Site-directed mutagenesis of the T4 endonuclease V gene: role of lysine-130

Journal Article · Tue Mar 22 04:00:00 EST 1988 · Biochemistry; (United States)

DOI:https://doi.org/10.1021/bi00406a006· OSTI ID:6840282

Recinos, A III; Lloyd, R S

The DNA sequence of the bacteriophage T4 denV gene which encodes the DNA repair enzyme endonuclease V was previously constructed behind the hybrid lambda promoter OLPR in a plasmid vector. The OLPR-denV sequence was subcloned in M13mp18 and used as template to construct site-specific mutations in the denV structural gene in order to investigate structure/function relationships between the primary structure of the protein and its various DNA binding and catalytic activities. The Lys-130 residue of the wild-type endonuclease V has been postulated to be associated with its apurinic endonuclease (AP-endonuclease) activity. The codon for Lys-130 was changed to His-130 or Gly-130, and each denV sequence was subcloned into a pEMBL expression vector. These plasmids were transformed into repair-deficient Escherichia coli (uvrA recA), and the following parameters were examined for cells or cell extracts: expression and accumulation of endonuclease V protein (K-130, H-130, or G-130); survival after UV irradiation; dimer-specific DNA binding; and kinetics of phosphodiester bond scission at pyrimidine dimer sites, dimer-specific N-glycosylase activity, and AP-endonuclease activity. The enzyme's intracellular accumulation was significantly decreased for G-130 and slightly decreased for H-130 despite normal levels of denV-specific mRNA for each mutant. On a molar basis, the endonuclease V gene products generally gave parallel levels of each of the catalytic and binding functions with K-130 greater than H-130 greater than G-130 much greater than control denV-.

• Research Organization: Vanderbilt Univ. School of Medicine, Nashville, TN (USA)
• OSTI ID: 6840282
• Journal Information: Biochemistry; (United States), Journal Name: Biochemistry; (United States) Vol. 27:6; ISSN BICHA
• 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
BIOCHEMICAL REACTION KINETICS
BIOLOGICAL FUNCTIONS
BIOLOGICAL RECOVERY
BIOLOGICAL REPAIR
CARBOXYLIC ACIDS
CELL CONSTITUENTS
DNA REPAIR
DNA SEQUENCING
DNA-ASE
ELECTROMAGNETIC RADIATION
ENDONUCLEASES
ENZYME ACTIVITY
ENZYMES
ESCHERICHIA COLI
ESTERASES
FUNCTIONS
GENE MUTATIONS
GENES
HYDROLASES
KINETICS
LYSINE
MICROORGANISMS
MUTAGENESIS
MUTATIONS
ORGANIC ACIDS
ORGANIC COMPOUNDS
PARASITES
PHOSPHODIESTERASES
PLASMIDS
PYRIMIDINE DIMERS
RADIATIONS
REACTION KINETICS
RECOVERY
REPAIR
STRUCTURAL CHEMICAL ANALYSIS
ULTRAVIOLET RADIATION
VIRUSES