Site-directed mutagenesis of the T4 endonuclease V gene

Mutations which enhance enzyme specific activity at low salt concentrations

Robert (Stephen) Lloyd, M. L. Augustine

Research output: Contribution to journalArticle

7 Citations (Scopus)

Abstract

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 (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 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.

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

Fingerprint

Mutagenesis
Site-Directed Mutagenesis
Pyrimidine Dimers
Aromatic Amino Acids
Salts
Genes
Codon
Mutation
Enzymes
DNA Repair Enzymes
Bacteriophage T4
Biochemistry
Bacteriophages
Terminator Codon
Oligonucleotides
Escherichia coli
Tyrosine
Assays
Plasmids
Repair

Keywords

  • Apurinic/apyrimidinic endonuclease
  • DNA repair
  • DNA repair enzymology
  • Glycosylase
  • Pyrimidine dimers
  • Ultraviolet light

ASJC Scopus subject areas

  • Biochemistry
  • Genetics
  • Structural Biology

Cite this

@article{1e3f83ad727e4bdaa6951d6a61ba62ad,
title = "Site-directed mutagenesis of the T4 endonuclease V gene: Mutations which enhance enzyme specific activity at low salt concentrations",
abstract = "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 (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 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.",
keywords = "Apurinic/apyrimidinic endonuclease, DNA repair, DNA repair enzymology, Glycosylase, Pyrimidine dimers, Ultraviolet light",
author = "Lloyd, {Robert (Stephen)} and Augustine, {M. L.}",
year = "1989",
language = "English (US)",
volume = "6",
pages = "128--138",
journal = "Proteins: Structure, Function and Genetics",
issn = "0887-3585",
publisher = "Wiley-Liss Inc.",
number = "2",

}

TY - JOUR

T1 - Site-directed mutagenesis of the T4 endonuclease V gene

T2 - Mutations which enhance enzyme specific activity at low salt concentrations

AU - Lloyd, Robert (Stephen)

AU - Augustine, M. L.

PY - 1989

Y1 - 1989

N2 - 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 (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 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.

AB - 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 (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 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.

KW - Apurinic/apyrimidinic endonuclease

KW - DNA repair

KW - DNA repair enzymology

KW - Glycosylase

KW - Pyrimidine dimers

KW - Ultraviolet light

UR - http://www.scopus.com/inward/record.url?scp=0024428795&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0024428795&partnerID=8YFLogxK

M3 - Article

VL - 6

SP - 128

EP - 138

JO - Proteins: Structure, Function and Genetics

JF - Proteins: Structure, Function and Genetics

SN - 0887-3585

IS - 2

ER -