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

Journal Article · Sun Jan 01 04:00:00 EST 1989 · Proteins; (USA)

DOI:https://doi.org/10.1002/prot.340060204· OSTI ID:7189997

Lloyd, R S; Augustine, M L ^[1]

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1. Vanderbilt Univ. School of Medicine, Nashville, TN (USA)

Previous structure/function analyses of the DNA repair enzyme, T4 endonuclease V, have suggested that the extreme carboxyl portion of the enzyme is associated with pyrimidine dimer-specific binding. 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 been investigated by oligonucleotide site-directed mutagenesis in which the codon for Tyr-129 has been altered to reflect conservative changes of Trp and Phe and more dramatic changes of Ser, a stop codon, deletion of the codon or introduction of a frameshift. Both changes to the aromatic amino acids resulted in proteins which accumulated well 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 Tyr-129----Trp mutant.

• OSTI ID: 7189997
• Journal Information: Proteins; (USA), Journal Name: Proteins; (USA) Vol. 6:2; ISSN PSFGE; ISSN 0887-3585
• Country of Publication: United States
• Language: English

Related Subjects

560130* -- Radiation Effects on Microorganisms
63 RADIATION, THERMAL, AND OTHER ENVIRON. POLLUTANT EFFECTS ON LIVING ORGS. AND BIOL. MAT.
AMINO ACIDS
BACTERIA
BACTERIOPHAGES
BIOLOGICAL EFFECTS
BIOLOGICAL RADIATION EFFECTS
BIOLOGICAL RECOVERY
BIOLOGICAL REPAIR
CARBOXYLIC ACIDS
CELL CONSTITUENTS
DNA REPAIR
DNA-ASE
ELECTROMAGNETIC RADIATION
ENDONUCLEASES
ENZYME ACTIVITY
ENZYMES
ESCHERICHIA COLI
ESTERASES
GENE MUTATIONS
GENES
GENETIC EFFECTS
GENETIC RADIATION EFFECTS
HYDROLASES
HYDROXY ACIDS
MICROORGANISMS
MUTAGENESIS
MUTATIONS
ORGANIC ACIDS
ORGANIC COMPOUNDS
PARASITES
PHOSPHODIESTERASES
PLASMIDS
PROTEIN STRUCTURE
PYRIMIDINE DIMERS
RADIATION EFFECTS
RADIATIONS
RADIOINDUCTION
RECOVERY
REPAIR
STRUCTURE-ACTIVITY RELATIONSHIPS
SURVIVAL TIME
TYROSINE
ULTRAVIOLET RADIATION
VIRUSES