Abstract
A uracil-DNA glycosylase has been purified over 1,000-fold from wheat germ, the first such repair activity isolated from a higher plant. The enzyme has a molecular weight of approximately 27,000 and is resistant to metal ion chelators, but inhibited by high concentrations of either mono or divalent cations. This glycosylase is unable to release uracil from the mononucleotides dUMP and dUTP or from wheat germ RNA. Twelve pyrimidine analogues which closely mimic uracil structurally and the nucleoside uridine were examined for their ability to inhibit glycosylase activity. However, only 5-azauracil and 6-aminouracil inhibited enzymatic release of uracil to the same degree as uracil itself. An inhibitor induced by bacteriophage T5 which inhibits Escherichia coli uracil-DNA glycosylase has been shown not to affect the glycosylase isolated from wheat germ, indicating that these two enzymes differ. The ability of the wheat germ uracil-DNA glycosylase to completely remove available uracil from synthetic DNA substrates in which thymine had been replaced by uracil in varying percentages was also examined and found not to depend on percentage of uracil in the substrates.
Highlights
Molecular weight of approximately 27,000 and is resist- This report details the characterization of one such uracilant to metal ion chelators, but inhibited by high con- D N A glvcosylase, purified over 1000-fold from wheat germ
Preparation of Crude Extract-All procedures were carried out at 0-4 "C unless otherwise stated. 500 g of wheat germ were suspended in 2 liters of buffer containing 0.01 M potassium phosphate, pH7.3, 15%glycerol, a n d 1mM dithiothreitol (PGD buffer) and homogenized for 1 min at high speed in a Waring blender
Inhibition by Wheat Germ RNA Species-The results described above indicate that while uracil is not released from ml) or wheat germ RNA (1.2 mg/ml) was added to the standardassay medium except that the T5 DNA substrate was replaced by labeled calf thymus DNA. 0-4,wheat germ RNA; 0--0,calf thymus DNA
Summary
Preparation of Crude Extract-All procedures were carried out at 0-4 "C unless otherwise stated. 500 g of wheat germ were suspended in 2 liters of buffer containing 0.01 M potassium phosphate, pH7.3, 15%glycerol, a n d 1mM dithiothreitol (PGD buffer) and homogenized for 1 min at high speed in a Waring blender. T h e 50-80% fraction was found to contain most uracil-DNA glycosylase activity and was redissolved75i0nml of PGD buffer and dialyzed against this buffer overnight to remove ammonium sulfate. Phosphocellulose Chromatography-The combined fractions containing uracil-DNA glycosylase activity were loaded onto a phosphocellulose column (2.7 X 30 cm) equilibrated with PGD buffer. Fractions containing peak activity were pooled, and the protein was precipitated witha saturating concentration of solid ammonium sulfate followed by centrifugation at 15,000 X g for 15min. A linear 0.01-0.4 M potassiumphosphategradientwasused.Peakglycosylase fractions were pooled, and the protein was precipitated with a saturating concentrationof solid ammonium sulfate followed by centrifugation. Second Phosphocellulose Chromatography-The concentrated enzyme was applied to a phosphocellulose column (1 X 3 cm) previously equilibrated with PGD buffer and washed with 5 column volumesof this buffer prior to elution of enzyme activity with a linear 0.01-0.4 M potassium phosphate gradient. Apurinicendonuclease activity was less than 0.1% of glycosylase activity after the final stage of purification
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