*4.5. Electrophoretic-Mobility-Shift Assay*

The affinity of APE1, APE1N∆35, and APE1N∆61 for AP-DNA was measured by electrophoretic-mobility-shift assay (EMSA). A desired protein (added at increasing from 0.05 to 2–6 µM concentrations) was incubated with FAM-labelled AP-DNA (50 nM) in a 10 µL mixture containing 50 mM Tris-HCl, pH 8.0, 100 mM NaCl, and 1 mM DTT at 4 ◦C for 30 min. After the addition of glycerol and bromophenol blue (to a final concentration of 5% and 0.1%, respectively), the incubation mixtures were electrophoresed at 4 ◦C on 5% nondenaturing PAG (polyacrylamide gel) in a 30 mM Tris-Borate-EDTA buffer. Gels were imaged on a Typhoon FLA 9500. Free and complexed DNA bands were quantified using Quantity One Basic software. To subtract free DNA co-migrated with the protein-bound DNA (due to partial overlapping of their migration zones as a result of DNA smearing) the portion of free DNA in the protein-containing samples (determined from radioactivity distribution between migration zones assigned to the free DNA and its complexes with proteins) was normalized to the portion of free DNA in the control sample without the protein. Data were fitted to a Hill equation: <sup>θ</sup> <sup>=</sup> <sup>θ</sup>∞/[1 <sup>+</sup> (EC50/C)<sup>n</sup> ], where θ is the portion of bound DNA (calculated as the complex amount divided by total DNA amount) at a given concentration (C) of the protein, θ<sup>∞</sup> is the maximal extent of DNA binding (i.e., the portion of DNA bound at saturating protein concentration), EC<sup>50</sup> is the protein concentration at which θ = θ∞/2, and n is the Hill coefficient.

**Supplementary Materials:** Supplementary materials can be found at http://www.mdpi.com/1422-0067/21/9/3122/ s1. Figure S1. Structures of DNA ligands used in the study; Figure S2. Influence of Polβ, XRCC1, and PARP1 on the AP endonuclease activity of APE1 and APE1N∆61. Figure S3. Scheme illustrating stages of BER subpathways.

**Author Contributions:** Conceptualization, N.M.; methodology, N.M. and I.V.; validation, N.M. and I.V.; investigation, N.M. and I.V.; resources, N.M. and I.V.; writing—original draft preparation, N.M.; writing—review and editing, N.M., I.V., and O.L.; project administration, N.M. and O.L.; funding acquisition, O.L. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Russian Science Foundation, grant number 19-14-00107 to O.L.

**Acknowledgments:** The authors are thankful to S.H. Wilson, A.A. Ishchenko, J.P. Radicella, and M. Satoh for providing us with recombinant plasmids coding for APE1, Polβ, XRCC1, and PARP1, and to A.-L. Haenni (Jacques Monod Institute, France) for careful reading of the manuscript and useful comments.

**Conflicts of Interest:** The authors declare no conflict of interest.
