2.4. Preparation of ds-Oligonucleotides with AP Sites

The radiolabeled, dry ds-oligos with dU in their sequence were incubated with uracil-DNA glycosylase (UDG) (5U, New England BioLabs Ipswich, MA, USA), which recognizes and removes dU from DNA. Reactions were performed in 20 μL of the reaction buffer (20 mM Tris-HCl, 1 mM EDTA, 1 mM DTT, pH 8.0 at 25 °C) at 37 °C for 30 min. The ds-oligos containing AP sites were purified by precipitation (see Section 2.8). Additionally, the AP sites formation was verified. The ds-oligos with AP sites were incubated with human apurinic/apyrimidinic endonuclease (APE1) (5U, New England BioLabs Ipswich, MA, USA) in 10 μL of the reaction buffer (50 mM potassium acetate, 20 mM Tris-acetate, 10 mM magnesium acetate, 1 mM DTT, pH 7.9 at 25 °C) at 37 °C for 30 min to generate SSBs visible as shorter DNA fragments with a length of 15–25 nucleotides. The purity and formation of SSBs were checked on 15% denaturing polyacrylamide gel (Figure S1B).