Figure 1.

a) Coomassie blue staining of purified WT and E96Q mutant APE1 proteins (0.5 µg protein) after SDS-PAGE (10% polyacrylamide); lane 1, marker; lane 2, WT APE1; and lane 3, E96Q proteins. b) AP-endonuclease activity was measured using 43-mer, THF- containing oligonucleotide. WT APE1 (0.1 nM) was incubated for 3 min at 37 °C as shown in lanes 2, 3, and 4 using a buffer containing 50 mM Tris-HCl (pH 8.0), 50 mM KCl and in absence or in presence of 2 mM Mg²⁺ respectively. The endonuclease activity of E96Q (1.0 nM) mutant APE1 was measured in the absence or in presence of 2, 5, and 10 mM Mg²⁺ as shown in lanes 5, 6, 7, and 8 respectively for 30 min at 37 °C. c) EMSA of THF-DNA binding by WT and E96Q (0.1 nM) mutant APE1 proteins. Lane 1, no protein; lane 2, WT APE1 without Mg²⁺; lanes 3 & 4, with 5 and 10 mM Mg²⁺ respectively; lane 5, no protein; lane 6, E96Q without Mg²⁺; lanes 7 & 8, with 5 and 10 mM Mg²⁺ respectively. While lanes, 2 & 6 supplemented with 2 mM EDTA. The data represent three or more independent experiments.