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.

Analysis of DNA looping interactions by type II restriction enzymes that require two copies of their recognition sites 1 1Edited by J. Karn

2001 ◽  
Vol 311 (3) ◽  
pp. 515-527 ◽  
Author(s):  
Susan E. Milsom ◽  
Stephen E. Halford ◽  
Michelle L. Embleton ◽  
Mark D. Szczelkun
Keyword(s):  
Restriction Enzymes ◽  
Dna Looping ◽  
Type Ii ◽  
Recognition Sites

scholarly journals Structure of the space of taboo-free sequences

2020 ◽  
Vol 81 (4-5) ◽  
pp. 1029-1057
Author(s):  
Cassius Manuel ◽  
Arndt von Haeseler
Keyword(s):  
Hamming Distance ◽  
Restriction Enzymes ◽  
Finite Alphabet ◽  
Sequence Evolution ◽  
Type Ii ◽  
Hamming Graph ◽  
Recognition Sites ◽  
Restriction Sites ◽  
Vertex Set ◽  
Hamming Graphs

Abstract Models of sequence evolution typically assume that all sequences are possible. However, restriction enzymes that cut DNA at specific recognition sites provide an example where carrying a recognition site can be lethal. Motivated by this observation, we studied the set of strings over a finite alphabet with taboos, that is, with prohibited substrings. The taboo-set is referred to as $$\mathbb {T}$$ T and any allowed string as a taboo-free string. We consider the so-called Hamming graph $$\varGamma _n(\mathbb {T})$$ Γ n ( T ) , whose vertices are taboo-free strings of length n and whose edges connect two taboo-free strings if their Hamming distance equals one. Any (random) walk on this graph describes the evolution of a DNA sequence that avoids taboos. We describe the construction of the vertex set of $$\varGamma _n(\mathbb {T})$$ Γ n ( T ) . Then we state conditions under which $$\varGamma _n(\mathbb {T})$$ Γ n ( T ) and its suffix subgraphs are connected. Moreover, we provide an algorithm that determines if all these graphs are connected for an arbitrary $$\mathbb {T}$$ T . As an application of the algorithm, we show that about $$87\%$$ 87 % of bacteria listed in REBASE have a taboo-set that induces connected taboo-free Hamming graphs, because they have less than four type II restriction enzymes. On the other hand, four properly chosen taboos are enough to disconnect one suffix subgraph, and consequently connectivity of taboo-free Hamming graphs could change depending on the composition of restriction sites.

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.

Transcriptional regulator Leu3 of Saccharomyces cerevisiae: separation of activator and repressor functions

1993 ◽  
Vol 13 (9) ◽  
pp. 5702-5709
Author(s):  
J Y Sze ◽  
E Remboutsika ◽  
G B Kohlhaw
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Binding ◽  
Rna Polymerase Ii ◽  
Dna Sequences ◽  
Transcriptional Activation ◽  
In Vitro Transcription ◽  
Test System ◽  
Metabolic Intermediate ◽  
Mutant Forms

The Leu3 protein of Saccharomyces cerevisiae binds to specific DNA sequences present in the 5' noncoding region of at least five RNA polymerase II-transcribed genes. Leu3 functions as a transcriptional activator only when the metabolic intermediate alpha-isopropylmalate is also present. In the absence of alpha-isopropylmalate, Leu3 causes transcription to be repressed below basal levels. We show here that different portions of the Leu3 protein are responsible for activation and repression. Fusion of the 30 C-terminal residues of Leu3 to the DNA-binding domain of the Gal4 protein created a strong cross-species activator, demonstrating that the short C-terminal region is not only required but also sufficient for transcriptional activation. Using a recently developed Leu3-responsive in vitro transcription assay as a test system for repression (J. Sze, M. Woontner, J. Jaehning, and G. B. Kohlhaw, Science 258:1143-1145, 1992), we show that mutant forms of the Leu3 protein that lack the activation domain still function as repressors. The shortest repressor thus identified had only about 15% of the mass of the full-length Leu3 protein and was centered on the DNA-binding region of Leu3. Implications of this finding for the mechanism of repression are discussed.

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 Transcriptional regulator Leu3 of Saccharomyces cerevisiae: separation of activator and repressor functions.

1993 ◽  
Vol 13 (9) ◽  
pp. 5702-5709 ◽  
Author(s):  
J Y Sze ◽  
E Remboutsika ◽  
G B Kohlhaw
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Binding ◽  
Rna Polymerase Ii ◽  
Dna Sequences ◽  
Transcriptional Activation ◽  
In Vitro Transcription ◽  
Test System ◽  
Metabolic Intermediate ◽  
Mutant Forms

The Leu3 protein of Saccharomyces cerevisiae binds to specific DNA sequences present in the 5' noncoding region of at least five RNA polymerase II-transcribed genes. Leu3 functions as a transcriptional activator only when the metabolic intermediate alpha-isopropylmalate is also present. In the absence of alpha-isopropylmalate, Leu3 causes transcription to be repressed below basal levels. We show here that different portions of the Leu3 protein are responsible for activation and repression. Fusion of the 30 C-terminal residues of Leu3 to the DNA-binding domain of the Gal4 protein created a strong cross-species activator, demonstrating that the short C-terminal region is not only required but also sufficient for transcriptional activation. Using a recently developed Leu3-responsive in vitro transcription assay as a test system for repression (J. Sze, M. Woontner, J. Jaehning, and G. B. Kohlhaw, Science 258:1143-1145, 1992), we show that mutant forms of the Leu3 protein that lack the activation domain still function as repressors. The shortest repressor thus identified had only about 15% of the mass of the full-length Leu3 protein and was centered on the DNA-binding region of Leu3. Implications of this finding for the mechanism of repression are discussed.

scholarly journals Functional Analyses of the EBNA1 Origin DNA Binding Protein of Epstein-Barr Virus

2000 ◽  
Vol 74 (11) ◽  
pp. 4939-4948 ◽  
Author(s):  
Derek F. J. Ceccarelli ◽  
Lori Frappier
Keyword(s):  
Dna Binding ◽  
Dna Sequences ◽  
Epstein Barr Virus ◽  
Dna Looping ◽  
Binding Ability ◽  
Barr Virus ◽  
Latently Infected ◽  
Infected Cells ◽  
Epstein Barr ◽  
Functional Analyses

ABSTRACT The EBNA1 protein of Epstein-Barr virus (EBV) governs the replication and segregation of the viral episomes in latently infected cells and transactivates the expression of other EBV latency proteins through direct interactions with DNA sequences in the EBV latent origin of replication, oriP. To better understand how EBNA1 controls these processes, we have assessed the contribution of various EBNA1 sequences to its replication, segregation, and transactivation functions. Here we show that EBNA1 residues 325 to 376 are responsible for the transactivation activity of EBNA1. This region coincides with the DNA looping domain previously shown to mediate interactions at a distance between DNA-bound EBNA1 molecules. The same residues mediate DNA segregation but have no apparent role in DNA replication, indicating that the replication and transcription activation activities of EBNA1 are distinct. The acidic C-terminal tail of EBNA1 was not found to contribute to replication, transactivation, or segregation. We have also investigated the functional significance of two structural motifs within the DNA binding and dimerization domains of EBNA1, the proline loop and the WF motif. Although the amino acids in these motifs do not directly contact the DNA, both of these motifs were found to contribute to EBNA1 functions by increasing the DNA-binding ability of EBNA1. Mechanisms by which DNA binding is stimulated by these motifs are discussed.

Restriction of single-stranded M13 DNA using synthetic oligonucleotides: the structural requirement of restriction enzymes

1987 ◽  
Vol 65 (1) ◽  
pp. 50-55
Author(s):  
Guo-Rong Qi ◽  
Pierre Wong ◽  
Robert Cedergren
Keyword(s):  
Dna Cleavage ◽  
Restriction Enzymes ◽  
Recognition Site ◽  
Duplex Dna ◽  
Structural Requirement ◽  
Single Stranded Dna ◽  
Dna Manipulation ◽  
Synthetic Oligonucleotides

A targeted ss (single stranded) DNA cleavage technique is reported which involves the use of synthetic oligomers complementary to the ss M13 DNA polylinker. BamHI, SmaI, and KpnI restriction enzymes were tested with a partial duplex DNA formed from ss M13 DNA and a nested series of fragments derived from a synthetic 21 -mer which were complementary to the polylinker region. These enzymes require up to two flanking nucleotides in addition to the hexameric recognition site for efficient cleavage. This technique could be useful for effecting unique cleavages of DNA with enzymes which generally give a large number of fragments and for strategies of ss DNA manipulation.

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