scholarly journals The EcoRI restriction endonuclease with bacteriophage λ DNA. Equilibrium binding studies

1980 ◽  
Vol 191 (2) ◽  
pp. 593-604 ◽  
Author(s):  
S E Halford ◽  
N P Johnson
Keyword(s):  
Restriction Endonuclease ◽  
Dna Sequences ◽  
Reaction Rates ◽  
Recognition Site ◽  
Binding Studies ◽  
Dna Molecule ◽  
Recognition Sites ◽  
Equilibrium Binding ◽  
Ecori Restriction ◽  
Binding Curve

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.

scholarly journals The EcoRI restriction endonuclease with bacteriophage λ DNA. Kinetic studies

1980 ◽  
Vol 191 (2) ◽  
pp. 581-592 ◽  
Author(s):  
S E Halford ◽  
N P Johnson ◽  
J Grinsted
Keyword(s):  
Restriction Endonuclease ◽  
Protein Interactions ◽  
Dna Sequences ◽  
Individual Recognition ◽  
Kinetic Studies ◽  
Restriction Enzymes ◽  
Reaction Rates ◽  
Recognition Site ◽  
Experimental Conditions ◽  
Ecori Restriction

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.

scholarly journals The reactions of the EcoRi and other restriction endonucleases

1979 ◽  
Vol 179 (2) ◽  
pp. 353-365 ◽  
Author(s):  
S E Halford ◽  
N P Johnson ◽  
J Grinsted
Keyword(s):  
Restriction Endonuclease ◽  
Dna Sequences ◽  
Recognition Site ◽  
Single Strand ◽  
Restriction Endonucleases ◽  
Dna Duplex ◽  
Phosphodiester Bond ◽  
Linear Dna ◽  
Ecori Restriction ◽  
Dna Substrates

The reaction of the EcoRI restriction endonuclease was studied with both the plasmid pMB9 and DNA from bacteriophage lambda as the substrates. With both circular and linear DNA molecules, the only reaction catalysed by the EcoRI restriction endonuclease was the hydrolysis of the phosphodiester bond within one strand of the recognition site on the DNA duplex. The cleavage of both strands of the duplex was achieved only after two independent reactions, each involving a single-strand scission. The reactivity of the enzyme for single-strand scissions was the same for both the first and the second cleavage within its recognition site. No differences were observed between the mechanism of action on supercoiled and linear DNA substrates. Other restriction endonucleases were tested against plasmid pMB9. The HindIII restriction endonuclease cleaved DNA in the same manner as the EcoRI enzyme. However, in contrast with EcoRI, the Sa/I and the BamHI restriction endonucleases appeared to cleave both strands of the DNA duplex almost simultaneously. The function of symmetrical DNA sequences and the conformation of the DNA involved in these DNA–protein interactions are discussed in the light of these observations. The fact that the same reactions were observed on both supercoiled and linear DNA substrates implies that these interactions do not involve the unwinding of the duplex before catalysis.

scholarly journals The SalGI restriction endonuclease. Enzyme specificity

1982 ◽  
Vol 203 (1) ◽  
pp. 93-98 ◽  
Author(s):  
A Maxwell ◽  
S E Halford
Keyword(s):  
Restriction Endonuclease ◽  
Dna Sequences ◽  
Dna Cleavage ◽  
Competitive Inhibition ◽  
Recognition Site ◽  
Enzyme Specificity ◽  
Recognition Sequence ◽  
Kinetics Of ◽  
Endonuclease Enzyme

We have analysed the kinetics of DNA cleavage in the reaction between the SalGI restriction endonuclease and plasmid pMB9. This reaction is subject to competitive inhibition by DNA sequences outside the SalGI recognition site; we have determined the Km and Vmax. for the reaction of this enzyme at its recognition site and the KI for its interaction at other DNA sequences. We conclude that the specificity of DNA cleavage by the enzyme is only partly determined by the discrimination it shows for binding at its recognition sequence compared with binding to other DNA sequences.

Hopping, jumping and looping by restriction enzymes

2001 ◽  
Vol 29 (4) ◽  
pp. 363-373 ◽  
Author(s):  
S. E. Halford
Keyword(s):  
Dna Binding ◽  
Dna Sequences ◽  
Dna Cleavage ◽  
Restriction Enzymes ◽  
Test System ◽  
Recognition Site ◽  
Dna Looping ◽  
Type Ii ◽  
Common View ◽  
Recognition Sites

Type II restriction endonucleases recognize specific DNA sequences and cleave both strands of the DNA at fixed locations at or near their recognition sites. Many of these enzymes are dimeric proteins that recognize, in symmetrical fashion, palindromic DNA sequences. They generally catalyse independent reactions at each recognition site on the DNA, although in some cases they act processively; cutting the DNA first at one site, then translocating along the DNA to another site and cutting that before leaving the DNA. The way in which the degree of processivity varies with the length of DNA between the sites can reveal the mechanism of translocation. In contrast with the common view that proteins move along DNA by ‘sliding’, the principal mode of transfer of the EcoRV endonuclease is by ‘hopping’ and ‘jumping’, i.e. the dissociation of the protein from one site followed by its re-association with another site in the same DNA molecule, either close to or distant from the original site. Other type II restriction enzymes require two copies of their recognition sites for their DNA cleavage reactions. Many of these enzymes, such as SfiI, are tetramers with two DNA-binding surfaces. SfiI has no activity when bound to just one recognition site, and instead both DNA-binding surfaces have to be filled before it becomes active. Although the two sites can be on separate DNA molecules, SfiI acts optimally with two sites on the same DNA, where it traps the DNA between the sites in a loop. SfiI thus constitutes a test system for the analysis of DNA looping.

Electron microscopic assays of restriction endonuclease cutting, exonuclease digestion, and hybridization

1984 ◽  
Vol 42 ◽  
pp. 686-687
Author(s):  
Mark Hannibal ◽  
Jacob Varkey ◽  
Michael Beer
Keyword(s):  
Electron Microscope ◽  
Low Temperature ◽  
Restriction Endonuclease ◽  
Double Helix ◽  
Test Material ◽  
Electron Microscopic ◽  
Exonuclease Iii ◽  
Dna Molecule ◽  
Linear Molecules ◽  
Exonuclease Digestion

Workman and Langmore have recently proposed a procedure for isolating particular chromatin fragments. The method requires restriction endonuclease cutting of the chromatin and a probe, their digestion with two exonucleases which leave complimentary single strand termini and low temperature hybridization of these. We here report simple electron microscopic monitoring of the four reactions involved.Our test material was ϕX-174 RF DNA which is cut once by restriction endonuclease Xho I. The conversion of circles to linear molecules was followed in Kleinschmidt spreads. Plate I shows a circular and a linear DNA molecule. The rate of cutting is shown in Figure 1.After completion of the endonuclease cutting, one portion of the DNA was treated with exonuclease III, an enzyme known to digest the 3' terminals of double helical DNA. Aliquots when examined in the electron microscope reveal a decreasing length of double helix and increasing bushes at the ends.

scholarly journals Kinetic mechanism of Na+-coupled aspartate transport catalyzed by GltTk

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Gianluca Trinco ◽  
Valentina Arkhipova ◽  
Alisa A. Garaeva ◽  
Cedric A. J. Hutter ◽  
Markus A. Seeger ◽  
...  
Keyword(s):  
Kinetic Mechanism ◽  
Protein Quantification ◽  
Detergent Solution ◽  
Sodium Ions ◽  
Binding Studies ◽  
Equilibrium Binding ◽  
Uptake Rates ◽  
Bona Fide ◽  
Inside Out

AbstractIt is well-established that the secondary active transporters GltTk and GltPh catalyze coupled uptake of aspartate and three sodium ions, but insight in the kinetic mechanism of transport is fragmentary. Here, we systematically measured aspartate uptake rates in proteoliposomes containing purified GltTk, and derived the rate equation for a mechanism in which two sodium ions bind before and another after aspartate. Re-analysis of existing data on GltPh using this equation allowed for determination of the turnover number (0.14 s−1), without the need for error-prone protein quantification. To overcome the complication that purified transporters may adopt right-side-out or inside-out membrane orientations upon reconstitution, thereby confounding the kinetic analysis, we employed a rapid method using synthetic nanobodies to inactivate one population. Oppositely oriented GltTk proteins showed the same transport kinetics, consistent with the use of an identical gating element on both sides of the membrane. Our work underlines the value of bona fide transport experiments to reveal mechanistic features of Na+-aspartate symport that cannot be observed in detergent solution. Combined with previous pre-equilibrium binding studies, a full kinetic mechanism of structurally characterized aspartate transporters of the SLC1A family is now emerging.

Molecular analyses of conjugative, gentamicin-resistance plasmids from staphylococcal clinical isolates

1988 ◽  
Vol 34 (9) ◽  
pp. 1050-1057 ◽  
Author(s):  
Ioannis Zorbas ◽  
Robert T. Hall ◽  
Sue L. Hall ◽  
William G. Barnes ◽  
Marvin Rogolsky
Keyword(s):  
Restriction Endonuclease ◽  
Clinical Isolates ◽  
Genetic Determinants ◽  
Conjugative Plasmids ◽  
Molecular Weights ◽  
Geographical Regions ◽  
Gentamicin Resistance ◽  
Ecori Restriction ◽  
High Degree ◽  
Endonuclease Digestion

Gentamicin-resistant Staphylococcus aureus and Staphylococcus epidermidis strains which were isolated from infants with staphylococcal bacteremia were analyzed for the presence of self-transmissible gentamicin-resistance (Gmr) plasmids. Conjugative GMr plasmids of approximately 43.8–63 kilobases (kb) were found in all S. aureus strains. Inter- and intra-species transfer of Gmr plasmids by conjugation was observed from S. aureus to S. aureus and to S. epidermidis recipient strains. However, neither inter- nor intra-species transfer of gentamicin resistance by conjugation was observed with nine out of nine S. epidermidis donor strains which were mated with either S. epidermidis or S. aureus recipient strains. These conjugative Gmr plasmids were unable to comobilize a smaller (15-kb) plasmid present in all but two S. aureus clinical isolates. Many of the conjugative Gmr plasmids also carried genetic determinants for kanamycin, tobramycin, neomycin, and ethidium bromide resistance, and for β-lactamase synthesis. EcoRI restriction endonuclease digests of the S. aureus Gmr conjugative plasmids revealed three different digestion patterns. Four EcoRI restriction endonuclease digestion fragments of 15, 11.4, 6.3, and 4.6 kb in size were common to all plasmids. These plasmids and conjugative Gmr staphylococcal plasmids from other geographical regions shared restriction digestion fragments of similar molecular weights. DNA hybridization with biotinylated S. aureus plasmid pIZ7814 DNA revealed a high degree of homology among these plasmids. A 50.9-kb plasmid from one of the nonconjugative S. epidermidis clinical isolates showed homology with the probe DNA but lacked a portion of a 6.3-kb fragment which was present in all conjugative plasmids and believed to carry much genetic information for conjugation.

scholarly journals Identification of Simian virus 40 promoter DNA sequences capable of conferring restriction endonuclease hypersensitivity.

1996 ◽  
Vol 70 (6) ◽  
pp. 3416-3422 ◽  
Author(s):  
R Hermansen ◽  
M A Sierra ◽  
J Johnson ◽  
M Friez ◽  
B Milavetz
Keyword(s):  
Restriction Endonuclease ◽  
Dna Sequences ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Promoter Dna
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