*4.2. Fluorescence Studies of Protein–Protein Interactions*

Fluorescent labelling of proteins on the terminal amino group using succinimidyl esters of 5(6)-carboxyfluorescein (FAM-SE) and 5(6)-carboxytetramethylrhodamine (TMR-SE) was performed as described [28]. The stoichiometry of protein labelling did not exceed 1 mole of dye per mole of protein, and FAM/TMR-labelled enzymes retained their specific activities (as detailed in Supplementary Materials, Table S1). Binding of proteins to each other was examined by fluorescence titration experiments. Fluorescence intensities of solutions of the FAM-labelled protein (APE1, APE1N∆35, or APE1N∆61) at a fixed concentration were measured in the absence and presence of varied concentrations of the partner (Polβ/PARP1/XRCC1), in a binding buffer containing 50 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), pH 8.0, 100 mM NaCl, and 4 mM DTT. Fluorescence intensity of the samples was measured in Corning 384-well black polypropylene assay plates using a CLARIOstar multifunctional microplate reader (BMG LABTECH GmbH, Germany); fluorescence probes were excited at 482 nm (482-16 filter), and the relative fluorescence intensities were detected at the emission maximum (530 nm, 530-40 filter). All measurements were carried out in duplicate for each specific condition, and the average values of fluorescence (with mean deviations not exceeding 2%) were taken for data analysis performed using MARS (Multivariate Adaptive Regression Splines) Data Analysis Software (BMG LABTECH GmbH, Germany). Data were plotted (F vs. C) and fitted by four-parameter logistic equation

$$\mathbf{F} = \mathbf{F}\_0 + (\mathbf{F}\_\infty - \mathbf{F}\_0)/[1 + (\mathbf{E}\mathbf{C}\_{50}\mathbf{/C})^\mathbf{n}] \tag{1}$$

where F is the fluorescence intensity of a solution containing the FAM-labelled protein and the binding partner at a given concentration (C), F<sup>0</sup> is the fluorescence of a solution of the labelled protein alone, F<sup>∞</sup> is the fluorescence of the labelled protein saturated with the partner, EC<sup>50</sup> is the concentration of the binding partner at which F – F<sup>0</sup> = (F<sup>∞</sup> – F0)/2, and n is the Hill coefficient denoting the slope of the nonlinear curve.

To detect protein–protein interactions by the FRET approach, the fluorescence intensity of the FAM-labelled protein (donor probe) was measured in the absence and presence of varied concentrations of the TMR-labelled protein (acceptor probe). Measurements were performed in two series: (1) Donor probe + unlabeled partner, and (2) donor probe + acceptor probe (FRET pair). FRET efficiency (E) was calculated from the fractional decrease of the donor fluorescence (Fd) due to the presence of the acceptor (Fda)

$$\mathbf{E} = \mathbf{1} - \mathbf{F}\_{\mathbf{da}} / \mathbf{F}\_{\mathbf{d}}.\tag{2}$$

In protein–protein binding experiments performed in the presence of model DNAs (prepared as described in Supplementary Materials), the FAM-labelled protein (40 nM) was premixed with the desired DNA, and fluorescence intensity was taken as a starting F<sup>0</sup> value. Conditions for the formation of ternary complexes were optimized by varying DNA concentration from 120 to 600 nM. Titration experiments were performed in binding buffer supplemented with 10 mM EDTA (ethylenediaminetetraacetic acid) (to suppress the AP-endonuclease and the 30 -50 -exonuclease activities of APE1 and its truncated forms).

### *4.3. AP Endonuclease Activity Assay*

The endonuclease activity of APE1 and APE1N∆61 was assayed in reaction mixtures (25–50 µL) containing 50 mM Tris-HCl, pH 8.0, 50 mM NaCl, 10 mM MgCl2, 1 mM DTT, 0.1 mg/mL BSA (bovine serum albumin), and 100 nM 5'-32P-labelled AP-DNA substrate (32 base-pair oligonucleotide with a synthetic AP site, prepared as described in Supplementary Materials). The reaction mixtures were preassembled on ice; when indicated, they were supplemented with Polβ, XRCC1, and PARP1 (at concentrations specified in the figure legends). Reaction was initiated by adding APE1 (APE1N∆61) to a final concentration of 0.5 nM; the reaction mixtures were incubated at 37 ◦C for 0.5–4 min and terminated by the addition of denaturing PAGE sample buffer and heating for 2 min at 90 ◦C. The reaction products were separated by electrophoresis in 20% denaturing polyacrylamide gels. The gels were imaged on a Typhoon FLA 9500, and the amounts of DNA substrate and product were quantified using Quantity One Basic software.
