Table 2.
Effects of BER DNA intermediates on protein–protein interactions.
| Labelled Protein a | DNA a | Protein Partner | EC50 b, nM | Effect on Affinity c | Effect on FRET Efficiency d |
|---|---|---|---|---|---|
| FAM-APE1 | AP-DNA | Polβ | 92 ± 7 | 1.1 | +0.05 * |
| FAM-APE1 | AP-DNA inc | Polβ | 170 ± 13 *** | 2.0 | –0.06 ** |
| FAM-APE1 | gap-DNA | Polβ | 110 ± 8 * | 1.3 | –0.11 ** |
| FAM-APE1NΔ35 | AP-DNA | Polβ | 91 ± 7 | 0.94 | –0.08 ** |
| FAM-APE1NΔ35 | AP-DNA inc | Polβ | 110 ± 10 | 1.1 | –0.0 8** |
| FAM-APE1NΔ61 | AP-DNA | Polβ | 110 ± 10 | 0.92 | –0.08 ** |
| FAM-APE1NΔ61 | AP-DNA inc | Polβ | 130 ± 11 | 1.1 | –0.09 ** |
| FAM-APE1NΔ61 | gap-DNA | Polβ | 140 ± 13 | 1.2 | –0.07 * |
| FAM-APE1 | AP-DNA | XRCC1 | 78 ± 7 | 1.0 | –0.01 |
| FAM-APE1 | gap-DNA | XRCC1 | 51 ± 4 ** | 0.67 | +0.08 ** |
| FAM-APE1NΔ35 | AP-DNA | XRCC1 | 68 ± 6 ** | 0.68 | –0.03 * |
| FAM-APE1NΔ35 | gap-DNA | XRCC1 | 65 ± 6 ** | 0.65 | –0.03 * |
| FAM-APE1NΔ61 | AP-DNA | XRCC1 | 99 ± 8 * | 0.76 | –0.06 ** |
| FAM-APE1NΔ61 | gap-DNA | XRCC1 | 97 ± 8 * | 0.75 | –0.05 ** |
| FAM-APE1 | AP-DNA | PARP1 | 76 ± 6 ** | 1.7 | +0.05 ** |
| FAM-APE1 | gap-DNA | PARP1 | 54 ± 4 | 1.2 | +0.02 |
| FAM-APE1NΔ35 | AP-DNA | PARP1 | 49 ± 4 | 0.86 | –0.03 |
| FAM-APE1NΔ35 | gap-DNA | PARP1 | 48 ± 4 | 0.84 | +0.03 |
| FAM-APE1NΔ61 | AP-DNA | PARP1 | 55 ± 5 * | 0.73 | –0.02 |
| FAM-APE1NΔ61 | gap-DNA | PARP1 | 54 ± 4 * | 0.72 | +0.04 * |
a Titration experiments performed at the constant concentrations of FAM-labelled protein (40 nM) and DNA (400 nM). Underlined data for FAM-APE1 (previously published [28]) reproduced to compare binding parameters of full-length and truncated forms. b Parameters derived from titration curves by fitting to four-parameter equation. Values are the mean (± SD) of at least three independent experiments. Values determined for each protein pair in the presence of DNA, which were statistically different from the respective value in the absence of DNA, are marked p < 0.05 (*), p < 0.01 (**), p < 0.001 (***). c Effect of DNA on protein–protein affinity determined as the ratio of EC50 values in the presence or absence of DNA. d Increase (+) or decrease (–) in FRET efficiency between FAM- and TMR (5(6)-carboxytetramethylrhodamine)-labelled proteins in the presence of DNA. Statistically significant changes in E values produced by DNA are marked p < 0.05 (*), p < 0.01 (**).