Imagequant software v5

UDG activity was determined in a reaction mixture (10 µl) that contained 25 mM Hepes–KOH (pH 8.0), 50 mM KCl, 1 mM DTT, 0.5 mM EDTA, 0.1 mg/ml BSA, and 1 nM 5′-end labeled duplex DNA substrates containing a uracil residue. The reactions were initiated by adding 50 ng cell extract and then incubated at 37 ℃. Aliquots of each reaction were withdrawn at the 0, 3, 5, 10, 20, and 30 min. The reaction was terminated by transferring the reaction mixture to 0 ℃. The apurinic/apyrimidinic (AP) site generated by either enzymatic uracil removal from the DNA substrates was hydrolyzed by adjusting the mixture to 100 mM NaOH and then incubating at 70 ℃ for 10 min. The reactions were terminated by adding the formamide-loading buffer (95% formamide, 20 mM EDTA, 0.01% bromophenol blue, and 0.01% xylene cyanol). The DNA products were resolved using electrophoresis in a 15% denaturing polyacrylamide gel containing 7 M urea in 90 mM Tris, 90 mM boric acid, and 2 mM EDTA. The gels were dried using a gel dryer (Bio-Rad, CA, USA), and the product was visualized using autoradiography and quantified using the ImageQuant software v5.2 (Molecular Dynamics, CA, USA). The percentage of cleaved uracil residues was calculated from the number of products divided by the sum of total products and substrates.

A reaction mixture containing 10 nM oligonucleotide substrate in the ligation buffer (5 mM MgCl₂, 40 mM Hepes–KOH (pH7.8), 0.5 mM DTT, 2 mM ATP, 0.36 mg/ml of BSA) and 1 μg of cell lysate was incubated at 37 ℃ for various periods. The reaction was terminated by the addition of formamide loading buffer (95% formamide, 20 mM EDTA, 0.01% bromophenol blue, 0.01% xylene cyanole) followed by incubation at 95 ℃ for 5 min. T4 ligase (New England Biolabs, UK) was used as a positive control, and no protein was added in the negative control. The DNA products were resolved using electrophoresis in a 15% denaturing polyacrylamide gel containing 7 M urea in TBE buffer (9 mM Tris, 9 mM boric acid, 0.2 mM EDTA).Gels were dried using a gel dryer (Bio-Rad, CA, USA), and the product was visualized using autoradiography and quantitated using the ImageQuant software v5.2 (Molecular Dynamics, CA, USA).