DNA glycosylase assay

To prepare the radiolabeled DNA substrate, the U-containing 25-mer oligonucleotide was 5′-end labeled with [μ-32P] ATP (specific activity 6,000 Ci/mmol; 1 Ci = 37 GBq, Amersham Pharmacia Biotech, Piscataway, NJ) and T4 polynucleotide kinase (United States Biochemical, Cleveland, OH) according to standard procedure. The ³²P-labeled oligomer was subsequently annealed to a complementary strand in a molar ratio of 1:1.5. A DNA cleavage assay was carried out as described previously^21,38. The reaction mixtures contained 2 nM ³²P-end labeled oligomer duplex in 10 mM HEPES-KOH (pH 7.4), 100 mM KCl, 0.1 mM DTT, and varying amounts of purified hUNG protein in a total volume of 10 μL. Metal ions were pre-incubated with hUNG for 10 min on ice prior to the addition of the substrate DNA. Further incubations were carried out for 30 min at 37 °C. In reactions using cell-free extracts, 0.5 μg of poly(dI-dC)-poly(dI-dC) (Amersham Pharmacia Biotech, Piscataway, NJ) was added to the reaction mixture as a nonspecific competitor. All glycosylase reactions were stopped by adding 5 μL alkaline buffer (300 mM NaOH, 90% formamide) followed by heating the samples at 95–100 °C for 3 min. Reaction products were resolved on a 7 M urea 12% denaturing PAGE with a 5′ ³²P-labeled 5-mer marker. The gel was subsequently dried and scanned with the Bio-Rad FX Molecular PhosphorImager (Hercules, CA). For band quantitation, Quantity One software (version 4.0.1) was used according to the manufacturer’s instructions.