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.     

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.     

The effects of base analogue substitutions on the cleavage by the EcoRI restriction endonuclease of octadeoxyribonucleotides containing modified EcoRI recognition sequences

It has been proposed that recognition of specific DNA sequences by proteins is accomplished by hydrogen bond formation between the protein and particular groups that are accessible in the major and minor grooves of the DNA. We have examined the DNA-protein interactions involved in the recognition of the hexameric DNA sequence, GAATTC, by the EcoRI restriction endonuclease by using derivatives of an oligodeoxyribonucleotide that contain a variety of base analogues. The base analogues hypoxanthine, 2-aminopurine, 2,6-diaminopurine, N6-methyladenine, 5-bromouracil, uracil, 5-bromocytosine, and 5-methylcytosine were incorporated as single substitutions into the octadeoxyribonucleotide d(pG-G-A-A-T-T-C-C). The effects of the substitutions on the interactions between the EcoRI endonuclease and its recognition sequence were monitored by determining the steady state kinetic values of the hydrolysis reaction. The substitutions resulted in effects that varied from complete inactivity to enhanced reactivity. The enzyme exhibited Michaelis-Menten kinetics with those substrates that were reactive, whereas octanucleotide analogues containing N6-methyladenine at either adenine position, uracil at the second thymine position, or 5-bromocytosine or 5-methylcytosine at the cytosine position were unreactive. The results are discussed in terms of possible effects on interactions between the enzyme and its recognition site during the reaction. An accompanying paper presents the results of a similar study using these oligonucleotides with the EcoRI modification methylase.     

Human cytomegalovirus DNA: BamHI, EcoRI and PstI restriction endonuclease cleavage maps

The cloned HindIII fragments of human cytomegalovirus (HCMV) strain AD169 DNA were mapped with respect to the BamHI, EcoRI and PstI restriction endonuclease cleavage sites. Composite restriction endonuclease cleavage maps for the entire virus genome were constructed using the previously established linkages between the HindIII fragments.     

Fluorescence stopped-flow kinetics of the cleavage of synthetic oligodeoxynucleotides by the EcoRI restriction endonuclease

We have investigated in fluorescence stopped-flow and temperature-jump experiments the EcoRI-catalyzed cleavage of synthetic palindromic tridecadeoxynucleotides which contain the EcoRI site but differ in the flanking sequences. The overall reaction can be resolved in several reactions which were analyzed by a nonlinear least-squares fitting procedure on the experimental data. The result of this analysis is a minimal scheme that describes the overall reaction in terms of the rate constants of the individual reactions. According to this scheme EcoRI and the tridecadeoxynucleotide substrates associate in the presence of Mg2+ in a nearly diffusion-controlled process. This is followed by a reaction which is or includes the cleavage of the first phosphodiester bond. There is no indication for a time-resolved conformational transition prior to catalysis. After cleavage of the first strand, dissociation of the nicked double strand can occur, which then rearranges to the original palindromic double-stranded substrate and is bound again by the enzyme. Alternatively, the nicked double strand can be cleaved in the second strand. This reaction is followed by product release from the enzyme. The magnitude of the individual rate constants depends on the substrate used; the differences explain the preference of EcoRI for substrates that contain AT as compared to GC base pairs next to the recognition site.     

Heparin inhibits EcoRI endonuclease cleavage of DNA at certain EcoRI sites.

Studies presented here demonstrate that heparin inhibits EcoRI endonuclease cleavage of DNA whereas related proteoglycans show no effect. The inhibition occurs at particular EcoRI sites that are near or overlap with palindromic sequences in the murine lambda 5 and Lyt-2 genes. Endogenous heparin from peritoneal mast cells co-isolates with DNA and inhibits digestion of peritoneal cell DNA at the inhibitable sites. Digestion of spleen DNA is inhibited at the same sites when commercial heparin is added prior to digestion. In both cases, the inhibition is abolished by pre-treating the DNA with heparinase. Thus, potential artifacts in restriction fragment length analyses could occur with DNA isolated either from cells that are naturally rich in heparin or from cells to which heparin has been added, e.g., as an anticoagulant.     

Modes of DNA cleavage by the EcoRV restriction endonuclease

The mechanism of action of the EcoRV restriction endonuclease at its single recognition site on the plasmid pAT153 was analyzed by kinetic methods. In reactions at pH 7.5, close to the optimum for this enzyme, both strands of the DNA were cut in a single concerted reaction: DNA cut in only one strand of the duplex was neither liberated from the enzyme during the catalytic turnover nor accumulated as a steady-state intermediate. In contrast, reactions at pH 6.0 involved the sequential cutting of the two strands of the DNA. Under these conditions, DNA cut in a single strand was an obligatory intermediate in the reaction pathway and a fraction of the nicked DNA dissociated from the enzyme during the turnover. The different reaction profiles are shown to be consistent with a single mechanism in which the kinetic activity of each subunit of the dimeric protein is governed by its affinity for Mg2+ ions. At pH 7.5, Mg2+ is bound to both subunits of the dimer for virtually the complete period of the catalytic turnover, while at pH 6.0 Mg2+ is bound transiently to one subunit at a time. The kinetics of the EcoRV nuclease were unaffected by DNA supercoiling.     

Accuracy of the EcoRI restriction endonuclease: binding and cleavage studies with oligodeoxynucleotide substrates containing degenerate recognition sequences

We have synthesized a series of 18 nonpalindromic oligodeoxynucleotides that carry all possible base changes within the recognition sequence of EcoRI. These single strands can be combined with their complementary single strands to obtain all possible EcoRI sequences (left), or they can be combined with a single strand containing the canonical sequence to obtain double strands with all possible mismatches within the recognition sequence (right): (sequence; see text) The rate of phosphodiester bond cleavage of these oligodeoxynucleotides by EcoRI was determined in single-turnover experiments under normal buffer conditions in order to find out to what extent the canonical recognition site can be distorted and yet serve as a substrate for EcoRI. Our results show that oligodeoxynucleotides containing mismatch base pairs are in general more readily attacked by EcoRI than oligodeoxynucleotides containing EcoRI sites and that the rates of cleavage of the two complementary strands of degenerate oligodeoxynucleotides are quite different. We have also determined the affinities of these oligodeoxynucleotides to EcoRI. They are higher for oligodeoxynucleotides carrying a mismatch within the EcoRI recognition site than for oligodeoxynucleotides containing an EcoRI site but otherwise do not correlate with the rate with which these oligodeoxynucleotides are cleaved by EcoRI. Our results allow details to be given for the probability of EcoRI making mistakes in cleaving DNA not only in its recognition sequence but also in sequences closely related to it. Due to the fact that the rates of cleavage in the two strands of a degenerate sequence generally are widely different, these mistakes are most likely not occurring in vivo, since nicked intermediates can be repaired by DNA ligase.     

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.     

Discrimination between DNA sequences by the EcoRV restriction endonuclease

The EcoRV restriction endonuclease cleaves not only its recognition sequence on DNA, GATATC, but also, at vastly reduced rates, a number of alternative DNA sequences. The plasmid pAT153 contains 12 alternative sites, each of which differs from the recognition sequence by one base pair. The EcoRV nuclease showed a marked preference for one particular site from among these alternatives. This noncognate site was located at the sequence GTTATC, and the mechanism of action of EcoRV at this site was analyzed. The mechanism differed from that at the cognate site in three respects. First, the affinity of the enzyme for the noncognate site was lower than that for the cognate site, but, by itself, this cannot account for the specificity of EcoRV as measured from the values of kcat/Km. Second, the enzyme had a lower affinity for Mg2+ when it was bound to the noncognate site than when it was bound to its cognate site: this appears to be a key factor in limiting the rates of DNA cleavage at alternative sites. Third, the reaction pathway at the noncognate site differed from that at the cognate site. At the former, the EcoRV enzyme cleaved first one strand of the DNA and then the other while at the latter, both strands were cut in one concerted reaction. The difference in reaction pathway allows DNA ligase to proofread the activity of EcoRV by selective repair of single-strand breaks at noncognate sites, as opposed to double-strand breaks at the cognate site. The addition of DNA ligase to reactions with EcoRV made no difference to product formation at the cognate site, but products from reactions at noncognate sites were no longer detected.     

Subunit assembly for DNA cleavage by restriction endonuclease SgrAI

The SgrAI endonuclease usually cleaves DNA with two recognition sites more rapidly than DNA with one site, often converting the former directly to the products cut at both sites. In this respect, SgrAI acts like the tetrameric restriction enzymes that bind two copies of their target sites before cleaving both sites concertedly. However, by analytical ultracentrifugation, SgrAI is a dimer in solution though it aggregates to high molecular mass species when bound to its specific DNA sequence. Its reaction kinetics indicate that it uses different mechanisms to cleave DNA with one and with two SgrAI sites. It cleaves the one-site DNA in the style of a dimeric restriction enzyme acting at an individual site, mediating neither interactions in trans, as seen with the tetrameric enzymes, nor subunit associations, as seen with the monomeric enzymes. In contrast, its optimal reaction on DNA with two sites involves an association of protein subunits: two dimers bound to sites in cis may associate to form a tetramer that has enhanced activity, which then cleaves both sites concurrently. The mode of action of SgrAI differs from all restriction enzymes characterised previously, so this study extends the range of mechanisms known for restriction endonucleases.     

DNA recognition and cleavage by the EcoP15 restriction endonuclease.

EcoP15 is a restriction-modification enzyme coded by the P15 plasmid of Escherichia coli. We have determined the sites recognized by this enzyme on pBR322 and simian virus 40 DNA. The enzyme recognizes the sequence:

In restriction, the enzyme cleaves the DNA 25 to 26 base-pairs 3′ to this sequence to leave single-stranded 5′ protrusions two bases long.     

Pentamidine-induced alteration in restriction endonuclease cleavage of plasmid DNA

We have used restriction enzymes and DNaseI as probes to determine the specificity of pentamidine binding to plasmid DNA. Cleavage of plasmid pAZ130 by EcoRI, EcoRV and ApaI is inhibited by pentamidine, cleavage by XbaI, NotI and AvaI is unaffected, while cleavage by XhoI, which recognizes the same sequence as AvaI, is stimulated. DNaseI footprinting of DNA containing these restriction sites revealed that pentamidine protection is not strictly limited to AT-rich regions. We suggest that perturbation of the DNA micro- environment by pentamidine binding is responsible for its effect on nucleases.     

In vivo restriction of Escherichia coli-transforming DNA by endonuclease R.M.EcoRI

Transformation of Escherichia coli K-12 for various chromosomal markers was accomplished by using AB1157 recBC+ strain as a recipient. The yield of transformants was reduced 10-fold, as compared with that obtained in JC7623 recBC sbcB recipient. Elimination of transformation has been obtained for arg, pro, his markers in AB1157 (pSA14) harbouring the R.M.EcoRI coding plasmid. Production of restriction endonuclease in this strain did not affect the efficiency of transformation for thr, leu markers. The presence of pSA25 which is isogenic to pSA14 but devoid of R.M.EcoRI genes has been irrelevant to transformation for leu, arg, pro, his, thr markers. Correlation between the restriction of transformed markers in vivo and in vitro is discussed.     

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.     

Preferential cleavage by restriction endonuclease HinfIII

The efficiency of endonucleolytic scission by restriction endonuclease HinfIII varies markedly for different recognition sites. The relative frequencies of cleavage at these sites have been determined on the basis of analysis of specific unit length linear molecules formed. The efficiency of restriction reaction depends also on the number of recognition sites in the DNA substrate. Cleavage by HinfIII in the absence or presence of S-adenosylmethionine is observed only when at least three recognition sites are present. HinfIII also shows preferential methylation of certain sites observable even for a substrate with one recognition site. The nucleotide sequences at sites cleaved or methylated at high frequency have been compared.     

DNA structural polymorphism modulates the kinetics of superhelical DNA cleavage by BamHI restriction endonuclease

A compartmental model developed by Hensley (Hensley, P., Nardone, G., Chirikjian, J.G., and Wastney, M. E., (1990) J. Biol. Chem. 265, 15300-15307) for analysis of the time courses of the cleavage of superhelical DNA substrates by the restriction endonuclease, BamHI, has been used to quantify the effects of changes in temperature, ionic strength, superhelical density, and the DNA substrate on the binding and strand cleavage processes. Studies reported here indicate that changes in topology may be introduced into the DNA substrate solely as a result of the plasmid preparation process and in the absence of covalent bond cleavage and ligation. These changes in topology have qualitatively different effects on the kinetics than those promoted by changes in the superhelical density. The former are removed by briefly warming the DNA prior to assay, suggesting that they are only kinetically stable, while the latter changes are not affected by heating. Increasing the [NaCl] from 0.01 M to 0.1 M increases the overall rate of plasmid cleavage by increasing both the rates of cleavage and enzyme DNA association. To describe the decrease in the overall cleavage rate observed in 0.15 M NaCl, an ionic strength-dependent rate-determining structural transition in the DNA substrate was incorporated into the model. The largest changes in the rate of the cleavage process resulted from changes in the DNA substrate. For the SV40 substrate compared to pBR322, the rate constants describing the two association processes and the first bond cleavage event were increased 6- to 7-fold. The rate of the second bond cleavage process was not affected. These changes may be due to differences in the flanking sequences.     

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.     

The time-resolved kinetics of superhelical DNA cleavage by BamHI restriction endonuclease

The kinetics of the cleavage of superhelical plasmid DNA (pBR322) by the restriction endonuclease, BamHI, have been analyzed in terms a compartmental model consistent with the chemistry first proposed by Rubin and Modrich (Rubin, R. A., and Modrich, P. (1978) Nucleic Acids Res. 5, 2991-2997) for analysis of the kinetics of the restriction endonuclease, EcoRI. The model was defined in terms of two compartments representing DNA substrate (bound and free), two compartments representing nicked intermediate (bound and free), one compartment representing linear product, and one compartment for free enzyme. A simultaneous analysis of concentration changes over time of the three DNA forms (superhelical, nicked, and linear) at six different enzyme concentrations was undertaken employing this compartmental model using SAAM (Simulation Analysis And Modeling) software. Results showed that rate constants characterizing the association of enzyme with superhelical DNA (6.0 x 10(5) M-1 s-1) and nicked DNA (2.8 x 10(5) M-1 s-1) were similar in magnitude and rate constants characterizing cleavage of the first (1.2 x 10(-2) s-1) and second phosphodiester bonds (3.1 x 10(-2) s-1) were also similar. The analysis yields a kinetically determined equilibrium constant of 12.9 nM for the dissociation of nicked intermediate from the enzyme. The rate constant describing the release of the nicked intermediate from the enzyme has a value of 3.7 x 10(-3) s-1. By comparing the value of this release rate constant to the value of the constant describing the second cleavage event, it can be determined that only 10% of the nicked intermediate bound to the enzyme is released as free nicked DNA and that 90% of the nicked intermediate is processed to the linear form without being released. Hence, most of the DNA is cleaved as the result of a single enzyme-DNA recognition event. No steady state assumptions were made in the analysis. The approach was to directly solve the differential equations which described the kinetic processes using an interactive method. This study demonstrates the usefulness of this approach for the analysis of kinetics of protein-DNA interactions for the restriction endonucleases.