Selection of mutations altering specificity in restriction-modification enzymes using the bacteriophage P22 challenge-phage system

Eric W. Fisher, Ming Te Yang, Shih tong Jeng, Jeffrey F. Gardner, Richard I. Gumport

Research output: Contribution to journalArticle

Abstract

A method for selecting mutants of site-specific DNA-binding proteins has been applied to the study of the EcoRI and RsrI restriction-modification enzymes. Catalytically inactive variants of both endonucleases are shown to function as pseudo-repressors in the bacteriophage P22 challenge-phage assay, and, upon further mutagenesis of the gene encoding R·EcoRI, a variant of that enzyme has been selected which appears to bind EcoRI-methylated GAATTC sequences to the exclusion of unmethylated sites: this specificity is the opposite of that belonging to the native enzyme. Variants of the EcoRI methylase have also been found that lack either catalytic activity or both binding and catalytic activities.

Original languageEnglish (US)
Pages (from-to)119-121
Number of pages3
JournalGene
Volume157
Issue number1-2
DOIs
StatePublished - May 19 1995

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Keywords

  • Pseudo-repressor
  • ant pseudo-operator
  • hydroxylamine
  • methyltransferase
  • mutagenesis

ASJC Scopus subject areas

  • Genetics

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