.     

Selective inhibition of restriction endonuclease cleavage by DNA intercalators

The preferred dye binding sites and the microenvironment of known nucleotide sequences within mitochondrial and plasmid pBR322 DNA was probed in a gross fashion with restriction endonucleases. The intercalating dyes, ethidium bromide and propidium iodide, do not inhibit a given restriction endonuclease equally at all of the restriction sites within a DNA molecule. The selective inhibition may be explained, in part, by the potential B to Z conformation transition of DNA flanking the restriction site and by preferred dye binding sites. Propidium iodide was found to be a more potent inhibitor than ethidium bromide and the inhibition is independent of the type of cut made by the enzyme.