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• W773037352 abstract "Divalent metal ions, normally Mg2+, are essential for both DNA cleavage by the EcoRV restriction endonuclease at its recognition sequence, GATATC1, and also for the enzymes discrimination between this particular sequence and all other sequences.2 In the absence of divalent metal ions, EcoRV demonstrates no catalytic activity, though it can still bind to DNA in a nonspecific manner with no preference in its recognition sequence.3 The complex of EcoRV and its cognate DNA, however, has a high affinity for Mg2+ due to the distortion of the bound DNA, creating a metal-binding site between the protein and the DNA.4 In contrast, EcoRV bound to nonspecific DNA has low affinity for Mg2+. In this case, the lack of distortion leaves the DNA too far away from the active site to allow a metal ion to be liganded by both protein and DNA.5 Thus, the metal ion effectively creates the specificity of EcoRV for its recognition site by locking the protein onto DNA at this sequence.6 Other metal ions in place of Mg2+ perturb both the activity and the specificity of EcoRV.7 In the presence of Mn2+, EcoRV has a lower reaction rate (kcat) at its recognition site but a higher rate at noncognate sites, with the result that the ratio of the DNA cleavage rate at cognate and noncognate sites alternates from a value of 1×106 with Mg2+ to 6 with Mn2+.8 The lack of discrimination with Mn2+ stems from both cognate and noncognate complexes having high affinities for this ion, but why the noncognate complex should have so much higher an affinity for Mn2+ than for Mg2+ is yet to be explained. The switch from Mg2+ to Mn2+ also perturbs both the mechanism and specificity of other restriction enzymes such as TaqI.9 The EcoRV endonuclease is thought to require two metal ions per active site in order to catalyze phosphodiester hydrolysis. One Mg2+ (or Mn2+) is located between Asp90 and Asp74 and the second ion between Asp74 and Glu45.2 It has also been shown that an Ile91 to Leu mutation switches metal ion specificty from Mg2+ to Mn2+.12 A mutant restriction endonuclease that recognizes a novel DNA sequence would be more useful than one recognizing a novel metal ion, but this is yet to be achieved.10 The conversion of a restriction enzyme to a new sequence specificity in vivo demands the parallel conversion of the modification methyltransferase to the same sequence.11 A mutant Ile91Leu EcoRV restriction endonuclease that switches the cofactor requirement from Mg2+ to Mn2+ suggests the circumvention of the need for a methyltransferase, inactivity in vivo coupled to high activity under non-physiological conditions in vitro.12 In a search for enzymes that recognize a novel DNA sequence or a novel metal ion, we carried out site directed mutagenesis of EcoRV restriction endonuclease. We report here the mutant enzymes that showed novel metal ion specificity. Several mutations were created by changing amino acid residues in the vicinity of the scissile phosphodiester bond in the EcoRV-DNA complex by site-directed mutagenesis. Table 1 shows the results of cleavage of plasmid pAT153 by mutant enzymes in the presence of a variety of added metal ions. Wild type enzyme is most active with Mg2+ as the cofactor than with any other divalent ion including Mn2+. Under standard conditions (10 mM MgCl2, 100mM NaCl, pH 7.5), all the mutants were much less active than wild type. Two mutant enzymes, however, showed different metal ion specificity. Asp90Cys mutant in which aspartate at position 90 was replaced by cystein was synthesized. Initially, we attempted to have a mutant that has a high activity with Zn2+ since the thiol group is known as a very good ligand for Zn2+. But the mutant Asp90Cys preferred Mn2+ over Zn2+ as the cofactor. The mutant enzyme showed 100 times less activity than wild type at standard reaction conditions. In the presence of Zn2+, the mutant enzyme showed activity that was close to that of the wild type enzyme. But it showed much lower activity with Zn2+ than with Mg2+ or Mn2+. The Mn2+ activity profile of the mutant enzyme showed a decrease in activity with increasing concentration of Mn2+ (data not shown). The same Mn2+ activity profile has been shown with the reported Ile91Leu mutant.12 Interestingly, the mutant enzyme showed higher sequence specificity than wild type enzyme. Plasmid pAT153 contains, in addition to one EcoRV recognition site, 12 noncognate sites that can be cleaved by EcoRV under star conditions with high concentration of the enzyme.13 The wild type EcoRV cuts noncognate sites at high concentration of enzyme or under star conditions, but Asp90Cys mutant enzyme did not cut noncognate sites even at high concentra-" @default.
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• W773037352 date "1999-01-01" @default.
• W773037352 modified "2023-09-23" @default.
• W773037352 title "Restriction Endonuclease EcoRV Mutants that Switch Metal Ion Requirement" @default.
• W773037352 hasPublicationYear "1999" @default.
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