A site-directed mutagenesis study to identify amino acid residues involved in the catalytic function of the restriction endonuclease EcoRV

Selent, Ursel; Rüter, T; Köhler, S. Eleonore; Liedtke, M; Thielking, Vera; Alves, Jürgen; Oelgeschläger, Thomas; Wolfes, Heiner; Peters, Frens; Pingoud, Alfred

doi:10.1021/bi00135a010

Cited by 155 publications

(158 citation statements)

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“…A recent report indicates that EcoRV can bind to DNA in the absence of Ca 2ϩ or Mg 2ϩ under different conditions (44); attempts to visualize a gel shift for PvuII in the absence of metal ions or in the presence of Mg 2ϩ for the catalytically inactive mutants have thus far been unsuccessful. Similar mutagenesis studies have been done on EcoRI (10 -13, 15, 45), EcoRV (18,20,23,24,28), and BamHI (26,27,30). These studies have anticipated or confirmed the predicted roles of residues identified as central from the crystal structures of the respective enzymes.…”

Section: Discussionmentioning

confidence: 62%

Catalytic and DNA Binding Properties of PvuII Restriction Endonuclease Mutants

Nastri

Evans

Walker

et al. 1997

Journal of Biological Chemistry

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The role of particular residues of the PvuII endonuclease in DNA binding and cleavage was studied by mutational analysis using a number of in vivo and in vitro approaches. While confirming the importance of residues predicted to be involved directly in function by the crystal structure, the analysis led to several striking results. Aspartate 34, which contacts the central base pair of the PvuII site (5 -CAGCTG-3 ) through the minor groove, plays a critical role in binding specificity. A D34G mutant binds with high affinity to any of the sequences in the set CANNTG, although its low level of cleavage activity acts only on the wild-type site. In addition, a His to Ala mutation at the residue that contacts the central G and is predicted to be blocked by PvuII methylation still requires the PvuII methylase to be maintained in vivo, arguing against this hypothesis as the only mechanism for methylation protection. Finally, four of the five mutations that reduce cleavage activity while still exhibiting binding in the gel shift assay are at residues that form DNA-or subunit-subunit contacts rather than in the catalytic center. This provides further evidence for a strong linkage between specific binding and catalysis.The structures of the five type II endonucleases determined by x-ray diffraction, EcoRI (1), BamHI (2, 3), EcoRV (4, 5), PvuII (6, 7) and Cfr10I (8), share substantial elements of similarity. Analysis of the enzymes complexed to DNA, where known, suggests a preliminary classification into two different groups (9). The endonucleases that produce 5Ј-overhanging ends, EcoRI and BamHI, approach the DNA from the major groove, where most of their base-specific contacts are made. The endonucleases that produce blunt-ended DNA products, EcoRV and PvuII, approach the DNA from the minor groove side and establish contacts in the major groove by wrapping around the DNA. Structure-function studies have been limited to a small number of type II enzymes. Years of studies have produced an extensive amount of information about EcoRI (10 -18), EcoRV (18 -24), and NaeI (25) and, more recently, BamHI (26,27). These studies have focused on the identification and analysis of residues involved in catalysis, testing alternative mechanisms of catalysis, and understanding how a specific DNA sequence is recognized. Attempts to alter the sequence specificity have proven to be difficult, with successful examples limited to mutants showing relaxed specificity (10,11,28) or displaying activity toward DNA substituted with unnatural bases (15,29). One explanation for this difficulty is that a change in specificity not only requires recognition of a different DNA sequence, but also requires that the linkage between recognition and catalysis be retained. A class of mutations that might illuminate this linkage is the catalytic mutations, where specific DNA binding is retained but catalysis is reduced (26, 30). In particular, mutants in this class that are outside the catalytic center might be deficient in the linkage between recognition and ca...

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Section: Discussionmentioning

confidence: 62%

Catalytic and DNA Binding Properties of PvuII Restriction Endonuclease Mutants

Nastri

Evans

Walker

et al. 1997

Journal of Biological Chemistry

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“…It is possible that the lysine residue functions as a general base or that it partly stabilizes the pentacovalent intermediate during the transition state to reduce the free energy of activation. A lysine residue close to the catalytic centre of EcoRV endonuclease is crucial for catalysis, and has been suggested to function as a general base or to compensate the extra negative charges of the pentacovalent phosphorus in the transition state (Selent et al 1992;Kostrewa & Winkler 1995). However, the residual cleavage activity of K118R suggests a more indirect role.…”

Section: Junction Specific Binding and Catalytically Competent Interamentioning

confidence: 99%

Mutational analysis on structure–function relationship of a Holliday junction specific endonuclease RuvC

et al. 1998

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“…In the cocrystal structures these residues (Asp-91, Glu-lll and Lys-ll3 in EcoRI, Asp-74, Asp-90 and Lys-92 in EcoRV, Asp-58, Glu-68 and Lys-70 in PvuII and Asp-94, Glu-lll and Glu-ll3 in BamHI) are in a similar position relative to the phosphodiester bond to be cleaved ( Fig. 1) and their importance for catalysis was verified by site directed mutagenesis experiments for EcoRI [10][11][12][13], EeoRV [14] and BamHI [15,16]. The similar architecture of the catalytic centres of the four enzymes suggests that they use the same catalytic mechanism for DNA cleavage.…”

Section: Introductionmentioning

confidence: 99%