Versadoc gel documenting system

To determine the effect of the studied amino acid substitutions on the stage of enzyme binding to a DNA substrate containing a gap, the electrophoretic mobility shift assay was used. The reaction was carried out in a buffer composed of 50 mM Tris-HCl pH 7.5, 50 mM KCl, 1 mM Na₂EDTA, 5 mM MgCl₂, 1 mM DTT, and 7% glycerol. The recombinant enzymes were serially diluted; for the Polβ L19P polymorphic variant, the reaction was carried out in the concentration range from 57 nM to 7.3 μM, and for the G66R polymorphic variant, from 55.5 nM to 7.1 μM. The samples were incubated for 15 min at room temperature and applied to a nondenaturing 10% polyacrylamide gel (PAAG; the ratio of acrylamide to N,N′-methylenebisacrylamide was 75:1).
To determine the dissociation constant, the resultant gel was visualized in a VersaDoc gel-documenting system (Bio-Rad Laboratories, Hercules, CA, USA). The results were processed using Gel-Pro Analyzer 4 software (Media Cybernetics, Rockville, MD, USA). Dissociation constant K_d for each enzyme–DNA complex was computed in OriginPro 8 software via the following equation:
where h is the Hill coefficient, Fu is the correction for background illumination, and Fb is the maximum intensity of the complex.

The dRP-lyase reaction substrate was generated using 3′-FAM-labeled 36 bp U-containing DNA substrate (5′-GCCTCGCAGCGGTCCAACCUTAGTCACCTCAATCCA-FAM/5′-TGGATTGAGGTGACTAGGGTTGGACGGCTGCGAGGC) treated with Udg and APE1 enzymes for 30 min at 37 °C in buffer containing 50 mM Tris-HCl pH 7.5, 50 mM KCl, 1 mM Na₂EDTA, 5 mM Mg²⁺, 1 mM DTT, and 7% glycerol. The reaction mixture was purified by gel filtration with Sephadex G-25 400 μL columns. The resulting dRP-substrate was mixed with WT Polβ, L19P, or G66R variants, supplemented with 50 mM Tris-HCl pH 7.5, 50 mM KCl, 1 mM Na₂EDTA, 5 mM Mg²⁺, 1 mM DTT, and 7% glycerol buffer, and incubated at 20 °C. The reaction was stopped by incubation on ice with 340 mM NaBH₄ for 30 min. The excess of NaBH₄ was deleted by gel filtration with Sephadex G-25 400 μL columns containing 7.5 M urea, 0.1% bromophenol blue, and 0.1% xylene cyanol dyes. The reaction product was separated in 20% TBE-urea PAAG at 500 V (constant V) for 5 h. The resulting gel was visualized in the VersaDoc gel-documenting system (Bio-Rad Laboratories, Hercules, CA, USA).