EcoRV restriction endonuclease binds all DNA sequences with equal affinity.

In the presence of MgCl2, the EcoRV restriction endonuclease cleaves its recognition sequence on DNA at least a million times more readily than any other sequence. In this study, the binding of the EcoRV restriction enzyme to DNA was examined in the absence of Mg2+. With each DNA fragment tested, several DNA-protein complexes were detected by electrophoresis through polyacrylamide. No differences were observed between isogenic DNA molecules that either contained or lacked the EcoRV recognition site. The number of complexes with each fragment varied with the length of the DNA. Three complexes were formed with a DNA molecule of 55 base pairs, corresponding to the DNA bound to 1, 2, or 3 molecules of the protein, while greater than 15 complexes were formed with a DNA of 381 base pairs. A new method was developed to analyze the binding of a protein to multiple sites on DNA. The method showed that the EcoRV enzyme binds to all DNA sequences, including the EcoRV recognition site, with the same equilibrium constant, though two molecules of the protein bind preferentially to adjacent sites on the DNA in a cooperative fashion. All of the complexes with a substrate that contained the EcoRV site dissociated upon addition of competitor DNA, but when the competitor was mixed with MgCl2, a fraction of the substrate was cleaved at the EcoRV site. The fraction cleaved was due mainly to the translocation of the enzyme from nonspecific sites on the DNA to the specific site.

[1]  W. Jencks,et al.  Binding energy, specificity, and enzymic catalysis: the circe effect. , 2006, Advances in enzymology and related areas of molecular biology.

[2]  S. Benkovic,et al.  Mechanism of DNA replication fidelity for three mutants of DNA polymerase I: Klenow fragment KF(exo+), KF(polA5), and KF(exo-). , 1991, Biochemistry.

[3]  J. Rosenberg.  Structure and function of restriction endonucleases , 1991 .

[4]  H. Blöcker,et al.  Crystallization of complexes of EcoRV endonuclease with cognate and non-cognate DNA fragments. , 1991, Journal of molecular biology.

[5]  S. Halford,et al.  Fidelity of DNA recognition by the EcoRV restriction/modification system in vivo. , 1990, Biochemistry.

[6]  M. Kurpiewski,et al.  The energetic basis of specificity in the Eco RI endonuclease--DNA interaction. , 1990, Science.

[7]  D. Williams,et al.  Interaction of the EcoRV restriction endonuclease with the deoxyadenosine and thymidine bases in its recognition hexamer d(GATATC). , 1990, Biochemistry.

[8]  A. Bergerat,et al.  The double role of methyl donor and allosteric effector of S-adenosyl-methionine for Dam methylase of E. coli. , 1990, Nucleic acids research.

[9]  S. Halford,et al.  Discrimination between DNA sequences by the EcoRV restriction endonuclease. , 1989, Biochemistry.

[10]  P. Modrich,et al.  The negative charge of Glu-111 is required to activate the cleavage center of EcoRI endonuclease. , 1989, The Journal of biological chemistry.

[11]  P. V. von Hippel,et al.  Facilitated Target Location in Biological Systems* , 2022 .

[12]  F. Seela,et al.  Analysis of the recognition mechanism involved in the EcoRV catalyzed cleavage of DNA using modified oligodeoxynucleotides. , 1988, Nucleic acids research.

[13]  Donald M. Crothers,et al.  DNA sequence determinants of CAP-induced bending and protein binding affinity , 1988, Nature.

[14]  H. Bading.  Determination of the molecular weight of DNA-bound protein(s) responsible for gel electrophoretic mobility shift of linear DNA fragments examplified with purified viral myb protein. , 1988, Nucleic acids research.

[15]  Annette J. Goodall,et al.  Modes of DNA cleavage by the EcoRV restriction endonuclease. , 1988, Biochemistry.

[16]  L. McLaughlin,et al.  Effects of functional group changes in the EcoRI recognition site on the cleavage reaction catalyzed by the endonuclease. , 1987, Biochemistry.

[17]  M. A. Shea,et al.  "Footprint" titrations yield valid thermodynamic isotherms. , 1986, Proceedings of the National Academy of Sciences of the United States of America.

[18]  P. V. von Hippel,et al.  On the specificity of DNA-protein interactions. , 1986, Proceedings of the National Academy of Sciences of the United States of America.

[19]  P. Modrich,et al.  Facilitated diffusion during catalysis by EcoRI endonuclease. Nonspecific interactions in EcoRI catalysis. , 1985, The Journal of biological chemistry.

[20]  L. Bougueleret,et al.  Overproduction of the EcoR V endonuclease and methylase. , 1985, Nucleic acids research.

[21]  C. Gualerzi,et al.  Linear diffusion of restriction endonucleases on DNA. , 1985, The Journal of biological chemistry.

[22]  A. Varshavsky,et al.  A protein binds to a satellite DNA repeat at three specific sites that would be brought into mutual proximity by DNA folding in the nucleosome , 1984, Cell.

[23]  A. Tsugita,et al.  Characterization of the genes coding for the Eco RV restriction and modification system of Escherichia coli. , 1984, Nucleic acids research.

[24]  C. Banner,et al.  The cleavage site for the restriction endonuclease EcoRV is 5'-GAT/ATC-3'. , 1984, Gene.

[25]  R. Burgess,et al.  Direct evidence for the preferential binding of Escherichia coli RNA polymerase holoenzyme to the ends of deoxyribonucleic acid restriction fragments. , 1983, Biochemistry.

[26]  P. Modrich,et al.  Thermodynamic parameters governing interaction of EcoRI endonuclease with specific and nonspecific DNA sequences. , 1983, The Journal of biological chemistry.

[27]  P. Modrich,et al.  Involvement of outside DNA sequences in the major kinetic path by which EcoRI endonuclease locates and leaves its recognition sequence. , 1982, Proceedings of the National Academy of Sciences of the United States of America.

[28]  G M Clore,et al.  Theoretical aspects of specific and non-specific equilibrium binding of proteins to DNA as studied by the nitrocellulose filter binding assay. Co-operative and non-co-operative binding to a one-dimensional lattice. , 1982, Journal of molecular biology.

[29]  D. Crothers,et al.  Equilibria and kinetics of lac repressor-operator interactions by polyacrylamide gel electrophoresis. , 1981, Nucleic acids research.

[30]  P. V. von Hippel,et al.  Diffusion-driven mechanisms of protein translocation on nucleic acids. 1. Models and theory. , 1981, Biochemistry.

[31]  M. M. Garner,et al.  A gel electrophoresis method for quantifying the binding of proteins to specific DNA regions: application to components of the Escherichia coli lactose operon regulatory system , 1981, Nucleic Acids Res..

[32]  S. Halford,et al.  The EcoRI restriction endonuclease with bacteriophage lambda DNA. Equilibrium binding studies. , 1980, The Biochemical journal.

[33]  D. Sherratt,et al.  Trans-complementable copy-number mutants of plasmid ColE1 , 1980, Nature.

[34]  A. C. Chang,et al.  Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid , 1978, Journal of bacteriology.

[35]  T. Lohman,et al.  Thermodynamic analysis of ion effects on the binding and conformational equilibria of proteins and nucleic acids: the roles of ion association or release, screening, and ion effects on water activity , 1978, Quarterly Reviews of Biophysics.

[36]  P. V. von Hippel,et al.  DNA "melting" proteins. IV. Fluorescence measurements of binding parameters for bacteriophage T4 gene 32-protein to mono-, oligo-, and polynucleotides. , 1976, The Journal of biological chemistry.

[37]  N. Seeman,et al.  Sequence-specific Recognition of Double Helical Nucleic Acids by Proteins (base Pairs/hydrogen Bonding/recognition Fidelity/ion Binding) , 2022 .

[38]  P. V. von Hippel,et al.  Theoretical aspects of DNA-protein interactions: co-operative and non-co-operative binding of large ligands to a one-dimensional homogeneous lattice. , 1974, Journal of molecular biology.

[39]  Bennett Sp,et al.  Recognition of DNA by type II restriction enzymes. , 1989 .

[40]  M. Fried.  Measurement of protein‐DNA interaction parameters by electrophoresis mobility shift assay , 1989, Electrophoresis.

[41]  P. Hagerman.  Flexibility of DNA. , 1988, Annual review of biophysics and biophysical chemistry.

[42]  S. Halford,et al.  Relaxed specificity of the EcoRV restriction endonuclease. , 1986, Gene.