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.     

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.     

The EcoRI restriction endonuclease with bacteriophage lambda DNA. Kinetic studies.

The kinetics of the reactions of the EcoRI restriction endonuclease at individual recognition sites on the DNA from bacteriophage lambda were found to differ markedly from site to site. Under certain conditions of pH and ionic strength, the rates for the cleavage of the DNA were the same at each recognition site. But under altered experimental conditions, different reaction rates were observed at each recognition site. These results are consistent with a mechanism in which the kinetic stability of the complex between the enzyme and the recognition site on the DNA differs among the sites, due to the effect of interactions between the enzyme and DNA sequences surrounding each recognition site upon the transition state of the reaction. Reactions at individual sites on a DNA molecule containing more than one recognition site were found to be independent of each other, thus excluding the possibility of a processive mechanism for the EcoRI enzyme. The consequences of these observations are discussed with regard to both DNA-protein interactions and to the application of restriction enzymes in the study of the structure of DNA molecules.     

Properties of 2'-fluorothymidine-containing oligonucleotides: interaction with restriction endonuclease EcoRV

2'-Fluorothymidine (Tf) was synthesized via an improved procedure with (diethylamino)sulfur trifluoride. The compatibility of the analogue with DNA synthesis via the phosphoramidite method was demonstrated after complete enzymatic digestion of the oligonucleotides d(Tf11T) and d(Tf3T), the sole products of which were 2'-fluorothymidine and thymidine in the expected ratio. The 2'-fluorothymidine was also incorporated into the EcoRV recognition sequence (underlined), within the complementary oligonucleotides d(CAAACCGATATCGTTGTG) and d(CACAACGATATCGGTTTG). Thermal melting characteristics of these duplexes showed a significant decrease in stability only when both of the thymidine residues in one of the strands were replaced. In contrast, when all of one strand of a duplex contained 2'-fluorothymidine, as in d(Tf11T).d(A12), a substantially higher Tm and cooperativity of melting was observed relative to the unmodified structure. EcoRV cleaved a duplex that contained a 2'-fluorothymidine at the scissile linkage in each strand at two-thirds of the rate obtained for the unmodified structure. A duplex containing two 2'-fluorothymidine residues in one strand and none in the other was cleaved at one-third of the rate in its unsubstituted strand, whereas the cleavage rate was reduced to 22% in its modified strand.     

The EcoRI restriction endonuclease with bacteriophage lambda DNA. Equilibrium binding studies.

The EcoRI restriction endonuclease was found by the filter binding technique to form stable complexes, in the absence of Mg2+, with the DNA from derivatives of bacteriophage lambda that either contain or lack EcoRI recognition sites. The amount of complex formed at different enzyme concentrations followed a hyperbolic equilibrium-binding curve with DNA molecules containing EcoRI recognition sites, but a sigmoidal equilibrium-binding curve was obtained with a DNA molecule lacking EcoRI recognition sites. The EcoRI enzyme displayed the same affinity for individual recognition sites on lambda DNA, even under conditions where it cleaves these sites at different rates. The binding of the enzyme to a DNA molecule lacking EcoRI sites was decreased by Mg2+. These observations indicate that (a) the EcoRI restriction enzyme binds preferentially to its recognition site on DNA, and that different reaction rates at different recognition sites are due to the rate of breakdown of this complex; (b) the enzyme also binds to other DNA sequences, but that two molecules of enzyme, in a different protein conformation, are involved in the formation of the complex at non-specific consequences; (c) the different affinities of the enzyme for the recognition site and for other sequences on DNA, coupled with the different protein conformations, account for the specificity of this enzyme for the cleavage of DNA at this recognition site; (d) the decrease in the affinity of the enzyme for DNA, caused by Mg2+, liberates binding energy from the DNA-protein complex that can be used in the catalytic reaction.     

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.     

Solubility of the EcoRI restriction endonuclease and its purification from an over-producing strain

The solubility of the EcoRI restriction endonuclease was measured in solutions of varying NaCl concentrations, at different temperatures and in the presence of DNA. The precipitation of the protein was enhanced by low NaCl concentrations, by elevated temperatures, and by the addition of DNA. These observations are discussed in relationship to the interaction of this protein with DNA. The purification of the EcoRI restriction enzyme from a strain of Escherichia coli that over-produces this enzyme was hampered by the insolubility of the protein, and hence the purification procedure was modified to optimize the recovery of active enzyme.     

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.     

The interaction of Na ions with synthetic polynucleotides

The interaction of Na⁺ with poly A, poly U, poly A·poly U, and Poly A·2 poly U has been investigated by means of potentiometry, by means of potentiometry, by means of a linked-function analysis of its effect on the binding of Mg⁺⁺ ions, and of K⁺ by means of an analysis of its effect on the sedimentation coefficients of the polymers. The last method was found to be inapplicable. The results of the other two methods were found to be consistent, except in the case of poly A where the existence of base stacking, influenced by the binding of Mg⁺⁺, significantly affects the linked-function analysis. The results are also consistent with the effects of the concentration of Na⁺ ions on the thermally induced conformational transitions of poly A·poly U and poly A·2 poly U, and with the extents of “binding” of Na⁺ to DNA measured by equilibrium and by transport methods. The interaction of Na⁺ with polynucleotides appears to be physically quite specific, although its thermodynamic basis is not clear. The extent of binding of Na⁺, Ψ, was found to be independent of the total Na⁺ concentration but a quadratic function of the extent of Mg⁺⁺ binding, θ. In the absence of Mg⁺⁺, Ψ = 0.35–0.38 for poly U, 0.40 for poly A, 0.59 for poly A·poly U, and 0.66 for poly A·2 poly U.     

Mutants of the EcoRI endonuclease with promiscuous substrate specificity implicate residues involved in substrate recognition.

The EcoRI restriction endonuclease cleaves DNA molecules at the sequence GAATTC. We devised a genetic screen to isolate EcoRI mutants with altered or broadened substrate specificity. In vitro, the purified mutant enzymes cleave both the wild-type substrate and sites which differ from this by one nucleotide (EcoRI star sites). These mutations identify four residues involved in substrate recognition and catalysis that are different from the amino acids proposed to recognize the substrate based on the EcoRI-DNA co-crystal structure. In fact, these mutations suppress EcoRI mutants altered at some of the proposed substrate binding residues (R145, R200). We argue that these mutations permit cleavage of additional DNA sequences either by perturbing or removing direct DNA-protein interactions or by facilitating conformational changes that allosterically couple substrate binding to DNA scission.     

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.     

Structural adaptations in the interaction of EcoRI endonuclease with methylated GAATTC sites.

We have studied the interaction of EcoRI endonuclease with oligonucleotides containing GAATTC sites bearing one or two adenine-N6-methyl groups, which would be in steric conflict with key protein side chains involved in recognition and/or catalysis in the canonical complex. Single-strand methylation of either adenine produces small penalties in binding free energy (deltadeltaG0(S) approximately +1.4 kcal/mol), but elicits asymmetric structural adaptations in the complex, such that cleavage rate constants are strongly inhibited and unequal in the two DNA strands. The dependences of cleavage rate constants on the concentration of the Mg2+ cofactor are unaltered. When either adenine is methylated on both DNA strands, deltadeltaG0(S) (approximately +4 kcal/mol) is larger than the expected sum of the deltadeltaG0(S) values for the single-strand methylations, because the asymmetric adaptations cannot occur. Cleavage rate constants are reduced by 600 000-fold for the biologically relevant GAmATTC/CTTmAAG site, but the GmAATTC/CTTAmAG site forms only a non-specific complex that cannot be cleaved. These observations provide a detailed thermodynamic and kinetic explanation of how single-strand and double-strand methylation protect against endonuclease cleavage in vivo. We propose that non-additive effects on binding and structural 'adaptations' are important in understanding how DNA methylation modulates the biological activities of non-catalytic DNA binding proteins.     

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.     

Cross-linking of bromodeoxyuridine-substituted oligonucleotides to the EcoRI and EcoRV restriction endonucleases

We have synthesized several self-complementary oligodeoxynucleotides which contain bromodeoxyuridine in various positions within and outside of the recognition sequence for the EcoRI and EcoRV restriction endonucleases. These oligodeoxynucleotides are cleaved in the presence of Mg2+ by their respective enzyme. Upon irradiation by long-wavelength ultraviolet light and in the absence of Mg2+ they are cross-linked in low yield to their enzymes, forming 1:1 and 1:2 (oligodeoxynucleotide:enzyme subunit) adducts. Cross-linking occurs with both specific and non-specific complexes. With EcoRI the site of cross-linking was determined to be at or close to Met-137, i.e. in a region of the molecule implicated by other studies from our laboratory [Scholtissek et al. (1986) J. Biol. Chem. 261, 2228-2234] in the binding and cleavage of the substrate.     

Application of phosphate-backbone-modified oligonucleotides in the studies on EcoRI endonuclease mechanism of action.

Chemical synthesis of oligodeoxyribonucleotides modified at a preselected internucleotide bond by the replacement of one of the two "nonbridging" oxygens by a sulfur atom or an ethoxy group yields model substrates for studies on DNA-protein interactions. Chromatographic (RP-HPLC) separation of the diastereomers of oligonucleotides containing EcoRI canonical sequence together with the assignment of the substituent orientation in the DNA molecule allowed study of the stereochemical aspects of DNA-EcoRI endonuclease interactions. The DNA segment involved in interactions between EcoRI protein and phosphate groups appeared to be larger than its canonical sequence, ...GAATTC..., and was extended to the nonamer. The modification of certain internucleotide bonds within this nonamer caused significant or complete protection against the nucleolytic action of EcoRI and, in some cases, manifested the diastereoselectivity of the enzyme. On the basis of the results of EcoRI-catalyzed hydrolysis of stereodefined phosphorothioate and phosphotriester substrates, we propose a model to explain this phenomenon at the molecular level.     

Analysis of Euglena gracilis chloroplast deoxyribonucleic acid with a restriction endonuclease, EcoRI.

Cleavage of chloroplast deoxyribonucleic acid (DNA) of Euglena gracilis Z with restriction endonuclease RI from Escherichia coli (EcoRI) yielded 23 bands upon electrophoresis in gels of agarose. Four of the bands contained twice the stoichiometric amount of DNA. One of these bands contained two similarly sized fragments. The sum of the molecular weight of the 24 different fragments equaled the molecular weight of the circular molecule. The restriction fragments had different buoyant densities, with four having distinctly heavy densities in CsCl. Restriction fragments with a high buoyant density were preferentially lost when broken chloroplast DNA was purified by equilibrium density gradient centrifugation. Hybridization of chloroplast ribosomal ribonucleic acid to intact chloroplast DNA determined that there are two cistrons for 16S and 23S ribosomal ribonucleic acid. These two cistrons are located on six restriction fragments, all of which have buoyant densities greater than the intact molecule of chloroplast DNA.     

Molecular recognition in the minor groove of the DNA helix. Studies on the synthesis of oligonucleotides and polynucleotides containing 3-deaza-2''-deoxyadenosine. Interaction of the oligonucleotides with the restriction endonuclease EcoRV.

An improved procedure for the preparation of 3-deaza-2'-deoxyadenosine (d3CA) is described which is suitable for the synthesis of gram quantities of this analogue. Using phosphoramidite chemistry d3CA has been incorporated into the Eco RV restiction endonuclease recognition sequence (underlined) present in the self-complementary dodecamer d(GACGATATCGTC). The modified oligonucleotides have been thoroughly characterised by nucleoside composition analysis, circular dichroism and thermal melting studies. Studies with Eco RV show that incorporation of d3CA into either the central or outer dA-dT base-pair results in a substantial reduction in the rate of cleavage. The two-step conversion of d3CA to 3-deaza-2'-deoxyadenosine-5'-O-triphosphate (d3CATP) via the 5'-O-tosylate is also described. d3CATP is not a substrate in the poly[d(AT)].poly[d(AT)] primed polymerisation for either E. coli DNA polymerase I or Micrococcus luteus DNA polymerase. In a more detailed kinetic analysis d3CATP was shown to be a competitive inhibitor of E. coli DNA polymerase I with respect to dATP.     

Complexes of silver ion with natural and synthetic polynucleotides

A study of silver-ion binding by nucleic acids and synthetic ribo and deoxyribopolynucleotides, has been carried out by means of potentiometric titration, thermal transition, and difference spectra. It is clearly demonstrated that a strong complex between Ag⁺ and nitrogen atoms of bases is made reversibly. Binding constants and site numbers are determined for each type of polynucleotide. Base reactivity varies strongly with chain length, and a cooperative phenomenon is found in each case. Two successive complexes with DNA are seen in all the three techniques, and they have the same characteristics as complexes with respectively poly-dGC and poly dAT. In the first complex, Ag⁺ is linked to four bases, provided two of them are a G-C pair. Calculated and experimental values of site numbers agree very well for DNA of different G-C content. Thermal stabilization occurs simultaneously, and the increase of melting temperature corresponds to calculated changes of stacking energy between base pairs. In the second complex a new ordered structure insensitive to temperature is formed, with simultaneous release of protons. The stoichiometry can be related to base sequence. Complexing with silver increases the resistance of TMV RNA to both temperature and ribonuclease; a tentative explanation is given in the latter case.