Cooperative Binding Properties of Restriction Endonuclease EcoRII with DNA Recognition Sites*

EcoRII is a member of the expanding group of type IIe restriction endonucleases that share the distinguishing feature of requiring cooperativity between two recognition sites in their substrate DNA. To determine the stoichiometry of the active DNA-enzyme complex and the mode of cooperative interaction, we have investigated the dependence ofEcoRII cleavage on the concentration of EcoRII dimers. Maximal restriction was observed at dimer/site ratios of 0.25 and 0.5. The molecular weight of the DNA-enzyme complex eluted from a gel filtration column also corresponds to a dimeric enzyme structure bound to two substrate sites. We conclude that oneEcoRII dimer is sufficient to interact cooperatively with two DNA recognition sites. A Lac repressor “barrier” bound between two normally reactive EcoRII sites did not inhibit restriction endonuclease activity, indicating that cooperativity between EcoRII sites is achieved by bending or looping of the intervening DNA stretch. Comparative cleavage of linear substrates with differently spaced interacting sites revealed an inverse correlation between cleavage rate and site distance. At the optimal distance of one helical turn, EcoRII cleavage is independent of the orientation of the recognition sequence in the DNA double strand.

[1]  M. Gellert,et al.  Communication between segments of DNA during site-specific recombination , 1987, Nature.

[2]  D. Krüger,et al.  An improved method for the detection of Dcm methylation in DNA molecules. , 1990, Gene.

[3]  F Wright,et al.  Codon usage in the G+C-rich Streptomyces genome. , 1992, Gene.

[4]  A. Jeltsch,et al.  Linear diffusion of the restriction endonuclease EcoRV on DNA is essential for the in vivo function of the enzyme. , 1996, The EMBO journal.

[5]  J. Wang,et al.  Action at a distance along a DNA. , 1988, Science.

[6]  T. Bickle,et al.  Biology of DNA restriction. , 1993, Microbiological reviews.

[7]  M. D. Topal,et al.  DNA topoisomerase and recombinase activities in Nae I restriction endonuclease. , 1995, Science.

[8]  Y. Buryanov,et al.  Interaction of EcoRII restriction and modification enzymes with synthetic DNA fragments , 1984, FEBS letters.

[9]  B. Müller-Hill,et al.  Lac repressor with the helix‐turn‐helix motif of lambda cro binds to lac operator. , 1992, The EMBO journal.

[10]  S. Halford,et al.  DNA cleavage at two recognition sites by the SfiI restriction endonuclease: salt dependence of cis and trans interactions between distant DNA sites. , 1995, Journal of molecular biology.

[11]  G. Butcher,et al.  Mechanism of Host Specificity in Malarial Infection , 1973, Nature.

[12]  A. Bhagwat,et al.  Interaction of EcoRII endonuclease with DNA substrates containing single recognition sites. , 1992, The Journal of biological chemistry.

[13]  K Rippe,et al.  Action at a distance: DNA-looping and initiation of transcription. , 1995, Trends in biochemical sciences.

[14]  M. D. Topal,et al.  Changing endonuclease EcoRII Tyr308 to Phe abolishes cleavage but not recognition: possible homology with the Int-family of recombinases. , 1993, Nucleic acids research.

[15]  P. Sadowski.  Site-specific recombinases: changing partners and doing the twist , 1986, Journal of bacteriology.

[16]  S. Halford,et al.  The SfiI restriction endonuclease makes a four-strand DNA break at two copies of its recognition sequence. , 1995, Journal of molecular biology.

[17]  M Reuter,et al.  EcoRII can be activated to cleave refractory DNA recognition sites. , 1988, Nucleic acids research.

[18]  Effects on NaeI-DNA recognition of the leucine to lysine substitution that transforms restriction endonuclease NaeI to a topoisomerase: a model for restriction endonuclease evolution. , 1996, Nucleic acids research.

[19]  R. Roberts,et al.  Primary sequence of the EcoRII endonuclease and properties of its fusions with beta-galactosidase. , 1990, The Journal of biological chemistry.

[20]  G. H. Beaven,et al.  Ultraviolet absorption spectra of proteins and amino acids. , 1952, Advances in protein chemistry.

[21]  M. D. Topal,et al.  Changing a leucine to a lysine residue makes NaeI endonuclease hypersensitive to DNA intercalative drugs. , 1996, Biochemistry.

[22]  A. Jeltsch,et al.  Pausing of the restriction endonuclease EcoRI during linear diffusion on DNA. , 1994, Biochemistry.

[23]  D. Krüger,et al.  Use of specific oligonucleotide duplexes to stimulate cleavage of refractory DNA sites by restriction endonucleases. , 1993, Analytical biochemistry.

[24]  T. Bickle,et al.  Type III restriction endonucleases translocate DNA in a reaction driven by recognition site‐specific ATP hydrolysis. , 1995, The EMBO journal.

[25]  D. Koshland.  The structural basis of negative cooperativity: receptors and enzymes. , 1996, Current opinion in structural biology.

[26]  S. Halford,et al.  Recombination by resolvase to analyse DNA communications by the SfiI restriction endonuclease. , 1996, The EMBO journal.

[27]  M. D. Topal,et al.  DNA cleavage by NaeI: protein purification, rate-limiting step, and accuracy. , 1994, Biochemistry.

[28]  M. D. Topal,et al.  Ability of DNA and spermidine to affect the activity of restriction endonucleases from several bacterial species. , 1991, Biochemistry.

[29]  P. Dröge.  Protein tracking‐induced supercoiling of DNA: A tool to regulate DNA transactions in vivo? , 1994, BioEssays : news and reviews in molecular, cellular and developmental biology.

[30]  M. D. Topal,et al.  NaeI endonuclease binding to pBR322 DNA induces looping. , 1991, Biochemistry.

[31]  D. Krüger,et al.  Hyperexpressed EcoRII renatured from inclusion bodies and native enzyme both exhibit essential cooperativity with two DNA sites. , 1995, Protein expression and purification.