Site‐directed mutagenesis of the T4 endonuclease V Gene: Mutations which enhance enzyme specific activity at low salt concentrations

R. Stephen Lloyd, Mary Lou Augustine

Research output: Contribution to journalArticle

7 Scopus citations

Abstract

Previous structure/function analyses of the DNA repairenzyme, T4 endonuclease V, have suggested that the extreme carboxyl portion of the enzyme is associated with pyrimidine dimer‐specific binding (Recinos and Lloyd, and Stump and Lloyd, Biochemistry 27:1832–1838 and 1839–1843, 1988, respectively). Within the final 11 amino acids there are 5 aromatic, 2 basic, and no acidic residues and it has been proposed that these residues stack with and electrostatically interact with the kinked DNA at the site of a pyrimidine dimer. The role of the tyrosine residue at position 129 has beeninvestigated by oligonucleotide site‐directed mutagenesis in which the codon for Tyr‐129 has been altered to reflect conservative changesof Trp and Phe and more dramatic changes of Ser‐ a stop, codon, deletion of the codon or introduction of a frameshift. Both changesto the aromatic amino acids resulted in proteins which accumulated will in E. coli and not only significantly enhanced the UV survival of repair, deficient cells but also complemented a defective denV gene within UV‐irradiated T4 phage. Partially purified preparations of the Tyr‐129 → Trp and Tyr‐129 → Phe mutants were assayed for their ability to processively incise UV‐irradiated plasmid DNA (a nicking reaction carried out at low 25 mM salt concentrations). The mutant enzymes Tyr‐129 → Phe and Tyr‐129 → Trp displayed a 1000% and 500% enhanced specific nicking activity, respectively. These reactions were also shown to be completely processive. Assays performed at higher (100 mM) salt concentrations reduced the specific activities of the mutant enzymes approximately to that of wild type for the Tyr‐129 → Phe mutant and to 20% that of wild type for the Tyr1‐29 → Trp mutant.

Original languageEnglish (US)
Pages (from-to)128-138
Number of pages11
JournalProteins: Structure, Function, and Bioinformatics
Volume6
Issue number2
DOIs
StatePublished - 1989

Keywords

  • DNA repair
  • DNA repair enzymology
  • apurinic/apyrimidinic endonuclease
  • glycosylase
  • pyrimidine dimers
  • ultravioletlight

ASJC Scopus subject areas

  • Structural Biology
  • Biochemistry
  • Molecular Biology

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