Selective metal binding to Cys-78 within endonuclease V causes an inhibition of catalytic activities without altering nontarget and target DNA binding

Journal Article · Wed Jun 05 04:00:00 EDT 1991 · Journal of Biological Chemistry; (United States)

OSTI ID:5552037

Prince, M A; Friedman, B; Gruskin, E A; Schrock, R.D. 3d.; Lloyd, R S ^[1]

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1. Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, TN (USA)

T4 endonuclease V is a pyrimidine dimer-specific DNA repair enzyme which has been previously shown not to require metal ions for either of its two catalytic activities or its DNA binding function. However, we have investigated whether the single cysteine within the enzyme was able to bind metal salts and influence the various activities of this repair enzyme. A series of metals (Hg2+, Ag+, Cu+) were shown to inactivate both endonuclease Vs pyrimidine dimer-specific DNA glycosylase activity and the subsequent apurinic nicking activity. The binding of metal to endonuclease V did not interfere with nontarget DNA scanning or pyrimidine dimer-specific binding. The Cys-78 codon within the endonuclease V gene was changed by oligonucleotide site-directed mutagenesis to Thr-78 and Ser-78 in order to determine whether the native cysteine was directly involved in the enzyme's DNA catalytic activities and whether the cysteine was primarily responsible for the metal binding. The mutant enzymes were able to confer enhanced ultraviolet light (UV) resistance to DNA repair-deficient Escherichia coli at levels equal to that conferred by the wild type enzyme. The C78T mutant enzyme was purified to homogeneity and shown to be catalytically active on pyrimidine dimer-containing DNA. The catalytic activities of the C78T mutant enzyme were demonstrated to be unaffected by the addition of Hg2+ or Ag+ at concentrations 1000-fold greater than that required to inhibit the wild type enzyme. These data suggest that the cysteine is not required for enzyme activity but that the binding of certain metals to that amino acid block DNA incision by either preventing a conformational change in the enzyme after it has bound to a pyrimidine dimer or sterically interfering with the active site residue's accessibility to the pyrimidine dimer.

• OSTI ID: 5552037
• Journal Information: Journal of Biological Chemistry; (United States), Journal Name: Journal of Biological Chemistry; (United States) Vol. 266:16; ISSN 0021-9258; ISSN JBCHA
• Country of Publication: United States
• Language: English

Related Subjects

560130 -- Radiation Effects on Microorganisms
560300* -- Chemicals Metabolism & Toxicology
63 RADIATION, THERMAL, AND OTHER ENVIRON. POLLUTANT EFFECTS ON LIVING ORGS. AND BIOL. MAT.
AMINO ACIDS
BACTERIA
BIOCHEMICAL REACTION KINETICS
BIOLOGICAL EFFECTS
BIOLOGICAL FUNCTIONS
BIOLOGICAL RADIATION EFFECTS
BIOLOGICAL RECOVERY
BIOLOGICAL REPAIR
CARBOXYLIC ACIDS
CATALYSIS
COPPER
CYSTEINE
DNA REPAIR
DNA-ASE
ELECTROMAGNETIC RADIATION
ELEMENTS
ENDONUCLEASES
ENZYME ACTIVITY
ENZYMES
ESCHERICHIA COLI
ESTERASES
HYDROLASES
INHIBITION
KINETICS
MERCURY
METABOLISM
METALS
MICROORGANISMS
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC SULFUR COMPOUNDS
PHOSPHODIESTERASES
PYRIMIDINE DIMERS
RADIATION EFFECTS
RADIATIONS
REACTION KINETICS
RECOVERY
REPAIR
SILVER
THIOLS
TRANSITION ELEMENTS
ULTRAVIOLET RADIATION