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

Participation of the N-terminal extension of human APE1 in binding the intact and incised AP site-containing DNA was demonstrated by chemical footprinting assay [17]. Recently, oligomerization of APE1 on undamaged DNA and its dependence on the presence of the N-terminal 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 APE1N∆35 and APE1N∆61. 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 APE1N∆35 and APE1N∆61 (Table 3). At concentrations of APE1/APE1N∆35/APE1N∆61 around the EC<sup>50</sup> 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 APE1N∆35 and APE1N∆61. 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 (mono- and/or oligomerization) depending on protein concentration. *Int. J. Mol. Sci.* **2020**, *21*, x 10 of 19

**Figure 7.** Comparison of APE1 and its N-terminally truncated forms in binding AP site-containing DNA. FAM-labelled AP-DNA was incubated with increasing concentrations of APE1/APE1NΔ35/APE1NΔ61 in the absence of metal ions. After incubation, protein–DNA complexes were separated from free DNA by native gel electrophoresis as described in Materials and Methods. Data are representative of three independent experiments. **Figure 7.** Comparison of APE1 and its N-terminally truncated forms in binding AP site-containing DNA. FAM-labelled AP-DNA was incubated with increasing concentrations of APE1/APE1N∆35/APE1N∆61 in the absence of metal ions. After incubation, protein–DNA complexes were separated from free DNA by native gel electrophoresis as described in Materials and Methods. Data are representative of three independent experiments.

**Table 3.** Parameters of AP-DNA binding by full-length and truncated forms of APE1.


APE1NΔ61 0.21 ± 0.03 0.84 ± 0.03 a Parameters derived from electrophoretic-mobility-shift-assay (EMSA) data as described in Materials and Methods; EC50 is the effective protein concentration, at which the extent of DNA <sup>a</sup> Parameters derived from electrophoretic-mobility-shift-assay (EMSA) data as described in Materials and Methods; EC<sup>50</sup> is the effective protein concentration, at which the extent of DNA binding is half of the maximal extent. Values are the mean (± SD) of at least three independent experiments.

binding is half of the maximal extent. Values are the mean (± SD) of at least three independent

BER is an exceptionally efficient process evolved by mammalian cells to correct the most abundant DNA lesions. The BER process can proceed along one of different subpathways that involve distinct enzymes and accessory proteins/cofactors (Figure S3). The efficient repair of damaged DNA via the multistep process of each of the subpathways requires the coordinated action of enzymes catalyzing the sequential individual reactions [2–4]. Coordination is facilitated by multiple protein–protein interactions, reviewed previously [13,44]. Physical interaction and functional interplay between two major BER enzymes, APE1 and Polβ, were shown by various approaches [13,31,38,41,45]. XRCC1 functioning as a nonenzymatic scaffold protein directly interacts with multiple enzymatic components of BER, using all structural domains and flexible linkers [13,46]. PARP1, responsible for assembling DNA repair complexes via automodification at sites of DNA damage, forms direct and PAR-mediated contacts with various BER proteins, and modulates the catalytic activities of BER enzymes [4,13,35,40,41,47,48]. Physical and functional interaction between APE1 and PARP1 detected by various in vitro techniques as well as ADP-ribosylation of APE1 catalyzed by PARP1 via the unusual mechanism controlled by the structure of damaged DNA provide evidence of functional assistance between the proteins during DNA repair [28,41,45,49–53]. Here, we explored the contribution of the N-terminal extension of human APE1 to the interaction with Polβ, XRCC1, and PARP1, using fluorescence-based quantitative techniques. The eukaryote-specific extension of APE1 (residues 1–61) is dispensable for endonuclease activity [16], but essential for DNA binding and acetylation-mediated modulation of the enzyme DNA repair activity in vitro and in vivo [17–19,21]. Our data, obtained under true equilibrium conditions, clearly showed that the removal of the entire N-terminal extension (APE1NΔ61) or its mammalian-conserved fragment (APE1NΔ35) decreased the affinity of APE1 for all three proteins to small but measurable extents (Table 1). The small effects produced by N-terminal truncation (less than two-fold) indicate that the C-terminal catalytic core of human APE1, highly conserved through evolution, mainly contributes to the interaction with proteins, and the N-terminal extension forms

## **3. Discussion**

BER is an exceptionally efficient process evolved by mammalian cells to correct the most abundant DNA lesions. The BER process can proceed along one of different subpathways that involve distinct enzymes and accessory proteins/cofactors (Figure S3). The efficient repair of damaged DNA via the multistep process of each of the subpathways requires the coordinated action of enzymes catalyzing the sequential individual reactions [2–4]. Coordination is facilitated by multiple protein–protein interactions, reviewed previously [13,44]. Physical interaction and functional interplay between two major BER enzymes, APE1 and Polβ, were shown by various approaches [13,31,38,41,45]. XRCC1 functioning as a nonenzymatic scaffold protein directly interacts with multiple enzymatic components of BER, using all structural domains and flexible linkers [13,46]. PARP1, responsible for assembling DNA repair complexes via automodification at sites of DNA damage, forms direct and PAR-mediated contacts with various BER proteins, and modulates the catalytic activities of BER enzymes [4,13,35,40,41,47,48]. Physical and functional interaction between APE1 and PARP1 detected by various in vitro techniques as well as ADP-ribosylation of APE1 catalyzed by PARP1 via the unusual mechanism controlled by the structure of damaged DNA provide evidence of functional assistance between the proteins during DNA repair [28,41,45,49–53].

Here, we explored the contribution of the N-terminal extension of human APE1 to the interaction with Polβ, XRCC1, and PARP1, using fluorescence-based quantitative techniques. The eukaryote-specific extension of APE1 (residues 1–61) is dispensable for endonuclease activity [16], but essential for DNA binding and acetylation-mediated modulation of the enzyme DNA repair activity in vitro and in vivo [17–19,21]. Our data, obtained under true equilibrium conditions, clearly showed that the removal of the entire N-terminal extension (APE1N∆61) or its mammalian-conserved fragment (APE1N∆35) decreased the affinity of APE1 for all three proteins to small but measurable extents (Table 1). The small effects produced by N-terminal truncation (less than two-fold) indicate that the C-terminal catalytic core of human APE1, highly conserved through evolution, mainly contributes to the interaction with proteins, and the N-terminal extension forms an additional low-affinity binding site. Our results contradict previous data that demonstrated that the first 35 residues are absolutely required for APE1–XRCC1 interaction assayed by far-Western blotting [27]. This discrepancy could have resulted from disadvantages of the far-Western blotting assay: A significantly lower isoelectric point of APE1N∆35 (6.69 vs. 8.33 for full-length APE1, Figure 1) might reduce efficiency of protein electrotransfer to nitrocellulose membrane [54]. The considerable part of the eukaryote-specific extension of APE1 is disordered and invisible in the crystal structure of the full-length protein [55]. Numerous studies showed that disordered protein regions are involved in dynamic interactions, contributing little to overall binding energy, but being essential for regulatory functions [56,57].

Disordered terminal tails characterized by clustering of positively charged residues are present predominantly in mammalian DNA-binding proteins [56]. They were proposed to have similar functions in the initial scanning of DNA by BER proteins via transient electrostatic binding, which is followed by specific binding of the lesion in the active site [56]. The N-terminus of human APE1 was shown to stabilize interaction with nonspecific RNA/DNA structures [18,20] and to increase the extent of APE1 oligomerization on undamaged long DNA [43]. Its involvement in binding a specific AP site-containing DNA substrate was demonstrated by protein footprinting with a lysine-reactive probe [16]. In our EMSA binding experiments, we detected a metastable, unique to the full-length APE1, complex with AP-DNA, which accumulated along with the most stable complex formed independently of N-terminal truncation (Figure 7). Compared to APE1, the truncated APE1N∆35 and APE1N∆61 forms were shown to form less stable monomeric and oligomeric complexes. These results provide the first quantitative evidence that the N-terminal extension forms an additional low-affinity DNA binding site in the whole protein and contributes to the stabilization of various forms of the APE1–DNA complex.

Previously, we demonstrated that the strength of APE1 interaction with Polβ, XRCC1, and PARP1, and the structure of APE1–protein complexes are modulated by model BER DNA intermediates to

different extents depending on the type of damaged DNA [28]. This finding motivated us to explore the involvement of eukaryote-specific extension in this modulation, particularly in light of its capacity to dynamically bind both the DNA and the protein partners. The distinct effects produced by various DNA intermediates on protein–protein affinity binding constants of the full-length APE1 turned out to not be distinguishable for the N-terminally truncated forms (Table 2). These results indicate that the N-terminal extension of human APE1 plays a primary role in the DNA-dependent modulation of the strength of APE1 interaction with both the downstream enzyme and the accessory proteins of BER. This role is obviously related to the coordination of enzymatic functions during BER that is governed by DNA-binding specificity and protein–protein interactions. The interaction between APE1 and Polβ is stronger in the presence of intact AP-DNA than in the complex mimicking a step after the APE1-catalyzed incision, suggesting that the incised DNA intermediate is more effectively passed to Polβ immediately during the incision step. The conformationally flexible N-terminus of APE1, being highly adaptable to both the protein and DNA structure, may differently modulate direct and DNA-mediated protein–protein interactions in the ternary complex with the specific DNA depending on the relative affinities of the protein partners for the DNA. Indeed, changes in FRET signals reflecting structural DNA-induced rearrangements of the APE1–Polβ complex are quite dissimilar for different DNA intermediates. Footprinting experiments showed that intact and incised AP-DNAs protect the N-terminus of APE1 in accordance with the different stabilities of the complexes, but their protective action was undetectable in the ternary complexes with Polβ [17], suggesting higher flexibility of the N-terminal extension in the APE1–DNA–Polβ complexes than in the respective APE1–DNA complexes. The product release is the rate-limiting step of the APE1-catalyzed AP site incision, and this limitation can be overcome by the stimulatory action of Polβ [39]. Involvement of Polβ and PARP1 in the dynamics of APE1 function in vivo was recently shown [58]. Here, we showed that the major function of APE1 is also activated by XRCC1 (Figure 5), previously detected to stimulate the 30 -diesterase activities of APE1 [27]. A comparison of the effects produced by Polβ, XRCC1, and PARP1 on the activity of APE1 and APE1N∆61 revealed more efficient regulation by all proteins (present separately) for the full-length enzyme. These results provide further evidence that the N-terminal extension of APE1 is involved in interactions with canonical BER proteins promoting the coordination of the process.

APE1 enhances the 5'-deoxyribose phosphate(dRp)-lyase and nucleotidyltransferase activities of Polβ [31,41]. Our comparison of APE1 and APE1N∆61 in modulating the catalytic activity of Polβ in DNA synthesis, tested on the canonical substrate of short-patch BER, revealed no dependence of the stimulatory function of APE1 on the integrity of its N-terminal portion (Figure 6). No appreciable difference between the two APE1 forms was detected when their effects were compared in the presence of XRCC1, capable of forming the ternary complex with these enzymes [28]. The question of whether the N-terminal extension of APE1 contributes to the functional coupling with Polβ upon the processing of other DNA intermediates, more efficient substrates of APE1 in the 30 -50 -exonuclease reaction [59], remains open for future research.

APE1 stimulates the catalytic activities of different DNA glycosylases, upstream BER enzymes, via accelerating product release [43,60–64]. The coordination mechanism via protein–protein interactions was proposed for TDG (thymine DNA glycosylase) and MYH (MutY homolog DNA glycosylase) [60,62]; binding sites for MYH do not involve the N-terminal extension of APE1 [62]. Other studies suggested no physical interaction between APE1 and DNA glycosylases. The importance of the N-terminal extension of APE1 for the stimulatory function is explained by its participation in protein oligomerization along the DNA, proposed to promote dissociation of DNA glycosylases from the complex with product [43,64]. Recently we detected and characterized new, never previously predicted [65,66], activity of APE1 in binding poly(ADP-ribose) (PAR) [53]. Both the N-terminal extension and the conserved catalytic core of APE1 were found to be involved in PAR binding, with the first contributing for the most part to the interaction with small linear polymers. We propose that the interaction of APE1 with PAR, found to be less efficient as compared to that of XRCC1, may contribute to assembling DNA repair complexes

during the PARP1-dependent processing of SSBs. Present and previous studies provide evidence that the N-terminal extension of APE1 may perform multiple, diverse functions in the coordination of BER.
