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Single turnovers of the EcoRI restriction endonuclease.

ABSTRACT: 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.

SUBMITTER: Halford SE

PROVIDER: S-EPMC1154373 | biostudies-other | 1983 May

REPOSITORIES: biostudies-other

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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

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.

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

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Diffusion of the restriction nuclease EcoRI along DNA.

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Effects of 2'-O-methyl nucleotide substitution on EcoRI endonuclease cleavage activities.

Project description:To investigate the effect of sugar pucker conformation on DNA-protein interactions, we used 2'-O-methyl nucleotide (2'-OMeN) to modify the EcoRI recognition sequence -TGAATTCT-, and monitored the enzymatic cleavage process using FRET method. The 2'-O-methyl nucleotide has a C3'-endo sugar pucker conformation different from the C2'-endo sugar pucker conformation of native DNA nucleotides. The initial reaction velocities were measured and the kinetic parameters, Km and Vmax were derived using Michaelis-Menten equation. Experimental results showed that 2'-OMeN substitutions for the EcoRI recognition sequence decreased the cleavage efficiency for A2, A3 and T4 substitutions significantly, and 2'-OMeN substitution for T5 residue inhibited the enzymatic activity completely. In contrast, substitutions for G1 and C6 could maintain the original activity. 2'-fluoro nucleic acid (2'-FNA) and locked nucleic acid (LNA) having similar C3'-endo sugar pucker conformation also demonstrated similar enzymatic results. This position-dependent enzymatic cleavage property might be attributed to the phosphate backbone distortion caused by the switch from C2'-endo to C3'-endo sugar pucker conformation, and was interpreted on the basis of the DNA-EcoRI structure. These 2'-modified nucleotides could behave as a regulatory element to modulate the enzymatic activity in vitro, and this property will have potential applications in genetic engineering and biomedicine.

| S-EPMC3806743 | biostudies-literature

Single molecule detection of DNA looping by NgoMIV restriction endonuclease.

Project description:Single molecule fluorescence resonance energy transfer (FRET) and fluorescence correlation spectroscopy were used to investigate DNA looping by NgoMIV restriction endonuclease. Using a linear double-stranded DNA (dsDNA) molecule labeled with a fluorescence donor molecule, Cy3, and fluorescence acceptor molecule, Cy5, and by varying the concentration of NgoMIV endonuclease from 0 to 3 x 10(-6) M, it was possible to detect and determine diffusion properties of looped DNA/protein complexes. FRET efficiency distributions revealed a subpopulation of complexes with an energy transfer efficiency of 30%, which appeared upon addition of enzyme in the picomolar to nanomolar concentration range (using 10(-11) M dsDNA). The concentration dependence, fluorescence burst size analysis, and fluorescence correlation analysis were all consistent with this subpopulation arising from a sequence specific interaction between an individual enzyme and a DNA molecule. A 30% FRET efficiency corresponds to a distance of approximately 65 A, which correlates well with the distance between the ends of the dsDNA molecule when bound to NgoMIV according to the crystal structure of this complex. Formation of the looped complexes was also evident in measurements of the diffusion times of freely diffusing DNA molecules with and without NgoMIV. At very high protein concentrations compared to the DNA concentration, FRET and fluorescence correlation spectroscopy results revealed the formation of larger DNA/protein complexes.

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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

Single molecular investigation of DNA looping and aggregation by restriction endonuclease BspMI.

Project description:DNA looping and aggregation induced by restriction endonuclease BspMI are studied by atomic force microscopy (AFM) and magnetic tweezers (MT). With Ca(2+) substituted for the normal enzyme cofactor Mg(2+) and enzyme concentration below the critical concentration of 6 units/mL, AFM images of DNA-BspMI complex show that the number of binding and looping events increases with enzyme concentration. At the critical concentration 6 of units/mL, all the BspMI binding sites are saturated. It is worth noting that nonspecific BspMI binding to DNA at saturation concentration represents more than 8% of the total BspMI-DNA complexes directly observed in AFM images. Furthermore, we used MT to prove that additional loops can form when enzyme concentration is higher than its saturation valueand the complex is incubated for a long time (>2 hrs). We ascribe this phenomenon to the aggregation of enzymes. The force spectroscopy of the BspMI-DNA complex shows that the pulling force required to open the loop of the complex at less than saturation concentration has a peak at about 3 pN, which is lower than the force required to open additional loops due to enzyme aggregation at higher than saturation concentration (>6 pN).

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Identification of a single HNH active site in type IIS restriction endonuclease Eco31I.

Project description:Type IIS restriction endonuclease Eco31I is a "short-distance cutter", which cleaves DNA strands close to its recognition sequence, 5'-GGTCTC(1/5). Previously, it has been proposed that related endonucleases recognizing a common sequence core GTCTC possess two active sites for cleavage of both strands in the DNA substrate. Here, we present bioinformatic identification and experimental evidence for a single nuclease active site. We identified a short region of homology between Eco31I and HNH nucleases, constructed a three-dimensional model of the putative catalytic domain and validated our predictions by random and site-specific mutagenesis. The restriction mechanism of Eco31I is suggested by analogy to the mechanisms of phage T4 endonuclease VII and homing endonuclease I-PpoI. We propose that residues D311 and N334 coordinate the cofactor. H312 acts as a general base-activating water molecule for the nucleophilic attack. K337 together with R340 and D345 are located in close proximity to the active center and are essential for correct folding of catalytic motif, while D345 together with R264 and D273 could be directly involved in DNA binding. We also predict that the Eco31I catalytic domain contains a putative Zn-binding site, which is essential for its structural integrity. Our results suggest that the HNH-like active site is involved in the cleavage of both strands in the DNA substrate. On the other hand, analysis of site-specific mutants in the region, previously suggested to harbor the second active site, revealed its irrelevance to the nuclease activity. Thus, our data argue against the earlier prediction and indicate the presence of a single conserved active site in type IIS restriction endonucleases that recognize common sequence core GTCTC.

| S-EPMC2754561 | biostudies-literature

Negative regulation of the EcoRI restriction enzyme gene is associated with intragenic reverse promoters.

Project description:Type II restriction-modification systems are expected to possess mechanisms for tight regulation of their expression to suppress the potential of lethal attack on their host bacteria when they establish and maintain themselves within them. Although the EcoRI restriction enzyme has been well characterized, regulation of its expression is still poorly understood. In this study, mutational analysis with lacZ gene fusion and primer extension assay identified a promoter for the transcription of the ecoRIR gene. Further analyses revealed that an intragenic region containing two overlapping reverse promoter-like elements acted as a negative regulator for ecoRIR gene expression. The activity of these putative reverse promoters was verified by transcriptional gene fusion, primer extension and in vitro transcription. Mutations in these reverse promoters resulted in increased gene expression in both translational and transcriptional gene fusions. An RNase protection assay revealed that the transcript level of the wild type relative to that of the reverse promoter mutant at the downstream regions was much lower than the level at the upstream regions. This suggests that these reverse promoter-like elements affect their downstream transcript level. The possible mechanisms of this kind of negative regulation, in addition to their possible biological roles, are discussed.

| S-EPMC2045195 | biostudies-literature

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