Table 1.
Binding parameters of PARP2 interaction with BER proteins determined by fluorescent titration.
| Labelled Protein 1 | Protein Partner | EC50 2, nM | E 3 |
|---|---|---|---|
| FAM-PARP2 | PARP2 | 152 ± 12 | |
| FAM-PARP2 | Polβ | 185 ± 18 * | |
| FAM-PARP2 | XRCC1 | 190 ± 18 * | |
| FAM-PARP2 | PARP1 | 146 ± 10 | |
| FAM-APE1 | PARP2 | 61 ± 8 *** | |
| AF-PARP2 | PARP1 | 79 ± 7 *** | |
| AF-PARP2 | Cy3-PARP1 | 76 ± 7 *** | 0.36 ± 0.04 |
| Cy3-PARP1 | PARP2 | 90 ± 6 *** | |
| Cy3-XRCC1 | PARP2 | 196 ± 10 ** |
1 Titration experiments were performed at a constant concentration of the labelled protein (40 nM). 2 EC50 value derived from the titration curves by fitting to the four-parameter equation is the half-maximal effective concentration of the protein partner, at which F − F0 = (F∞ − F0)/2. Values are the mean (± SD) of 3–5 independent experiments. Values for PARP2–protein complexes, which are statistically different from that for the FAM-PARP2–PARP1 complex (underlined): p < 0.05 (*), p < 0.01 (**), p < 0.001 (***); t-test. 3 Efficiency of FRET calculated from the experimentally determined fractional decrease of the fluorescence intensity: E = 1 − Fda/Fd, where Fda and Fd are fluorescence intensities of the AF-labelled PARP2 (donor) measured in the presence of Cy3-labelled (acceptor) or unlabelled PARP1, respectively. Relative intensity of fluorescence excited at 482 nm was monitored at 530 nm. Fda values were corrected for the contribution of the Cy3-labelled PARP1 (measured by titration of the unlabelled PARP2 with the Cy3-labelled PARP1). Values are the mean (± SD) of three independent experiments.