*4.2. Fluorescence Anisotropy Assay*

PARP-1 protein was obtained from the insect cells using baculovirus expression system. A suspension of Hi5 cells in serum-free medium (2·10<sup>6</sup> cells/mL) was infected with baculovirus (10 pfu/mL) containing cDNA of PARP-1, a kind gift of V. Schreiber (Strasbourg, France). Insect cells were then collected by centrifugation during 10 min at 1000× *g*. The purification of PARP-1 was performed according to the earlier described protocol [48]. The fluorescein-labeled DNA duplex used in kinetics experiments is provided in Table S2.

Real-time measurements of the PARP-1 activity were based on recently developed fluorescent method [27]. The reaction mixture contained a buffer (50 mM Tris-HCl, pH 8.0, 50 mM NaCl, 1 mM DTT, 5 mM MgCl2), 200 nM PARP-1, 100 nM DNA, and 7-MG (0–450 µM). The reaction was started by adding NAD<sup>+</sup> (100 µM). Fluorescence anisotropy measurements were performed at 25 ◦C using the CLARIOstar multifunctional microplate reader (BMG LABTECH, Ortenberg, Germany). The fluorescent probes were excited at 495 nm, and the fluorescence intensity was detected at 520 nm. To determine the IC<sup>50</sup> value, the experiment was done in triplicate. To determine the type of enzyme inhibition, reaction mixtures containing the buffer, 7-MG (0–400 µM) and NAD<sup>+</sup> (0–2500 µM) were used, and the reaction was started by adding a mixture of PARP-1 (200 nM) and DNA (100 nM).

Anisotropy was calculated using formula:

$$\mathbf{A} = \frac{(\mathbf{I}\_{\parallel} - \mathbf{I}\_{\perp})}{(\mathbf{I}\_{\parallel} + \mathbf{2}\mathbf{I}\_{\perp})} \tag{1}$$

where Ik—fluorescence intensity parallel to the plane-polarized exited light, I⊥—fluorescence intensity perpendicular to the light.

### *4.3. spFRET Microscopy*

Mononucleosomes were assembled using 147 or 167 bp DNA fragments containing strong nucleosome positioning sequence 603 (147 bp-long) [49,50]. Nucleosomes P147 contained only the 603 sequence, while P167 included an additional 20-bp linker. Cy3 and Cy5 fluorophore labels (Biotech Industry Ltd., Moscow, Russia) were introduced at +13 and +91 bp (from the beginning of the 603 sequence) to enable spFRET-microscopy detection of changes in the nucleosome structure. Label positions were selected in the neighboring DNA gyres based on the nucleosome crystal structure [51,52]. Sequences of DNA template and fluorescently labeled oligonucleotide primers are shown in Table S3. Nucleosomes were assembled with donor chromatin (lacking H1 histone) from chicken erythrocytes [53,54] using salt dialysis against buffers (10 mM Tris pH 7.5, 0.2 mM EDTA, 0.1% NP-40, 5 mM 2-mercaptoethanol) with decreasing concentrations of NaCl (1, 0.5, 0.2, 0.01 M ). The nucleosome assembly was controlled by a native polyacrylamide gel (4%) electrophoresis. Fluorescently-labeled nucleosomes (1 nM) were incubated with PARP-1 for 20 min in a buffer containing 50 mM Tris-HCl pH 8.0, 40 mM NaCl, 1mM DTT at 25 ◦C in siliconized tubes. To activate PARP-1, NAD<sup>+</sup> was added to final concentration of 100 µM and the mixture was incubated for 15 min. In experiments with 7-MG, PARP-1 was preincubated with 450 µM of the inhibitor for 15 min and mixed with the nucleosomes.

Fluorescence of single nucleosomes and their complexes was measured during their free diffusion through the focus of a laser beam (wavelength of 514.5 nm) with the LSM 710 ConfoCor 3 confocal microscope (Zeiss, Oberkochen, Germany) as described elsewhere [32]. To characterize the distance between Cy3 (donor) and Cy5 (acceptor) dyes in neighboring gyres, a proximity ratio EPR was calculated for each nucleosome:

$$E\_{PR} = \frac{(I\_{Aa} - \alpha \times I\_{Dd})}{[I\_{Aa} + (1 - \alpha) \times I\_{Dd}]} \tag{2}$$

where *IAa* indicates the fluorescent intensity of the Cy5 label (Cy5 channel), *IDd* indicates intensity of the Cy3 label (Cy3 channel) and α is a coefficient of spectral cross-talk calculated as:

$$\alpha = \frac{I\_{Da}}{I\_{Dd}} \tag{3}$$

where *IDa* is a fluorescent intensity of the Cy3 in the Cy5 channel. Data collected from at least 2000 individual particles have been used to plot a relative frequency distribution of nucleosomes by EPR.

**Supplementary Materials:** Supplementary materials can be found at http://www.mdpi.com/1422-0067/21/6/2159/s1.

**Author Contributions:** Conceptualization, D.N.; investigation, N.M., T.K., S.P., A.L. and M.K.; methodology, A.F., T.K. and D.N.; writing—original draft preparation, project administration, and funding acquisition, D.N. and N.G.; supervision and writing—review and editing, V.Š., O.L. and V.M.S. All authors have read and agreed to the published version of the manuscript.

**Funding:** Fluorescence anisotropy studies were funded by the Russian Science Foundation, grant number 19-74-10072; spFRET analysis and molecular modeling were funded by the Russian Foundation for Basic Research, grant number 17-00-00163 (17-00-00132, 17-00-00097).

**Acknowledgments:** The research was carried out using the equipment of the shared research facilities of HPC computing resources at Lomonosov Moscow State University.

**Conflicts of Interest:** The authors declare no conflict of interest.
