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.

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.

Efficient repair of HO-induced chromosomal breaks in Saccharomyces cerevisiae by recombination between flanking homologous sequences

1988 ◽  
Vol 8 (9) ◽  
pp. 3918-3928 ◽  
Author(s):  
N Rudin ◽  
J E Haber
Keyword(s):  
Strand Break ◽  
Recognition Site ◽  
Spontaneous Recombination ◽  
Chromosomal Breaks ◽  
A Site ◽  
Flanking Regions ◽  
Kinetics Of ◽  
Endonuclease Enzyme ◽  
Ho Gene

Novel recombinational repair of a site-specific double-strand break (DSB) in a yeast chromosome was investigated. When the recognition site for the HO endonuclease enzyme is embedded in nonyeast sequences and placed between two regions of homology, expression of HO endonuclease stimulates recombination between the homologous flanking regions to yield a deletion, the apparent product of an intrachromosomal exchange between direct repeats. This deletion-repair event is very efficient, thus preventing essentially all the potential lethality due to the persistence of a DSB. Interestingly, unlike previous studies involving spontaneous recombination between chromosomal repeats, the recombination events stimulated by HO-induced DSBs are accompanied by loss of the sequences separating the homologous regions greater than 99.5% of the time. Repair is dependent on the RAD52 gene. The deletion-repair event provides an in vivo assay for the sensitivity of any particular recognition site to HO cleavage. By taking advantage of a galactose-inducible HO gene, it has been possible to follow the kinetics of this event at the DNA level and to search for intermediates in this reaction. Deletion-repair requires approximately 45 min and is inhibited when cycloheximide is added after HO endonuclease cleavage.

scholarly journals The SalGI restriction endonuclease. Mechanism of DNA cleavage

1982 ◽  
Vol 203 (1) ◽  
pp. 85-92 ◽  
Author(s):  
A Maxwell ◽  
S E Halford
Keyword(s):  
Restriction Endonuclease ◽  
Linear Form ◽  
Dna Cleavage ◽  
Tertiary Structure ◽  
Recognition Site ◽  
Optimal Conditions ◽  
Supercoiled Dna ◽  
Linear Forms ◽  
Open Circle ◽  
Concerted Reaction

The cleavage of supercoiled DNA of plasmid pMB9 by restriction endonuclease SalGI has been studied. Under the optimal conditions for this reaction, the only product is the linear form of the DNA, in which both strands of the duplex have been cleaved at the SalGI recognition site. DNA molecules cleaved in one strand at this site were found to be poor substrates for the SalGI enzyme. Thus, both strands of the DNA appear to be cleaved in a concerted reaction. However, under other conditions, the enzyme cleaves either one or both strands of the DNA; the supercoiled substrate is then converted to either open-circle or linear forms, the two being produced simultaneously rather than consecutively. We propose a mechanism for the SalGI restriction endonuclease which accounts for the reactions of this enzyme under both optimal and other conditions. These reactions were unaffected by the tertiary structure of the DNA.

scholarly journals Efficient repair of HO-induced chromosomal breaks in Saccharomyces cerevisiae by recombination between flanking homologous sequences.

1988 ◽  
Vol 8 (9) ◽  
pp. 3918-3928 ◽  
Author(s):  
N Rudin ◽  
J E Haber
Keyword(s):  
Strand Break ◽  
Recognition Site ◽  
Spontaneous Recombination ◽  
Chromosomal Breaks ◽  
A Site ◽  
Flanking Regions ◽  
Kinetics Of ◽  
Endonuclease Enzyme ◽  
Ho Gene

Novel recombinational repair of a site-specific double-strand break (DSB) in a yeast chromosome was investigated. When the recognition site for the HO endonuclease enzyme is embedded in nonyeast sequences and placed between two regions of homology, expression of HO endonuclease stimulates recombination between the homologous flanking regions to yield a deletion, the apparent product of an intrachromosomal exchange between direct repeats. This deletion-repair event is very efficient, thus preventing essentially all the potential lethality due to the persistence of a DSB. Interestingly, unlike previous studies involving spontaneous recombination between chromosomal repeats, the recombination events stimulated by HO-induced DSBs are accompanied by loss of the sequences separating the homologous regions greater than 99.5% of the time. Repair is dependent on the RAD52 gene. The deletion-repair event provides an in vivo assay for the sensitivity of any particular recognition site to HO cleavage. By taking advantage of a galactose-inducible HO gene, it has been possible to follow the kinetics of this event at the DNA level and to search for intermediates in this reaction. Deletion-repair requires approximately 45 min and is inhibited when cycloheximide is added after HO endonuclease cleavage.

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

1990 ◽  
Vol 265 (25) ◽  
pp. 15300-15307 ◽  
Author(s):  
P. Hensley ◽  
G. Nardone ◽  
J.G. Chirikjian ◽  
M.E. Wastney
Keyword(s):  
Restriction Endonuclease ◽  
Dna Cleavage ◽  
Time Resolved ◽  
Kinetics Of

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.

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