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Alfred Pingoud - One of the best experts on this subject based on the ideXlab platform.
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The rotation-coupled sliding of EcoRV
Nucleic acids research, 2012Co-Authors: Jasmina Dikić, Alfred Pingoud, Carolin Menges, Samuel Clarke, Michael Kokkinidis, Wolfgang Wende, Pierre DesbiollesAbstract:It has been proposed that certain type II restriction enzymes (REs), such as EcoRV, track the helical pitch of DNA as they diffuse along DNA, a so-called rotation-coupled sliding. As of yet, there is no direct experimental observation of this phenomenon, but mounting indirect evidence gained from single-molecule imaging of RE–DNA complexes support the hypothesis. We address this issue by conjugating fluorescent labels of varying size (organic dyes, proteins and quantum dots) to EcoRV, and by fusing it to the engineered Rop protein scRM6. Single-molecule imaging of these modified EcoRVs sliding along DNA provides us with their linear diffusion constant (D1), revealing a significant size dependency. To account for the dependence of D1 on the size of the EcoRV label, we have developed four theoretical models describing different types of motion along DNA and find that our experimental results are best described by rotation-coupled sliding of the protein. The similarity of EcoRV to other type II REs and DNA binding proteins suggests that this type of motion could be widely preserved in other biological contexts.
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Sliding and jumping of single EcoRV restriction enzymes on non-cognate DNA
Nucleic Acids Research, 2008Co-Authors: Isabelle Bonnet, Alfred Pingoud, Wolfgang Wende, Andreas Biebricher, Pierre-louis Porté, Claude Loverdo, Olivier Bé Nichou, Christophe Escudé, Pierre DesbiollesAbstract:The restriction endonuclease EcoRV can rapidly locate a short recognition site within long non-cognate DNA using 'facilitated diffusion'. This process has long been attributed to a sliding mechanism, in which the enzyme first binds to the DNA via nonspecific interaction and then moves along the DNA by 1D diffusion. Recent studies, however , provided evidence that 3D translocations (hop-ping/jumping) also help EcoRV to locate its target site. Here we report the first direct observation of sliding and jumping of individual EcoRV molecules along nonspecific DNA. Using fluorescence micro-scopy, we could distinguish between a slow 1D diffusion of the enzyme and a fast translocation mechanism that was demonstrated to stem from 3D jumps. Salt effects on both sliding and jumping were investigated, and we developed numerical simulations to account for both the jump frequency and the jump length distribution. We deduced from our study the 1D diffusion coefficient of EcoRV, and we estimated the number of jumps occurring during an interaction event with nonspecific DNA. Our results substantiate that sliding alternates with hop-ping/jumping during the facilitated diffusion of EcoRV and, furthermore, set up a framework for the investigation of target site location by other DNA-binding proteins.
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On the possibilities and limitations of rational protein design to expand the specificity of restriction enzymes: a case study employing EcoRV as the target.
Protein engineering, 2000Co-Authors: Thomas Lanio, Albert Jeltsch, Alfred PingoudAbstract:The restriction endonuclease EcoRV has been characterized in structural and functional terms in great detail. Based on this detailed information we employed a structure-guided approach to engineer variants of EcoRV that should be able to discriminate between differently flanked EcoRV recognition sites. In crystal structures of EcoRV complexed with d(CGGGATATCCC)(2) and d(AAAGATATCTT)(2), Lys104 and Ala181 closely approach the two base pairs flanking the GATATC recognition site and thus were proposed to be a reasonable starting point for the rational extension of site specificity in EcoRV [Horton,N.C. and Perona,J.J. (1998) J. Biol. Chem., 273, 21721-21729]. To test this proposal, several single (K104R, A181E, A181K) and double mutants of EcoRV (K104R/A181E, K104R/A181K) were generated. A detailed characterization of all variants examined shows that only the substitution of Ala181 by Glu leads to a considerably altered selectivity with both oligodeoxynucleotide and macromolecular DNA substrates, but not the predicted one, as these variants prefer cleavage of a TA flanked site over all other sites, under all conditions tested. The substitution of Lys104 by Arg, in contrast, which appeared to be very promising on the basis of the crystallographic analysis, does not lead to variants which differ very much from the EcoRV wild-type enzyme with respect to the flanking sequence preferences. The K104R/A181E and K104R/A181K double mutants show nearly the same preferences as the A181E and A181K single mutants. We conclude that even for the very well characterized restriction enzyme EcoRV, properties that determine specificity and selectivity are difficult to model on the basis of the available structural information.
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crosslinking the EcoRV restriction endonuclease across the dna binding site reveals transient intermediates and conformational changes of the enzyme during dna binding and catalytic turnover
The EMBO Journal, 1998Co-Authors: Claudia Schulze, Albert Jeltsch, Ingo Franke, Claus Urbanke, Alfred PingoudAbstract:EcoRV completely encircles bound DNA with two loops, forming the entry and exit gate for the DNA substrate. These loops were crosslinked generating CL-EcoRV which binds and releases linear DNA only slowly, because threading linear DNA into and out of the DNA-binding 'tunnel' of CL-EcoRV is not very effective. If the crosslinking reaction is carried out with a circular bound DNA, CL-EcoRV is hyperactive towards the trapped substrate which is cleaved very quickly but not very accurately. CL-EcoRV also binds to, but does not cleave, circular DNA when added from the outside, because it cannot enter the active site. Based on these results a two-step binding model is proposed for EcoRV: initial DNA binding occurs at the outer side of the loops before the gate opens and then the DNA is transferred to the catalytic center.
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Crosslinking the EcoRV restriction endonuclease across the DNA‐binding site reveals transient intermediates and conformational changes of the enzyme during DNA binding and catalytic turnover
The EMBO journal, 1998Co-Authors: Claudia Schulze, Albert Jeltsch, Ingo Franke, Claus Urbanke, Alfred PingoudAbstract:EcoRV completely encircles bound DNA with two loops, forming the entry and exit gate for the DNA substrate. These loops were crosslinked generating CL-EcoRV which binds and releases linear DNA only slowly, because threading linear DNA into and out of the DNA-binding 'tunnel' of CL-EcoRV is not very effective. If the crosslinking reaction is carried out with a circular bound DNA, CL-EcoRV is hyperactive towards the trapped substrate which is cleaved very quickly but not very accurately. CL-EcoRV also binds to, but does not cleave, circular DNA when added from the outside, because it cannot enter the active site. Based on these results a two-step binding model is proposed for EcoRV: initial DNA binding occurs at the outer side of the loops before the gate opens and then the DNA is transferred to the catalytic center.
Stephen E. Halford - One of the best experts on this subject based on the ideXlab platform.
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DNA cleavage by the EcoRV restriction endonuclease: pH dependence and proton transfers in catalysis.
Journal of molecular biology, 1999Co-Authors: Neil P. Stanford, Stephen E. Halford, Geoffrey S. BaldwinAbstract:To characterise the pH dependence of phosphodiester hydrolysis by the EcoRV endonuclease in the presence of Mn2+, single turnover reactions on a 12 bp DNA substrate were examined by stopped-flow and quench-flow methods between pH 6.0 and 8.5. At each pH value, the apparent rate constants for phosphodiester hydrolysis increased hyperbolically with the concentration of MnCl2, thus allowing values to be determined for the intrinsic rate constant at saturation with Mn2+ and the equilibrium dissociation constant for Mn2+. The equilibrium constants showed no systematic variation across the pH range tested, while the rate constants increased steeply with increasing pH up to an asymptote above pH 7.5. At low pH conditions, the gradient of a plot of log (rate constant) against pH approached a value of 2. DNA cleavage by EcoRV thus requires the de-protonation of two acidic groups. To determine whether aspartate 36 is one of the groups, mutants of EcoRV were made with other amino acid residues at position 36. Glutamate caused a partial loss of activity, while all other replacements gave near-zero activities. In contrast to wild-type EcoRV, the mutant with glutamate required the de-protonation of only one acidic group for DNA cleavage. A mechanism for EcoRV is proposed in which the water molecule that hydrolyses the phosphodiester bond is de-protonated by two Bronsted bases, probably the ionised forms of aspartate 36 and glutamate 45.
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An isoleucine to leucine mutation that switches the cofactor requirement of the EcoRV restriction endonuclease from magnesium to manganese.
Biochemistry, 1996Co-Authors: Ib Vipond, Byung-jo Moon, Stephen E. HalfordAbstract:The EcoRV restriction endonuclease cleaves DNA at its recognition sequence more readily with Mg2+ as the cofactor than with Mn2+ but, at noncognate sequences that differ from the EcoRV site by one base pair, Mn2+ gives higher rates than Mg2+. A mutant of EcoRV, in which an isoleucine near the active site was replaced by leucine, showed the opposite behavior. It had low activity with Mg2+, but, in the presence of Mn2+ ions, it cleaved the recognition site faster than wild-type EcoRV with either Mn2+ or Mg2+. The mutant was also more specific for the recognition sequence than the native enzyme: the noncognate DNA cleavages by wild-type EcoRV and Mn2+ were not detected with the mutant. Further mutagenesis showed that the protein required the same acidic residues at its active site as wild-type EcoRV. The Ile→Leu mutation seems to perturb the configuration of the metal-binding ligands at the active site so that the protein has virtually no affinity for Mg2+ yet it can still bind Mn2+ ions, though the latter ...
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Site-Directed Mutagenesis of Ile91 of Restriction Endonuclease EcoRV: Dramatic Consequences on the Activity and the Properties of the Enzyme
Journal of Biochemistry and Molecular Biology, 1996Co-Authors: Byung-jo Moon, I. Barry Vipond, Stephen E. HalfordAbstract:Ile91 of restriction endonuclease EcoRV, which has not been known to take part directly in catalytic activity, was substituted with Leu by site-directed mutagenesis. The Ile91Leu mutant shows over 1000-fold less activity than the wild type EcoRV under standard reaction condition. The metal ion dependency of the reaction was altered. In contrast to the wild type EcoRV, the mutant prefers to as the cofactor. In buffer the mutant is as active as the wild type enzyme in buffer. Like the wild type enzyme, the mutant shows an unspecific binding of DNA in gel shift experiments. In contrast to the wild type enzyme, the mutant did not cleave at noncognate sites of DNA under star condition.
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Divalent metal ions at the active sites of the EcoRV and EcoRI restriction endonucleases.
Biochemistry, 1995Co-Authors: Ib Vipond, Geoff S. Baldwin, Stephen E. HalfordAbstract:Restriction enzymes cannot cleave DNA without a metal ion cofactor. The specificities of the EcoRV and EcoRI endonucleases for metals were studied by measuring DNA cleavage rates with several metal ions and with combinations of metal ions. Both EcoRV and EcoRI had optimal activities with Mg2+, were less active with several other ions including Mn2+, and had virtually no activity with Ca2+. But the activities of EcoRV and EcoRI with either Mg2+ or Mn2+ were perturbed by Ca2+. For EcoRI, both Mg2+- and Mn(2+)-dependent activities, at both cognate and noncognate sites, were all inhibited by Ca2+. The activity of EcoRV at its recognition site with Mg2+ was also inhibited by Ca2+. But the Mn(2+)-dependent reaction at the EcoRV recognition site was stimulated by Ca2+. EcoRV activities at noncognate sites with either Mg2+ or Mn2+ displayed a biphasic response to Ca2+: stimulation at low concentrations of Ca2+ and inhibition at high concentrations. These observations, together with the known structures of the proteins, indicate that EcoRI needs only one metal ion per active site and is inactive when Mg2+ is displaced by Ca2+, while EcoRV needs two and that the displacement of one by Ca2+ can enhance activity. We propose a mechanism for phosphodiester hydrolysis by EcoRV that involves two metal ions.
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structure function correlation for the EcoRV restriction enzyme from non specific binding to specific dna cleavage
Molecular Microbiology, 1993Co-Authors: Ib Vipond, Stephen E. HalfordAbstract:Summary The EcoRV restriction endonuclease cleaves DNA at its recognition sequence at least a million times faster than at any other DNA sequence. The only cofactor it requires for activity is Mg2+: but in binding to DNA in the absence of Mg2+, the EcoRV enzyme shows no specificity for its recognition site. Instead, the reason why EcoRV cuts one DNA sequence faster than any other is that the rate of cleavage is controlled by the binding of Mg2+ to EcoRV-DNA complexes: the complex at the recognition site has a high affinity for Mg2+, while the complexes at other DNA sequences have low affinities for Mg2+. The structures of the EcoRV endonuclease, and of its complexes with either 8pecific or non-specific DNA, have been solved by X-ray crystallography. In the specific complex, the protein interacts with the bases in the recognition sequence and the DNA takes up a highly distorted structure. In the non-specific complex with an unrelated DNA sequence, there are virtually no interactions with the bases and the DNA retains a B-like structure. Since the free energy changes for the formation of specific and non-specific complexes are the same, the energy from the specific interactions balances that required for the distortion of the DNA. The distortion inserts the phosphate at the scissile bond into the active site of the enzyme, where it forms part of the binding site for Mg2+. Without this distortion, the EcoRV–DNA complex would be unable to bind Mg2+ and thus unable to cleave DNA. The specificity of the EcoRV restriction enzyme is therefore governed, not by DNA binding as such, but by its ability to organize the structure of the DNA to which it is bound.
Albert Jeltsch - One of the best experts on this subject based on the ideXlab platform.
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On the possibilities and limitations of rational protein design to expand the specificity of restriction enzymes: a case study employing EcoRV as the target.
Protein engineering, 2000Co-Authors: Thomas Lanio, Albert Jeltsch, Alfred PingoudAbstract:The restriction endonuclease EcoRV has been characterized in structural and functional terms in great detail. Based on this detailed information we employed a structure-guided approach to engineer variants of EcoRV that should be able to discriminate between differently flanked EcoRV recognition sites. In crystal structures of EcoRV complexed with d(CGGGATATCCC)(2) and d(AAAGATATCTT)(2), Lys104 and Ala181 closely approach the two base pairs flanking the GATATC recognition site and thus were proposed to be a reasonable starting point for the rational extension of site specificity in EcoRV [Horton,N.C. and Perona,J.J. (1998) J. Biol. Chem., 273, 21721-21729]. To test this proposal, several single (K104R, A181E, A181K) and double mutants of EcoRV (K104R/A181E, K104R/A181K) were generated. A detailed characterization of all variants examined shows that only the substitution of Ala181 by Glu leads to a considerably altered selectivity with both oligodeoxynucleotide and macromolecular DNA substrates, but not the predicted one, as these variants prefer cleavage of a TA flanked site over all other sites, under all conditions tested. The substitution of Lys104 by Arg, in contrast, which appeared to be very promising on the basis of the crystallographic analysis, does not lead to variants which differ very much from the EcoRV wild-type enzyme with respect to the flanking sequence preferences. The K104R/A181E and K104R/A181K double mutants show nearly the same preferences as the A181E and A181K single mutants. We conclude that even for the very well characterized restriction enzyme EcoRV, properties that determine specificity and selectivity are difficult to model on the basis of the available structural information.
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Crosslinking the EcoRV restriction endonuclease across the DNA‐binding site reveals transient intermediates and conformational changes of the enzyme during DNA binding and catalytic turnover
The EMBO journal, 1998Co-Authors: Claudia Schulze, Albert Jeltsch, Ingo Franke, Claus Urbanke, Alfred PingoudAbstract:EcoRV completely encircles bound DNA with two loops, forming the entry and exit gate for the DNA substrate. These loops were crosslinked generating CL-EcoRV which binds and releases linear DNA only slowly, because threading linear DNA into and out of the DNA-binding 'tunnel' of CL-EcoRV is not very effective. If the crosslinking reaction is carried out with a circular bound DNA, CL-EcoRV is hyperactive towards the trapped substrate which is cleaved very quickly but not very accurately. CL-EcoRV also binds to, but does not cleave, circular DNA when added from the outside, because it cannot enter the active site. Based on these results a two-step binding model is proposed for EcoRV: initial DNA binding occurs at the outer side of the loops before the gate opens and then the DNA is transferred to the catalytic center.
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crosslinking the EcoRV restriction endonuclease across the dna binding site reveals transient intermediates and conformational changes of the enzyme during dna binding and catalytic turnover
The EMBO Journal, 1998Co-Authors: Claudia Schulze, Albert Jeltsch, Ingo Franke, Claus Urbanke, Alfred PingoudAbstract:EcoRV completely encircles bound DNA with two loops, forming the entry and exit gate for the DNA substrate. These loops were crosslinked generating CL-EcoRV which binds and releases linear DNA only slowly, because threading linear DNA into and out of the DNA-binding 'tunnel' of CL-EcoRV is not very effective. If the crosslinking reaction is carried out with a circular bound DNA, CL-EcoRV is hyperactive towards the trapped substrate which is cleaved very quickly but not very accurately. CL-EcoRV also binds to, but does not cleave, circular DNA when added from the outside, because it cannot enter the active site. Based on these results a two-step binding model is proposed for EcoRV: initial DNA binding occurs at the outer side of the loops before the gate opens and then the DNA is transferred to the catalytic center.
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Protein engineering of the restriction endonuclease EcoRV--structure-guided design of enzyme variants that recognize the base pairs flanking the recognition site.
European journal of biochemistry, 1998Co-Authors: Sylvia Schöttler, Christian Wenz, Albert Jeltsch, Thomas Lanio, Alfred PingoudAbstract:We generated variants of the restriction endonuclease EcoRV that discriminate between recognition sites with different flanking sequences. This was achieved by designing new contacts to the bases in the major groove of the DNA preceding and following the EcoRV recognition site. We selected Ala181 as the starting point for the extension of the site specificity of EcoRV because, according to the structure of the specific EcoRV · DNA complex, this residue is involved in a water mediated contact with the bases flanking the recognition sequence on the 5′ side. A substitution of this alanine residue by other amino acid residues changes the protein-DNA interface in this region and potentially creates new contacts, such that EcoRV variants could have an extended specificity, i.e. a greater selectivity for EcoRV sites within a particular sequence context. EcoRV variants with naturally occuring amino acid residues at position 181 were produced and their selectivity analyzed with oligodeoxynucleotide and plasmid substrates that differ only in the base pairs immediately flanking the EcoRV site. Some variants, having amino acid residues with long or bulky side chains at position 181 showed altered preferences for the base pairs flanking the recognition sequence with oligodeoxynucleotide substrates without loosing their catalytic efficiency. One variant, A181K, is able to discriminate between purine and pyrimidine bases on the 5′ side of the recognition sequence, probably by means of a new hydrogen bond to the N7 of the purine base. Another variant, A181E, strongly prefers a thymine base on the 5′ side of the recognition sequence, presumably due to a hydrogen bond formed between the protonated glutamic acid residue and the O4 of thymine.
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Kinetic Characterization of Linear Diffusion of the Restriction Endonuclease EcoRV on DNA
Biochemistry, 1998Co-Authors: Albert Jeltsch, Alfred PingoudAbstract:We have examined the kinetic parameters of linear diffusion of EcoRV on DNA. The data were analyzed by Monte Carlo simulations in which the efficiency of recognition of EcoRV sites during linear diffusion, the efficiency of linear diffusion, and the behavior of enzymes at the ends of linear DNA is explicitly treated. The analysis of the dependence of linear diffusion on the concentrations of NaCl and MgCl2 shows that linear diffusion is maximal at 50 mM NaCl under all concentrations of MgCl2 tested and increases with increasing concentrations of Mg2+ up to 10 mM, the highest concentration used in the test. Under these conditions, EcoRV scans 2 × 106 bp during one binding event with a velocity of about 1.7 × 106 bp s-1. The enzyme tends to overlook cleavage sites at 1 mM but not at 10 mM MgCl2. This result confirms the thermodynamic finding that EcoRV does not bind very specifically to DNA in the absence of Mg2+. It demonstrates that there is a Mg2+-dependent continuous transition between a nonspecific and...
Ib Vipond - One of the best experts on this subject based on the ideXlab platform.
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An isoleucine to leucine mutation that switches the cofactor requirement of the EcoRV restriction endonuclease from magnesium to manganese.
Biochemistry, 1996Co-Authors: Ib Vipond, Byung-jo Moon, Stephen E. HalfordAbstract:The EcoRV restriction endonuclease cleaves DNA at its recognition sequence more readily with Mg2+ as the cofactor than with Mn2+ but, at noncognate sequences that differ from the EcoRV site by one base pair, Mn2+ gives higher rates than Mg2+. A mutant of EcoRV, in which an isoleucine near the active site was replaced by leucine, showed the opposite behavior. It had low activity with Mg2+, but, in the presence of Mn2+ ions, it cleaved the recognition site faster than wild-type EcoRV with either Mn2+ or Mg2+. The mutant was also more specific for the recognition sequence than the native enzyme: the noncognate DNA cleavages by wild-type EcoRV and Mn2+ were not detected with the mutant. Further mutagenesis showed that the protein required the same acidic residues at its active site as wild-type EcoRV. The Ile→Leu mutation seems to perturb the configuration of the metal-binding ligands at the active site so that the protein has virtually no affinity for Mg2+ yet it can still bind Mn2+ ions, though the latter ...
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Specific DNA recognition by EcoRV restriction endonuclease induced by calcium ions.
Biochemistry, 1995Co-Authors: Ib VipondAbstract:In the presence of Mg2+, the EcoRV restriction endonuclease cleaves DNA specifically at its recognition sequence, but in the absence of divalent metal ions, it binds DNA without any specificity: gel-shift experiments had revealed multiple EcoRV-DNA complexes, due to the binding of one, two, three, or more molecules of protein per molecule of DNA, with the same equilibrium constant for each association. In this study, the binding of EcoRV to DNA was measured by gel shift in the presence of Ca2+, an ion that perturbs the Mg(2+)-dependent activity of EcoRV but that fails to support DNA cleavage. With Ca2+, and at a lower concentration of EcoRV protein than that required for binding in the absence of divalent metal ions, a single complex was observed with DNA containing the EcoRV recognition site. This complex was not formed with DNA that had been methylated at the EcoRV site nor with an isogenic DNA lacking the EcoRV recognition site. The single complex thus is due to the specific binding of EcoRV to its recognition site on the DNA. From gel shifts with a permuted set of DNA fragments, the degree of DNA bending by EcoRV at its recognition site was estimated to be 53 degrees +/- 4 degrees. This angle is similar to that seen in the crystal structure of the cognate DNA-protein complex. Calcium ions thus appear to mimic the role of Mg2+ in generating a specific protein-metal-DNA complex, but in contrast to Mg2+, Ca2+ gives a stable ternary complex in which the DNA-bound nuclease cannot cleave the DNA.
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Divalent metal ions at the active sites of the EcoRV and EcoRI restriction endonucleases.
Biochemistry, 1995Co-Authors: Ib Vipond, Geoff S. Baldwin, Stephen E. HalfordAbstract:Restriction enzymes cannot cleave DNA without a metal ion cofactor. The specificities of the EcoRV and EcoRI endonucleases for metals were studied by measuring DNA cleavage rates with several metal ions and with combinations of metal ions. Both EcoRV and EcoRI had optimal activities with Mg2+, were less active with several other ions including Mn2+, and had virtually no activity with Ca2+. But the activities of EcoRV and EcoRI with either Mg2+ or Mn2+ were perturbed by Ca2+. For EcoRI, both Mg2+- and Mn(2+)-dependent activities, at both cognate and noncognate sites, were all inhibited by Ca2+. The activity of EcoRV at its recognition site with Mg2+ was also inhibited by Ca2+. But the Mn(2+)-dependent reaction at the EcoRV recognition site was stimulated by Ca2+. EcoRV activities at noncognate sites with either Mg2+ or Mn2+ displayed a biphasic response to Ca2+: stimulation at low concentrations of Ca2+ and inhibition at high concentrations. These observations, together with the known structures of the proteins, indicate that EcoRI needs only one metal ion per active site and is inactive when Mg2+ is displaced by Ca2+, while EcoRV needs two and that the displacement of one by Ca2+ can enhance activity. We propose a mechanism for phosphodiester hydrolysis by EcoRV that involves two metal ions.
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structure function correlation for the EcoRV restriction enzyme from non specific binding to specific dna cleavage
Molecular Microbiology, 1993Co-Authors: Ib Vipond, Stephen E. HalfordAbstract:Summary The EcoRV restriction endonuclease cleaves DNA at its recognition sequence at least a million times faster than at any other DNA sequence. The only cofactor it requires for activity is Mg2+: but in binding to DNA in the absence of Mg2+, the EcoRV enzyme shows no specificity for its recognition site. Instead, the reason why EcoRV cuts one DNA sequence faster than any other is that the rate of cleavage is controlled by the binding of Mg2+ to EcoRV-DNA complexes: the complex at the recognition site has a high affinity for Mg2+, while the complexes at other DNA sequences have low affinities for Mg2+. The structures of the EcoRV endonuclease, and of its complexes with either 8pecific or non-specific DNA, have been solved by X-ray crystallography. In the specific complex, the protein interacts with the bases in the recognition sequence and the DNA takes up a highly distorted structure. In the non-specific complex with an unrelated DNA sequence, there are virtually no interactions with the bases and the DNA retains a B-like structure. Since the free energy changes for the formation of specific and non-specific complexes are the same, the energy from the specific interactions balances that required for the distortion of the DNA. The distortion inserts the phosphate at the scissile bond into the active site of the enzyme, where it forms part of the binding site for Mg2+. Without this distortion, the EcoRV–DNA complex would be unable to bind Mg2+ and thus unable to cleave DNA. The specificity of the EcoRV restriction enzyme is therefore governed, not by DNA binding as such, but by its ability to organize the structure of the DNA to which it is bound.
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Mechanism of action of restriction endonuclease EcoRV
Nucleic Acids and Molecular Biology, 1993Co-Authors: Stephen E. Halford, Christian L. M. Vermote, Jd Taylor, Ib VipondAbstract:Type II restriction/modification (R/M) systems, such as EcoRV, consist of two enzymes that act at the same DNA sequence; a modification methyltransferase and a restriction endonuclease (Bennett and Halford 1989; Wilson and Murray 1991). For EcoRV, the recognition sequence is GATATC (Kholmina et al. 1980). The EcoRV modification enzyme methylates the first adenine within this sequence (Nwosu et al. 1988) while, in the presence of Mg2+ ions, the EcoRV restriction enzyme cleaves DNA specifically at this site (Schildkraut et al. 1984). The endonuclease cuts both strands at the centre of the sequence, to leave blunt-ended DNA fragments. However, prior methylation of the recognition site blocks restriction activity. The basic tenet of R/M systems is that, in vivo, the restriction enzyme cuts only DNA molecules that have not been exposed previously to the methyltransferase. Hence, they enable the cell to degrade foreign DNA entering the cell without destroying host DNA. E. coli strains carrying the EcoRV system show this phenotype (Bougueleret et al. 1984).
Wolfgang Wende - One of the best experts on this subject based on the ideXlab platform.
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The rotation-coupled sliding of EcoRV
Nucleic acids research, 2012Co-Authors: Jasmina Dikić, Alfred Pingoud, Carolin Menges, Samuel Clarke, Michael Kokkinidis, Wolfgang Wende, Pierre DesbiollesAbstract:It has been proposed that certain type II restriction enzymes (REs), such as EcoRV, track the helical pitch of DNA as they diffuse along DNA, a so-called rotation-coupled sliding. As of yet, there is no direct experimental observation of this phenomenon, but mounting indirect evidence gained from single-molecule imaging of RE–DNA complexes support the hypothesis. We address this issue by conjugating fluorescent labels of varying size (organic dyes, proteins and quantum dots) to EcoRV, and by fusing it to the engineered Rop protein scRM6. Single-molecule imaging of these modified EcoRVs sliding along DNA provides us with their linear diffusion constant (D1), revealing a significant size dependency. To account for the dependence of D1 on the size of the EcoRV label, we have developed four theoretical models describing different types of motion along DNA and find that our experimental results are best described by rotation-coupled sliding of the protein. The similarity of EcoRV to other type II REs and DNA binding proteins suggests that this type of motion could be widely preserved in other biological contexts.
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Sliding and jumping of single EcoRV restriction enzymes on non-cognate DNA
Nucleic Acids Research, 2008Co-Authors: Isabelle Bonnet, Alfred Pingoud, Wolfgang Wende, Andreas Biebricher, Pierre-louis Porté, Claude Loverdo, Olivier Bé Nichou, Christophe Escudé, Pierre DesbiollesAbstract:The restriction endonuclease EcoRV can rapidly locate a short recognition site within long non-cognate DNA using 'facilitated diffusion'. This process has long been attributed to a sliding mechanism, in which the enzyme first binds to the DNA via nonspecific interaction and then moves along the DNA by 1D diffusion. Recent studies, however , provided evidence that 3D translocations (hop-ping/jumping) also help EcoRV to locate its target site. Here we report the first direct observation of sliding and jumping of individual EcoRV molecules along nonspecific DNA. Using fluorescence micro-scopy, we could distinguish between a slow 1D diffusion of the enzyme and a fast translocation mechanism that was demonstrated to stem from 3D jumps. Salt effects on both sliding and jumping were investigated, and we developed numerical simulations to account for both the jump frequency and the jump length distribution. We deduced from our study the 1D diffusion coefficient of EcoRV, and we estimated the number of jumps occurring during an interaction event with nonspecific DNA. Our results substantiate that sliding alternates with hop-ping/jumping during the facilitated diffusion of EcoRV and, furthermore, set up a framework for the investigation of target site location by other DNA-binding proteins.
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Intra- vs Intersubunit Communication in the Homodimeric Restriction Enzyme EcoRV: Thr 37 and Lys 38 Involved in Indirect Readout Are Only Important for the Catalytic Activity of Their Own Subunit†
Biochemistry, 1998Co-Authors: Frank Stahl, Wolfgang Wende, Christian Wenz, And Albert Jeltsch, Alfred PingoudAbstract:EcoRV is a dimer of two identical subunits which together form one binding site for the double-stranded DNA substrate. Concerted cleavage of both strands of the duplex requires intersubunit communication to synchronize the two catalytic centers of EcoRV. Here we address the question of how contacts to the DNA backbone trigger conformational changes which lead to the activation of both catalytic centers. The structure of the specific EcoRV−DNA complex shows that a region including amino acids Thr 37 and Lys 38 is involved in interactions with the DNA backbone and is a candidate for intersubunit communication. Homodimeric EcoRV T37A and K38A variants have a 1000-fold reduced catalytic activity. To examine whether Thr 37 and Lys 38 of one subunit affect the catalytic center in the same subunit and/or in the other subunit, we have produced heterodimeric variants containing a Thr 37 → Ala or Lys 38 → Ala substitution in one subunit combined with a wild type (wt) subunit (wt/T37A and wt/K38A) or with a subunit ...
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The mechanism of DNA cleavage by the type II restriction enzyme EcoRV: Asp36 is not directly involved in DNA cleavage but serves to couple indirect readout to catalysis.
Biological chemistry, 1998Co-Authors: Frank Stahl, Wolfgang Wende, Albert Jeltsch, Alfred PingoudAbstract:Three different mechanisms have been proposed to describe DNA cleavage by the type II restriction endonuclease EcoRV, which differ in the number and function of metal ions directly involved in catalysis and the different roles assigned to amino acid residues in the active sites and a phosphate group of the substrate. There are only four acidic amino acid residues close to the scissile bond: the essential Asp74 and Asp90, the non-essential Glu45, and Asp36. We show here that Asp36 can be exchanged for alanine, with only minor effects on the cleavage rate of the nearby phosphodiester bond, excluding that Asp36 could be directly involved in catalysis. Hence, the two versions of the two-metal-ion mechanism are not compatible with the experimental data, because too few ligands for two metal ions are present near the active site of EcoRV. Our result, thus, supports the one-metal-ion mechanism for EcoRV. We suggest that Asp36 has an allosteric effect by which specific contacts between one strand of the DNA and one subunit of the enzyme trigger the activation of one catalytic center. Given the similar structures of the active sites of EcoRV, EcoRI, BamHI, PvuII and FokI, as well as the occurrence of a characteristic catalytic motif in several other restriction enzymes, we conclude that these enzymes most likely share a similar mechanism of DNA cleavage, whose characteristic feature is the involvement of only one Mg2+ ion in catalysis.
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Introduction of asymmetry in the naturally symmetric restriction endonuclease EcoRV to investigate intersubunit communication in the homodimeric protein.
Proceedings of the National Academy of Sciences of the United States of America, 1996Co-Authors: Frank Stahl, Wolfgang Wende, Albert Jeltsch, Alfred PingoudAbstract:Abstract Type II restriction endonucleases are dimers of two identical subunits that together form one binding site for the double-stranded DNA substrate. Cleavage within the palindromic recognition site occurs in the two strands of the duplex in a concerted manner, due to the action of two catalytic centers, one per subunit. To investigate how the two identical subunits of the restriction endonuclease EcoRV cooperate in binding and cleaving their substrate, heterodimeric versions of EcoRV with different amino acid substitutions in the two subunits were constructed. For this purpose, the EcoRV gene was fused to the coding region for the glutathione-binding domain of the glutathione S-transferase and a His6-tag, respectively. Upon cotransformation of Escherichia coli cells with both gene fusions stable homo- and heterodimers of the EcoRV variants are produced, which can be separated and purified to homogeneity by affinity chromatography over Ni-nitrilotriacetic acid and glutathione columns. A steady-state kinetic analysis shows that the activity of a heterodimeric variant with one inactive catalytic center is decreased by 2-fold, demonstrating that the two catalytic centers operate independently from each other. In contrast, heterodimeric variants with a defect in one DNA-binding site have a 30- to 50-fold lower activity, indicating that the two subunits of EcoRV cooperate in the recognition of the palindromic DNA sequence. By combining a subunit with an inactive catalytic center with a subunit with a defect in the DNA-binding site, EcoRV heterodimers were produced that only nick DNA specifically within the EcoRV recognition sequence.
