Fluorescently labeled human apurinic/apyrimidinic endonuclease APE1 reveals effects of DNA polymerase β on the APE1–DNA interaction

Genomic DNA in the live cell constantly undergoes various molecular lesions due to endogenous and exogenous factors [1]. The most common type of DNA damage is a single-base lesion that involves the loss of a base through spontaneous hydrolysis [2], base oxidation [3], [4], [5], [6], or alkylation [7], [8], [9], [10] during physiological metabolism or modification by endogenous DNA-damaging agents. These types of DNA lesion are mutagenic and therefore are repaired in live cells. These lesions can arise both in single-stranded and in double-stranded DNA [11]. In the single-stranded DNA context, they can be repaired by homologous recombination [12], [13] and template switching [14], [15], [16] mechanisms. By contrast, in double-stranded DNA, the single-base lesions are processed mainly by BER [17], [18], [19], [20], [21].

BER is a multistep process initiated by spontaneous or DNA glycosylase–initiated base loss. In the last case, DNA glycosylase recognizes and removes a damaged base, thereby generating an apurinic/apyrimidinic (AP) site [22], [23]. Subsequently, the damaged DNA strand is incised by apurinic/apyrimidinic endonuclease 1 (APE1) on the 5′ side of the AP site to generate 3′-hydroxyl and 5′-deoxyribose phosphate (dRP) groups at the ends [24], [25], [26], [27]. Then, DNA polymerase β (Polβ) adds a single nucleotide to the 3′-hydroxyl and removes the dRP group by means of intrinsic dRP lyase activity [28], [29], [30], [31], [32]. Alternatively, Polβ can insert 2–12 nucleotides thus creating a multinucleotide 5′-flap that is consequently cleaved off by flap endonuclease 1 (FEN1) [33], [34]. The first subpathway including only single-nucleotide replacement is referred to as short-patch BER. Another subpathway including ≥2-nucleotide replacement and involving FEN1 for cleaving off the flap is called long-patch BER. At the final step of the BER pathway, the integrity of the DNA backbone is restored by a DNA ligase. Aside from the aforementioned repair enzymes, accessory proteins such as X-ray repair cross-complimenting protein 1 (XRCC1) [35], [36], [37], [38], [39], [40], proliferating cell nuclear antigen (PCNA) [41], [42], and poly(ADP-ribose) polymerase 1 (PARP1) [43] are believed to participate in the coordination of BER processes.

According to the “passing-the-baton” model of BER, DNA intermediates in BER are processed and then passed from one enzyme to another in a coordinated fashion [44], [45], [46]. Step-by-step coordination of BER events is thought to be facilitated by multiple protein–protein interactions comprising BER enzymes and accessory proteins [43], [47], [48], [49], [50], [51]. Therefore, investigating not only protein–DNA but also protein–protein interactions is required for a complete understanding of the BER mechanism.

APE1 is one of BER enzymes that recognizes and incises a DNA strand containing an AP site [52], [53]. Although the interaction of APE1 with DNA has been studied quite extensively, the interactions of this enzyme with other BER proteins and its participation in the coordination of BER processes are still debated. The last 2 decades saw a number of studies in this field: it has been found that APE1 stimulates catalytic activity of many human DNA repair glycosylases, including uracil DNA glycosylase (UNG) [54], [55], thymine DNA glycosylase (TDG) [56], [57], [58], alkyladenine DNA glycosylase (AAG) [59], [60], methyl-CpG-binding domain 4 (MBD4) [51], adenine DNA glycosylase MutY and MYH [61], [62], [63], [64], and oxoguanine DNA glycosylase (OGG1) [51], [65], [66], [67], [68]. This effect has been explained: APE1 passively (not forming specific protein–protein interactions) and/or actively (forming specific interactions with glycosylase) displaces glycosylase from its complex with the product thereby raising the product-release rate of DNA glycosylase. This mechanism is still unclear and was discussed and clarified in recent and latest articles [68], [69]. Besides, APE1 is reported to stimulate the dRP lyase activity of Polβ [70], [71]. In turn, APE1 endonuclease activity is enhanced by some DNA glycosylases [50], Polβ [50], [72], and XRCC1 [37], [50], [72]. The mutual enhancement of each other’s enzymatic activities by APE1 and aforementioned proteins can materialize due to either direct [73], [74] or DNA-mediated [51] protein–protein interactions. Indeed, it has been demonstrated that APE1 and DNA form ternary complexes with Polβ [70], [75] and OGG1 [69]. The above data indicate that APE1 interacts with many other BER proteins, and further investigation is necessary to understand how exactly these proteins modulate each other’s enzymatic activities to coordinate all BER processes.

A powerful tool for research into protein–protein interactions is a fluorescence-based approach, when a protein is labeled by a relatively small-molecule fluorescent dye. Such a fluorescently labeled protein can form natural contacts with DNA and other proteins, and the local environment of the fluorophore can thus change, leading either to alterations in own fluorescence intensity or to the formation of a Förster resonance energy transfer (FRET) pair with a quencher located in the partner molecule. Such an approach has been used to analyze direct protein–protein contacts in BER under equilibrium conditions [73] and revealed new features of APE1–OGG1 interactions in a recent work [69]. Thus, protein–protein and protein–DNA interactions can be monitored in real time by the measurement of the fluorescent signal via the stopped-flow technique under pre–steady-state conditions. Therefore, the aim of the present study was to create an experimental system based on fluorescently labeled APE1 that would allow us to analyze the protein–protein and protein–DNA interactions in BER. It turned out that the obtained labeled APE1 variants change fluorescence intensity when associating with DNA substrates containing an AP site thus enabling us to monitor kinetics of the enzyme–DNA interaction. It was revealed that Polβ specifically affects the interaction of APE1 with its substrate and its product. The mechanisms of these protein–protein interactions in BER are discussed at the end of the paper.

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