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The amino acid Asp-59 was proposed to be involved in EcoRI catalyzed DNA cleavage (Cheng et al., EMBO J. 13, 3927-35, 1994). We have tested this hypothesis by site directed mutagenesis experiments. The four mutants D59A, D59E, D59G, and D59N bind with similar stability to the specific recognition sequence as wild type EcoRI. The D59E mutant cleaves DNA as fast as the wild type enzyme. Specific...
The catalytic center of the restriction endonuclease (ENase) EcoRI is structurally homologous to that of EcoRV,Bam HI and PvuII. Each of these ENases contains a short motif of three to four amino acid (aa) residues which are positioned in a similar orientation to the scissile phosphodiester bond. We have mutated these aa (Pro 90 , Asp 91 , Glu 111 and...
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