Activation of restriction endonuclease EcoRII does not depend on the cleavage of stimulator DNA.

The restriction endonuclease EcoRII is unable to cleave DNA molecules when recognition sites are very far apart. The enzyme, however can be activated in the presence of DNA molecules with a high frequency of EcoRII sites or by oligonucleotides containing recognition sites: Addition of the activator molecules stimulates cleavage of the refractory substrate. We now show that endonucleolysis of the stimulator molecules is not a necessary prerequisite of enzyme activation. A total EcoRII digest of pBR322 DNA or oligonucleotide duplexes with simulated EcoRII ends (containing the 5' phosphate group), as well as oligonucleotide duplexes containing modified bases within the EcoRII site, making them resistant to cleavage, are all capable of enzyme activation. For activation EcoRII requires the interaction with at least two recognition sites. The two sites may be on the same DNA molecule, on different oligonucleotide duplexes, or on one DNA molecule and one oligonucleotide duplex. The efficiency of functional intramolecular cooperation decreases with increasing distance between the sites. Intermolecular site interaction is inversely related to the size of the stimulator oligonucleotide duplex. The data are in agreement with a model whereby EcoRII simultaneously interacts with two recognition sites in the active complex, but cleavage of the site serving as an allosteric activator is not necessary.     

Site-dependent cleavage of pBR322 DNA by restriction endonuclease HinfI.

Cleavage of pBR322 DNA I by the restriction endonuclease HinfI is preferentially inhibited at specific HinfI cleavage sites. These sites in pBR322 DNA I have been identified and ordered with respect to the frequency with which they are cleaved. The HinfI site most resistant to cleavage in pBR322 DNA I is unique in that runs of G-C base pairs are immediately adjacent on both sites. Two differently permuted linear (DNA III) species were produced by cleavage with two different restriction endonucleases, PstI and AvaI. Only one of these linear molecules, that produced by PstI, exhibits the same preferential cleavage pattern as DNA I. The second linear species, that arising from AvaI digestion, shows pronounced relative inhibition of cleavage at the HinfI sites nearest the ends of the molecule (100 to 120 base pairs away, respectively). This result suggest that proximity to the termini of a linear DNA molecule might also influence preferential cleavage. The possibility of formation of stem-loop structures does not appear to influence preferential cleavage by HinfI.     

Interaction of EcoRII endonuclease with DNA substrates containing single recognition sites.

EcoRII is unusual among type II restriction enzymes in that, while it cleaves substrates such as pBR322 and bacteriophage lambda that contain several recognition sites for the enzyme efficiently, substrates such as the genomes of bacteriophages T3 and T7 which contain a small number of recognition sites are cut poorly by it. Interestingly, pBR322, or a short DNA duplex containing a single site for the enzyme, can activate the enzyme to cleave resistant substrates. We show here that, at low concentrations, activator short duplexes are themselves cleaved poorly by the enzyme. Further, the reaction shows substrate cooperativity, and at high concentrations, the duplexes are both activators and good substrates for the enzyme. This supports the model that the activation of EcoRII involves binding of more than one DNA molecule and provides a simple system to study the mechanism of activation. Using a gel mobility shift assay, we show that the enzyme forms sequence-specific, methylation-sensitive complexes with the duplexes in the absence of activating DNA. Therefore, resistance of the short duplexes to the enzyme at low concentrations cannot be due to an inability of the enzyme to bind the duplexes. Interestingly, these complexes are stable in the presence of Mg2+, the cofactor for the enzyme, and the complexes obtained in the presence of Mg2+ do not contain DNA that is cleaved by the enzyme. The inefficient step in the action of EcoRII on resistant substrates must occur subsequent to initial substrate binding and it is this step that the activating DNA must regulate.     

High-frequency transduction of pBR322 by cytosine-substituted T4 bacteriophage: evidence for encapsulation and transfer of head-to-tail plasmid concatemers

Transduction of plasmid pBR322 by cytosine-substituted T4 phages has been studied. Three T4 phage mutants which substitute cytosine for all of hydroxymethylcytosine residues in the DNA, were shown to transduce pBR322 at frequencies of 2 × 10^?2 to 4 × 10^?3 transductants per singly infected cell. Also, three T4 phage strains which partially substitute cytosine for hydroxymethylcytosine, transduced pBR322 at frequencies of 2 × 10^?3 to 2 × 10^?4. The transduction frequencies of pBR322 we attained are at least 10-fold higher than those reported by G. G. Wilson, K. Young, and G. J. Edlin (1979, Nature (London)280, 80–82). We found that multiplicity of infection in preparation of the transducing phage is the most important factor affecting the frequency of pBR322 transduction. When a lysate made at a multiplicity of infection ranging from 0.5 to 0.05 was used as the donor phage, transduction frequency of pBR322 was 10- to 40-fold higher than that of high-m.o.i. lysate. The transduction frequency was not affected by either restriction systems or amber suppressors of the recipient cells. However, no pBR322-containing transductant was obtained when either recA or polA mutants were used as the recipients. DNA from T4dC phage containing pBR322-transducing particles was analyzed on agarose gel electrophoresis after cleavage with restriction endonucleases. It was suggested that the pBR322 DNA in the T4dC phage particles exists as head-to-tail concatemers.     

Sites of reaction of Escherichia coli DNA gyrase on pBR322 in vivo as revealed by oxolinic acid-induced plasmid linearization

pBR322 DNA, linearized by lysis of an oxolinic acid-treated culture of Escherichia coli strain DK6recA- (pBR322) with sodium dodecyl sulfate, was purified, treated with DNA polymerase in the presence of the four deoxynucleoside triphosphates, and ligated to DNA linkers containing the XhoI recognition sequence. Most of the drug-resistant colonies resulting from transformation of E. coli with this material bore plasmids that appeared by restriction enzyme analysis to differ from pBR322 only by the introduction of an XhoI site. The XhoI sites in plasmids from 93 transformants were distributed unevenly around the pBR322 map. Maxam-Gilbert DNA sequence analysis of 36 of these plasmids, labeled at the 5' termini of the XhoI sites, revealed that 29 of them contained, in addition to the XhoI linker, a duplication of four base-pairs of the pBR322 sequence surrounding the linker. Therefore, oxolinic acid-induced linearization must have resulted in 5'-terminal extensions of four bases, the configuration known to result from oxolinic acid-induced DNA cleavage by DNA gyrase in vitro. The sequence data thus allowed the determination of the precise point at which linearization occurred, apparently by the abortion of a gyrase-DNA covalent intermediate that existed in vivo. When the 19 different sites of the 29 plasmids were compared, the following set of rules could be derived: (formula; see text) where N is any nucleotide, R is a purine, and Y is a pyrimidine. Cleavage occurred at the line between the eighth and ninth positions from the left. The parenthetical G and T were preferred secondarily to T and G, respectively, whereas T and G in the 13th position from the left were equally preferred. Several of these rules are similar to those proposed previously based on several in vitro gyrase cleavage sites. Some of our rules show dyad symmetry around the axis midway between the cleavage points in the two strands, while others are distinctly asymmetric.     

Construction and characterization of new cloning vehicles. IV. Deletion derivatives of pBR322 and pBR325

In vitro recombinant DNA experiments involving restriction endonuclease fragments derived from the plasmids pBR322 and pBR325 resulted in the construction of two new cloning vehicles. One of these plasmids, designated pBR327, was obtained after an EcoRII partial digestion of pBR322. The plasmid pBR327 confers resistance to tetracycline and ampicillin, contains 3273 base pairs (bp) and therefore is 1089 bp smaller than pBR322. The other newly constructed vector, which has been designated pBR328, confers resistance to chloramphenicol as well as the two former antibiotics. This plasmid contains unique HindIII, BamHI and SalI sites in the tetracycline resistance gene, unique PvuI and PstI sites in the ampicillin resistance gene and unique EcoRI, PvuII and BalI sites in the chloramphenicol resistance gene. The pBR328 plasmid contains approx. 4900 bp.     

Differential reactivities at restriction enzyme sites

A method has been developed to measure the rates of digestion by restriction enzymes at individual sites. This involves a simple arithmetical treatment of the integrated areas from a densitometer scan of an ethidium bromide stained gel. We have used this method to study the digestion by HpaI, HincII and SalI of pBR322 and phi X174 DNA, and the effect of various DNA binding ligands. One of the two HpaI sites in phi X174 DNA is much more sensitive to inhibition by ligands such as netropsin, which display a preference for AT base pairs, than is the other site. Inspection of the sequences flanking the restriction sites shows that the former contains a much higher proportion of AT base-pairs than dose the latter. The opposite phenomenon is observed with the two HincII sites in pBR322. This illustrates the importance of neighbouring sequences in the interaction between restriction enzymes and their cleavage sites in DNA.     

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.     

Oligonucleotide duplexes containing CC(A/T)GG stimulate cleavage of refractory DNA by restriction endonuclease EcoRII

Some DNA species are resistant towards the restriction endonuclease EcoRII despite the presence of unmodified recognition sites. We show that 14 base-pair oligonucleotide duplexes containing the EcoRII recognition site 5'-CC(A/T)GG are cleaved by this enzyme and are able to stimulate EcoRII cleavage of such resistant DNA molecules (e.g. DNA of bacterial virus T3). A direct correlation between the concentration of oligonucleotide duplex molecules and the degree of EcoRII digestion of the primarily resistant DNA is observed. This indicates a stoichiometric rather than a catalytic mode of enzyme activation. An excess of DNA devoid of EcoRII sites ('non-site' DNA, e.g. MvaI-digested T7 DNA) does not interfere with the activity of EcoRII.     

Simultaneous binding of three recognition sites is necessary for a concerted plasmid DNA cleavage by EcoRII restriction endonuclease

According to the current paradigm type IIE restriction endonucleases are homodimeric proteins that simultaneously bind to two recognition sites but cleave DNA at only one site per turnover: the other site acts as an allosteric locus, activating the enzyme to cleave DNA at the first. Structural and biochemical analysis of the archetypal type IIE restriction enzyme EcoRII suggests that it has three possible DNA binding interfaces enabling simultaneous binding of three recognition sites. To test if putative synapsis of three binding sites has any functional significance, we have studied EcoRII cleavage of plasmids containing a single, two and three recognition sites under both single turnover and steady state conditions. EcoRII displays distinct reaction patterns on different substrates: (i) it shows virtually no activity on a single site plasmid; (ii) it yields open-circular DNA form nicked at one strand as an obligatory intermediate acting on a two-site plasmid; (iii) it cleaves concertedly both DNA strands at a single site during a single turnover on a three site plasmid to yield linear DNA. Cognate oligonucleotide added in trans increases the reaction velocity and changes the reaction pattern for the EcoRII cleavage of one and two-site plasmids but has little effect on the three-site plasmid. Taken together the data indicate that EcoRII requires simultaneous binding of three rather than two recognition sites in cis to achieve concerted DNA cleavage at a single site. We show that the orthodox type IIP enzyme PspGI which is an isoschisomer of EcoRII, cleaves different plasmid substrates with equal rates. Data provided here indicate that type IIE restriction enzymes EcoRII and NaeI follow different mechanisms. We propose that other type IIE restriction enzymes may employ the mechanism suggested here for EcoRII.     

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.     

Deoxyribonucleic Acid Adenine and Cytosine Methylation in Salmonella typhimurium and Salmonella typhi

The methylations of adenine in the sequence -GATC- and of the second cytosine in the sequence - [Formula: see text] - were studied in Salmonella typhimurium and in Salmonella typhi. The study was carried out by using endonucleases which restrict the plasmid pBR322 by cleavage at the sequences -GATC- (DpnI and MboI) and - [Formula: see text] - (EcoRII). The restriction patterns obtained for this plasmid isolated from transformed S. typhimurium and S. typhi were compared with those of pBR322 isolated from Escherichia coli K-12. In E. coli K-12, adenines at the sequence -GATC- and the second cytosines at - [Formula: see text] - are met hylated by enzymes coded for by the genes dam and dem, respectively. From comparison of the restriction patterns obtained, it is concluded that S. typhimurium and S. typhi contain genes responsible for deoxyribonucleic acid methylation equivalent to E. coli K-12 genes dam and dcm.     

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.     

Sequence requirements for mammalian topoisomerase II mediated DNA cleavage stimulated by an ellipticine derivative.

Various antitumor drugs stabilize DNA topoisomerase II-DNA transient covalent complexes. The complexes distribution along pBR322 DNA was shown previously to depend upon the nature of the drug (Tewey et al. (1984) Science 226, 466-468). The position in pBR322 of DNA cleavage by calf DNA topoisomerase II for 115 such sites stabilized by an ellipticine derivative and the relative frequency of cleavage at most of these sites were determined. The nucleotide sequence surrounding the 25 strongest sites was analyzed and the following ellipticine specific consensus sequence was deduced: 5'-ANCNT(A/G)T.NN(G/C)N(A/G)-3' where cleavage occurs at the indicated mark. A thymine is always present at the 3' end of at least one strand of the strong cleavage sites, and the dinucleotide AT or GT at the 3' end of the break plays a major role in the complex stabilisation. The predictive value of cleavage of the consensus was tested for two regions of SV40 DNA and cleavage was indeed detected at the majority of the sites matching the consensus. Some complexes stabilized by ellipticine are resistant to salt dissociation and this property seems to be correlated with the presence of symmetrical sequences in the cleavage site with a center of symmetry staggered relatively to the center of symmetry of cleavage.     

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.     

Nucleosome "phasing" and cruciform structures in circular supercoiled pBR322 DNA

Cruciform structures have been detected in pBR322 supercoiled DNA, both in its naked state and when complexed with histone octamer, using S1 endonuclease cleavage and EcoRI restriction. An inspection of the DNA sequence shows that the S1-hypersensitive sites are very near to AT-rich regions of pBR322 genome. A nucleosome "phasing" in these regions, as found on AT-rich regions of SV40 DNA (15), has been shown by restriction enzymes analysis. On the basis of these results it can be proposed that cruciform structures protrude on the nucleosome surface. This model explains the reason why these structures, which need high superhelical density, can exist in supercoiled DNA partially relaxed by nucleosome formation.     

Two new restriction endonucleases DraII and DraIII from Deinococcus radiophilus.

In addition to recently characterized DraI (1), two new Type II restriction endonucleases, DraII and DraIII, with novel site-specificities were isolated and purified from Deinococcus radiophilus ATCC 27603. DraII and DraIII recognize the hepta- and nonanucleotide sequences (sequence in text) The cleavage sites within both strands are indicated by arrows. The recognition sequences were established by mapping of the cleavage sites on pBR322 (DraII) and fd109 RF DNA (DraIII). The sequence specifities were confirmed by computer-assisted restriction analyses of the generated fragment patterns of the sequenced DNA's of the bacteriophages lambda, phi X174 RF, M13mp8 RF and fd109 RF, the viruses Adeno2 and SV40, and the plasmids pBR322 and pBR328. The cleavage positions within the recognition sequences were determined by sequencing experiments.     

Mapping of DNA gyrase cleavage sites in vivo oxolinic acid induced cleavages in plasmid pBR322

We have developed a procedure which permits the mapping of DNA gyrase cleavage sites in vivo. Addition of oxolinic acid, an inhibitor of DNA gyrase, to growing cells of Escherichia coli containing the plasmid pBR322 resulted in double-strand cleavage of DNA, and allowed the isolation of significant quantities of linearized plasmid DNA after lysis of treated cells with sodium dodecyl sulfate. Initially the linear product was purified from agarose gels, cleaved by restriction endonucleases, and then subjected to Southern hybridization analysis using defined DNA probes. A number of distinct cleavage sites, used with varying degrees of efficiency, were identified within pBR322 using this simple procedure. To achieve greater resolution and to improve sensitivity, we then employed an electroblotting procedure to transfer DNA fragments from acrylamide gels onto nylon membranes. This alternative method does not require the isolation of the linearized product before performing the mapping procedure. The improved resolution obtained from acrylamide gels and the superior binding properties of the nylon membranes have allowed us to accurately map 74 distinct oxolinic acid-induced cleavage sites within pBR322. The significance of these findings in light of previously reported studies in vitro, as well as the possible role of such sites during illegitimate recombination, are discussed.     

Restriction enzyme cleavage of ultraviolet-damaged DNA

SV40 and pBR322 DNAs damaged by ultraviolet light were cleaved abnormally by several restriction enzymes because of damage to pyrimidines in the recognition sequences. The use of a tandemly duplicated plasmid provided a particularly sensitive target molecule for detecting pyrimidine dimers and other possible photoproducts. The relative efficiency with which cleavage was blocked (HindIII greater than TaqI greater than EcoRI greater than BamI greater than SalI much greater than Hha I, Hae III) corresponds approximately to the relative frequency of pyrimidine dimer formation in the recognition sequences, but at a slightly higher frequency in potential sites for the non-cyclobutane T-C product. The pyrimidine dimers appear to have a range of influence that extends 1 to 3 basepairs along the DNA molecule. These effects provide clues to the way DNA damage from mutagens and carcinogens can interfere with specific enzyme-DNA interactions.