*2.3. Influence of N-Terminal Truncation of APE1 on Functional Cooperation between BER Proteins*

Numerous studies showed that the enzymatic activities of APE1 on various DNA intermediates of BER are modulated by protein partners: The AP-endonuclease activity by Polβ and PARP1, and the 30 -50 -exonuclease activity by Polβ, PARP1, and XRCC1 [31,38–42]. The nucleotidyltransferase activity of Polβ is stimulated by APE1 and XRCC1, and inhibited by PARP1 [37–39,41,42]. In light of our results described above, we explored the possible involvement of the N-terminal extension of APE1 in the functional coupling of BER proteins to each other. First, we examined the influence of Polβ, XRCC1, and PARP1 on the AP endonuclease activity of APE1N∆61 in comparison with the full-length enzyme (Figure 5). The activity of APE1 and APE1N∆61 was enhanced by XRCC1 and inhibited by PARP1 in a concentration-dependent manner (Figure 5A). The effects observed for the full-length APE1 exceeded those for the truncated form at all tested concentrations of XRCC1 and PARP1. Stimulation of the AP endonuclease activity by Polβ shown previously by others [39] was detectable only for the full-length APE1 (Figure 5B). The stimulating effects produced by XRCC1 and Polβ present separately or together were fully suppressed by the addition of PARP1. The differences in effects detected for APE1 and APE1N∆61 in the presence of all binary combinations of the protein partners were statistically insignificant. At the same time, the inhibiting effect on the activity of APE1 detected in the presence of the ternary protein combination was higher than that for APE1N∆61. Thus, the N-terminal extension of APE1 being not essential for the major catalytic activity of APE1 may potentially contribute to the regulation of this activity by the BER protein partners. in effects detected for APE1 and APE1NΔ61 in the presence of all binary combinations of the protein partners were statistically insignificant. At the same time, the inhibiting effect on the activity of APE1 detected in the presence of the ternary protein combination was higher than that for APE1NΔ61. Thus, the N-terminal extension of APE1 being not essential for the major catalytic activity of APE1 may potentially contribute to the regulation of this activity by the BER protein partners.

Polβ present separately or together were fully suppressed by the addition of PARP1. The differences

*Int. J. Mol. Sci.* **2020**, *21*, x 8 of 19 Numerous studies showed that the enzymatic activities of APE1 on various DNA intermediates of BER are modulated by protein partners: The AP-endonuclease activity by Polβ and PARP1, and the 3*′*-5*′*-exonuclease activity by Polβ, PARP1, and XRCC1 [31,38–42]. The nucleotidyltransferase activity of Polβ is stimulated by APE1 and XRCC1, and inhibited by PARP1 [37–39,41,42]. In light of our results described above, we explored the possible involvement of the N-terminal extension of APE1 in the functional coupling of BER proteins to each other. First, we examined the influence of Polβ, XRCC1, and PARP1 on the AP endonuclease activity of APE1NΔ61 in comparison with the full-length enzyme (Figure 5). The activity of APE1 and APE1NΔ61 was enhanced by XRCC1 and inhibited by PARP1 in a concentration-dependent manner (Figure 5A). The effects observed for the full-length APE1 exceeded those for the truncated form at all tested concentrations of XRCC1 and

**Figure 5.** Comparison of effects produced by Polβ, XRCC1, and PARP1 on the AP endonuclease activity of APE1 and APE1NΔ61. Activity in incision of AP-DNA was measured in the absence or presence of Polβ, XRCC1, and PARP1, added separately at varied concentrations (indicated on the X axis, (**A**) or in different combinations at a constant concentration of each (200 nM) (**B**). In each set of experiments, activities were determined as initial rates of AP-DNA incision normalized to that of APE1/APE1NΔ61 in the absence of other proteins (taken as 100%). One representative experiment of the activity measurements is shown in Figure S2. Data are the mean (± SD) of three independent measurements. Effects detected for APE1NΔ61, which were statistically different from those for APE1, are marked *p* < 0.05 (\*), *p* < 0.01 (\*\*). We next compared APE1 and APE1NΔ61 in modulating the nucleotidyltransferase activity of **Figure 5.** Comparison of effects produced by Polβ, XRCC1, and PARP1 on the AP endonuclease activity of APE1 and APE1N∆61. Activity in incision of AP-DNA was measured in the absence or presence of Polβ, XRCC1, and PARP1, added separately at varied concentrations (indicated on the X axis, (**A**) or in different combinations at a constant concentration of each (200 nM) (**B**). In each set of experiments, activities were determined as initial rates of AP-DNA incision normalized to that of APE1/APE1N∆61 in the absence of other proteins (taken as 100%). One representative experiment of the activity measurements is shown in Figure S2. Data are the mean (± SD) of three independent measurements. Effects detected for APE1N∆61, which were statistically different from those for APE1, are marked *p* < 0.05 (\*), *p* < 0.01 (\*\*).

of products of gap-filling and strand-displacement DNA synthesis produced by the full-length and truncated forms of APE1 were very similar. No appreciable difference between the two APE1 forms was detected when their influence on the Polβ-catalyzed DNA synthesis in the presence of XRCC1, a strong binding partner of Polβ [28], was compared. Data suggested that the N-terminal extension of APE1 is not essential for the stimulation of Polβ-catalyzed DNA repair synthesis. We next compared APE1 and APE1N∆61 in modulating the nucleotidyltransferase activity of Polβ, using gap-DNA as the substrate (Figure 6). The concentration-dependent effects on the yields of products of gap-filling and strand-displacement DNA synthesis produced by the full-length and truncated forms of APE1 were very similar. No appreciable difference between the two APE1 forms was detected when their influence on the Polβ-catalyzed DNA synthesis in the presence of XRCC1, a strong binding partner of Polβ [28], was compared. Data suggested that the N-terminal extension of APE1 is not essential for the stimulation of Pol *Int. J. Mol. Sci.* **2020**, *21*, x β-catalyzed DNA repair synthesis. 9 of 19

Polβ, using gap-DNA as the substrate (Figure 6). The concentration-dependent effects on the yields

**Figure 6.** Comparison of effects produced by APE1 and APE1NΔ61 on Polβ activity. Polβ activity in DNA synthesis was measured on gap-DNA in the absence or presence of APE1, APE1NΔ61, and XRCC1, added separately at varied concentrations (100–500 nM) or together at a constant concentration (100 nM APE1/APE1NΔ61 and 100 nM XRCC1). Histograms present relative amounts of substrate (S) and products of gap-filling (S+1) and strand-displacement synthesis (S+2 to S+10). Reaction was performed as described in Materials and Methods. Data are representative of three independent experiments with very similar results. (mono- and/or oligomerization) depending on protein concentration. **Figure 6.** Comparison of effects produced by APE1 and APE1N∆61 on Polβ activity. Polβ activity in DNA synthesis was measured on gap-DNA in the absence or presence of APE1, APE1N∆61, and XRCC1, added separately at varied concentrations (100–500 nM) or together at a constant concentration (100 nM APE1/APE1N∆61 and 100 nM XRCC1). Histograms present relative amounts of substrate (S) and products of gap-filling (S+1) and strand-displacement synthesis (S+2 to S+10). Reaction was performed as described in Materials and Methods. Data are representative of three independent experiments with very similar results.

extension was revealed by electron microscopy analysis [43]. However, there are no quantitative data on the relative contribution of the N-terminal extension and the conserved catalytic core to the protein affinity for the AP-DNA substrate. Here, we compared the full-length and truncated forms of APE1 in their modes and strength of AP-DNA binding using electrophoretic-mobility-shift assay (EMSA). Three types of complexes with different electrophoretic mobilities were detected for APE1 (Figure 7), while only two of them, designated as Complexes 1 and 3, could be visualized for APE1NΔ35 and APE1NΔ61. Fast migrating Complex 1 obviously represents a monomeric protein– DNA complex that is stabilized by interactions between AP-DNA and the conserved catalytic portion of APE1. Slow migrating Complex 3 most likely results from oligomerization of the monomeric complex via protein–protein interactions as previously proposed [43]. Complex 2 with intermediate mobility, formed exclusively by the full-length protein, is obviously stabilized by interactions between DNA and the N-terminal extension of APE1; the positively charged N-terminus may additionally neutralize the negative charge of DNA, thereby decreasing complex mobility. Another possibility is the dimerization of APE1 on the DNA, which is highly unstable in the absence of the N-terminus to be detected by the nonequilibrium EMSA technique. The affinity of APE1 for DNA estimated from the protein-concentration dependence of the total amount of bound DNA is 1.8-fold higher as compared to those of APE1NΔ35 and APE1NΔ61 (Table 3). At concentrations of APE1/APE1NΔ35/APE1NΔ61 around the EC50 value, the only detectable complex was Complex 1, indicating a lower stability of Complexes 2 and 3. Oligomerization was visualized at lower concentrations of APE1 as compared to those of APE1NΔ35 and APE1NΔ61. Our data provide quantitative evidence that the N-terminal extension of APE1 contributes to the additional stabilization of the complex with AP-DNA, and may control the mode of protein–DNA association

Participation of the N-terminal extension of human APE1 in binding the intact and incised AP

*2.4. Contribution of N-Terminal Extension of APE1 to AP-DNA Binding* 

experiments.

**3. Discussion** 
