Uracil-DNA glycosylase of thermophilic Thermothrix thiopara

Abstract

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.