An endonuclease specific for apurinic, apyrimidinic (AP) sites in DNA was purified nearly to homogeneity from the extremely thermophilic bacterium Thermothrix thiopara. The enzyme has a molecular weight of approximately 26,000. It cleaves neither native nor UV-or y-irradiated DNAs and has no contaminating exonuclease or uracil-DNA glycosylase activities. The enzyme has no cofactor requirement and is not inhibited by EDTA or N'-ethylmaleimide. It shows maximal activity at 70°C and a pH between 7.5 and 9.0. The Arrhenius activation energy of the reaction is 17 kJ/mol, and the apparent Km for AP sites is 38 nM. The rate of heat inactivation of the enzyme followed first-order kinetics, with a half-life of 10 min at 700C but about 150 min in the presence of 0.5 M ahtmonium sulfate or 0.5 mg of bovine serum albumin per ml at the same temperature. One cell of T. thiopara contains sufficient AP endonuclease activity for hydrolysis of about 10' phosphodiester bonds per, h at 70TC. An extract of these bacteria does not contain detectable Mg-dependent AP endonuclease activity, and the above-mentioned enzyme appears to be the main AP endonuclease of T. thiopara. Apurinic, apyrimidinic (AP) sites in DNA are formed by spontaneous hydrolysis of base-sugar bonds or by the action of DNA glycosylases (13, 15). The mechanism of repair of
An activity which released free uracil from dUMP-containing DNA was purified approximately 1,700-fold from extracts of Thermothrix thiopara, the first such activity to be isolated from extremely thermophilic bacteria. The enzyme appeared homogeneous, according to the results of sodium dodecyl sulfatepolyacrylamide gel electrophoresis. It had a native molecular weight of 26,000 and existed as a monomer protein in water solution. The enzyme had an optimal activity at 70°C, between pH 7.5 and 9.0, and in the presence of 0.2% Triton X-100. It had no cofactor requirement and was not inhibited by EDTA, but it was sensitive to N-ethylmaleimide. The purified enzyme did not contain any nuclease that acted on native or depurinated DNA. The Arrhenius activation energy was 76 kJ/mol between 30 and 50°C and 11 kJ/mol between 50 and 70°C. The rate of heat inactivation of the enzyme followed first-order kinetics with a half-life of 2 min at 70°C. Ammonium sulfate and bovine serum albumin protected the enzyme from heat inactivation. One T. thiopara cell contains enough activity to release about 2 x 108 uracil residues from DNA during one generation time at 70°C.
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