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

ABSTRACT: 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.

SUBMITTER: Halford SE

PROVIDER: S-EPMC1162251 | biostudies-other | 1980 Nov

REPOSITORIES: biostudies-other

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

Project description: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.

| S-EPMC1162250 | biostudies-other

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

Project description: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.

| S-EPMC1163435 | biostudies-other

Single turnovers of the EcoRI restriction endonuclease.

Project description:Single turnovers of the EcoRI restriction endonuclease, cleaving its recognition site on the covalently closed form of plasmid pMB9, were examined. Two methods were used to monitor the progress of the reactions: one involved quenching the reaction at various times followed by the electrophoretic separation of the products cleaved in one and in both strands of the duplex; the other employed a stopped-flow fluorimeter to measure the amount of ethidium bromide bound to the DNA as it changes when the DNA, cleaved in at least one strand, dissociates from the enzyme. Two procedures were used to initiate the reactions. For some, one solution containing the enzyme was mixed with a second containing both DNA and MgCl2: in these reactions, the fluorescence changed at the same rate as the cleavage of the first strand of the duplex. Other reactions were started by the addition of MgCl2 to a pre-equilibrium of enzyme and DNA: here, both strands of the DNA were cleaved faster than before, with the fluorescence signal now occurring at the same time as the cleavage of the second strand. The different kinetics from the two assays and the two mixing procedures are consistent with the rates of these reactions being controlled by protein conformational changes. These may affect either one subunit alone within the dimeric EcoRI enzyme, allowing the enzyme to cleave only one strand of the DNA in each turnover. Alternatively, both subunits of the dimer may change, so that the enzyme then cleaves both strands during the life-time of one enzyme-DNA complex.

| S-EPMC1154373 | biostudies-other

Structural and thermodynamic basis for enhanced DNA binding by a promiscuous mutant EcoRI endonuclease.

Project description:Promiscuous mutant EcoRI endonucleases bind to the canonical site GAATTC more tightly than does the wild-type endonuclease, yet cleave variant (EcoRI(*)) sites more rapidly than does wild-type. The crystal structure of the A138T promiscuous mutant homodimer in complex with a GAATTC site is nearly identical to that of the wild-type complex, except that the Thr138 side chains make packing interactions with bases in the 5'-flanking regions outside the recognition hexanucleotide while excluding two bound water molecules seen in the wild-type complex. Molecular dynamics simulations confirm exclusion of these waters. The structure and simulations suggest possible reasons why binding of the A138T protein to the GAATTC site has DeltaS degrees more favorable and DeltaH degrees less favorable than for wild-type endonuclease binding. The interactions of Thr138 with flanking bases may permit A138T, unlike wild-type enzyme, to form complexes with EcoRI(*) sites that structurally resemble the specific wild-type complex with GAATTC.

| S-EPMC3366113 | biostudies-literature

Diffusion of the restriction nuclease EcoRI along DNA.

Project description:Many specific sequence DNA binding proteins locate their target sequence by first binding to DNA nonspecifically, then by linearly diffusing or hopping along DNA until either the protein dissociates from the DNA or it finds the recognition sequence. We have devised a method for measuring one-dimensional diffusion along DNA based on the ratio of the dissociation rate of protein from DNA fragments containing one specific binding site to the dissociation rate from DNA fragments containing two specific binding sites. Our extensive measurements of dissociation rates and specific-nonspecific relative binding constants of the restriction nuclease EcoRI enable us to determine the diffusion rate of nonspecifically bound protein along the DNA. By varying the distance between the two binding sites, we confirm a linear diffusion mechanism. The sliding rate is relatively insensitive to salt concentration and osmotic pressure, indicating that the protein moves smoothly along the DNA probably following the helical phosphate-sugar backbone of DNA. We calculate a diffusion coefficient for EcoRI of 3 x 10(4) bp(2) s(-)(1) EcoRI is able to diffuse approximately 150 bp, on average, along the DNA in 1 s. This diffusion rate is about 2000-fold slower than the diffusion of free protein in solution. A factor of 40-50 can be accounted for by rotational friction resulting from following the helical path of the DNA backbone. Two possibilities could account for the remaining activation energy: salt bridges between the DNA and the protein are transiently broken, or the water structure at the protein-DNA interface is disrupted as the two surfaces move past each other.

| S-EPMC2813319 | biostudies-literature

Domain structure and DNA binding regions of beta protein from bacteriophage lambda.

Project description:beta protein from bacteriophage lambda promotes a single-strand annealing reaction that is central to Red-mediated recombination at double-strand DNA breaks and chromosomal ends. beta protein binds most tightly to an intermediate of annealing formed by the sequential addition of two complementary oligonucleotides. Here we have characterized the domain structure of beta protein in the presence and absence of DNA using limited proteolysis. Residues 1-130 form an N-terminal "core" domain that is resistant to proteases in the absence of DNA, residues 131-177 form a central region with enhanced resistance to proteases upon DNA complex formation, and the C-terminal residues 178-261 of beta protein are sensitive to proteases in both the presence and absence of DNA. We probed the DNA binding regions of beta protein further using biotinylation of lysine residues and mass spectrometry. Several lysine residues within the first 177 residues of beta protein are protected from biotinylation in the DNA complex, whereas none of the lysine residues in the C-terminal portion are protected. The results lead to a model for the domain structure and DNA binding of beta protein in which a stable N-terminal core and a more flexible central domain come together to bind DNA, whereas a C-terminal tail remains disordered. A fragment consisting of residues 1-177 of beta protein maintains normal binding to sequentially added complementary oligonucleotides and has significantly enhanced binding to single-strand DNA.

| S-EPMC1950251 | biostudies-literature

Solution parameters modulating DNA binding specificity of the restriction endonuclease EcoRV.

Project description:The DNA binding stringency of restriction endonucleases is crucial for their proper function. The X-ray structures of the specific and non-cognate complexes of the restriction nuclease EcoRV are considerably different suggesting significant differences in the hydration and binding free energies. Nonetheless, the majority of studies performed at pH 7.5, optimal for enzymatic activity, have found a < 10-fold difference between EcoRV binding constants to the specific and nonspecific sequences in the absence of divalent ions. We used a recently developed self-cleavage assay to measure EcoRV-DNA competitive binding and to evaluate the influence of water activity, pH and salt concentration on the binding stringency of the enzyme in the absence of divalent ions. We find the enzyme can readily distinguish specific and nonspecific sequences. The relative specific-nonspecific binding constant increases strongly with increasing neutral solute concentration and with decreasing pH. The difference in number of associated waters between specific and nonspecific DNA-EcoRV complexes is consistent with the differences in the crystal structures. Despite the large pH dependence of the sequence specificity, the osmotic pressure dependence indicates little change in structure with pH. The large osmotic pressure dependence means that measurement of protein-DNA specificity in dilute solution cannot be directly applied to binding in the crowded environment of the cell. In addition to divalent ions, water activity and pH are key parameters that strongly modulate binding specificity of EcoRV.

| S-EPMC3156093 | biostudies-literature

The DNA maturation domain of gpA, the DNA packaging motor protein of bacteriophage lambda, contains an ATPase site associated with endonuclease activity.

Project description:Terminase enzymes are common to double-stranded DNA (dsDNA) viruses and are responsible for packaging viral DNA into the confines of an empty capsid shell. In bacteriophage lambda the catalytic terminase subunit is gpA, which is responsible for maturation of the genome end prior to packaging and subsequent translocation of the matured DNA into the capsid. DNA packaging requires an ATPase catalytic site situated in the N terminus of the protein. A second ATPase catalytic site associated with the DNA maturation activities of the protein has been proposed; however, direct demonstration of this putative second site is lacking. Here we describe biochemical studies that define protease-resistant peptides of gpA and expression of these putative domains in Escherichia coli. Biochemical characterization of gpA-DeltaN179, a construct in which the N-terminal 179 residues of gpA have been deleted, indicates that this protein encompasses the DNA maturation domain of gpA. The construct is folded, soluble and possesses an ATP-dependent nuclease activity. Moreover, the construct binds and hydrolyzes ATP despite the fact that the DNA packaging ATPase site in the N terminus of gpA has been deleted. Mutation of lysine 497, which alters the conserved lysine in a predicted Walker A "P-loop" sequence, does not affect ATP binding but severely impairs ATP hydrolysis. Further, this mutation abrogates the ATP-dependent nuclease activity of the protein. These studies provide direct evidence for the elusive nucleotide-binding site in gpA that is directly associated with the DNA maturation activity of the protein. The implications of these results with respect to the two roles of the terminase holoenzyme, DNA maturation and DNA packaging, are discussed.

| S-EPMC2082050 | biostudies-literature

Rational engineering of type II restriction endonuclease DNA binding and cleavage specificity.

Project description:The type II restriction endonucleases are indispensible tools for molecular biology. Although enzymes recognizing nearly 300 unique sequences are known, the ability to engineer enzymes to recognize any sequence of choice would be valuable. However, previous attempts to engineer new recognition specificity have met limited success. Here we report the rational engineering of multiple new type II specificities. We recently identified a family of MmeI-like type II endonucleases that have highly similar protein sequences but different recognition specificity. We identified the amino-acid positions within these enzymes that determine position specific DNA base recognition at three positions within their recognition sequences through correlations between their aligned amino-acid residues and aligned recognition sequences. We then altered the amino acids at the identified positions to those correlated with recognition of a desired new base to create enzymes that recognize and cut at predictable new DNA sequences. The enzymes so altered have similar levels of endonuclease activity compared to the wild-type enzymes. Using simple and predictable mutagenesis in this family it is now possible to create hundreds of unique new type II restriction endonuclease specificities. The findings suggest a simple mechanism for the evolution of new DNA specificity in Nature.

| S-EPMC2731914 | biostudies-literature

Analyzing the forces binding a restriction endonuclease to DNA using a synthetic nanopore.

Project description:Restriction endonucleases are used prevalently in recombinant DNA technology because they bind so stably to a specific target sequence and, in the presence of cofactors, cleave double-helical DNA specifically at a target sequence at a high rate. Using synthetic nanopores along with molecular dynamics (MD), we have analyzed with atomic resolution how a prototypical restriction endonuclease, EcoRI, binds to the DNA target sequence--GAATTC--in the absence of a Mg(2+) ion cofactor. We have previously shown that there is a voltage threshold for permeation of DNA bound to restriction enzymes through a nanopore that is associated with a nanonewton force required to rupture the complex. By introducing mutations in the DNA, we now show that this threshold depends on the recognition sequence and scales linearly with the dissociation energy, independent of the pore geometry. To predict the effect of mutation in a base pair on the free energy of dissociation, MD is used to qualitatively rank the stability of bonds in the EcoRI-DNA complex. We find that the second base in the target sequence exhibits the strongest binding to the protein, followed by the third and first bases, with even the flanking sequence affecting the binding, corroborating our experiments.

| S-EPMC2709577 | biostudies-literature

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