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

• Published in: The Journal of biological chemistry 1991-06, Vol.266 (16), p.10686-10693
• Main Authors: M A Prince, B Friedman, E A Gruskin, R D Schrock, 3rd, R S Lloyd
• Format: Article
• Language: English
• Subjects: 560130 - Radiation Effects on Microorganisms; 560300 - Chemicals Metabolism & Toxicology; AMINO ACIDS; Analytical, structural and metabolic biochemistry; BACTERIA; BIOCHEMICAL REACTION KINETICS; Biological and medical sciences; BIOLOGICAL EFFECTS; BIOLOGICAL FUNCTIONS; BIOLOGICAL RADIATION EFFECTS; BIOLOGICAL RECOVERY; BIOLOGICAL REPAIR; Blotting, Western; CARBOXYLIC ACIDS; CATALYSIS; COPPER; CYSTEINE; Cysteine - metabolism; Deoxyribonuclease (Pyrimidine Dimer); DNA - metabolism; DNA Glycosylases; DNA REPAIR; DNA-ASE; DNA-Binding Proteins - metabolism; ELECTROMAGNETIC RADIATION; ELEMENTS; Endodeoxyribonucleases - antagonists & inhibitors; Endodeoxyribonucleases - isolation & purification; Endodeoxyribonucleases - metabolism; ENDONUCLEASES; ENZYME ACTIVITY; ENZYMES; Enzymes and enzyme inhibitors; ESCHERICHIA COLI; Escherichia coli - enzymology; Escherichia coli - radiation effects; ESTERASES; Fundamental and applied biological sciences. Psychology; HYDROLASES; INHIBITION; KINETICS; Mercuric Chloride - metabolism; MERCURY; METABOLISM; METALS; Metals - metabolism; MICROORGANISMS; N-Glycosyl Hydrolases - antagonists & inhibitors; ORGANIC ACIDS; ORGANIC COMPOUNDS; ORGANIC SULFUR COMPOUNDS; phage T4; PHOSPHODIESTERASES; PYRIMIDINE DIMERS; Pyrimidines - metabolism; RADIATION EFFECTS; RADIATION, THERMAL, AND OTHER ENVIRON. POLLUTANT EFFECTS ON LIVING ORGS. AND BIOL. MAT; RADIATIONS; REACTION KINETICS; RECOVERY; REPAIR; SILVER; Substrate Specificity; THIOLS; TRANSITION ELEMENTS; ULTRAVIOLET RADIATION; Viral Proteins
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(18)99278-1

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 by virtue of its ability to function in the presence of metal-chelating agents. 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.