Table 2.

Effects of model DNAs on the PARP2 interaction with proteins.

Labelled Protein 1	DNA 2	Protein Partner	EC50 3, nM	Effect on Binding Affinity 4	Effect on FRET Efficiency 5
FAM-PARP2	gap-DNA	PARP2	101 ± 9 **	1.5 (+)
FAM-PARP2	gap-DNA	Polβ	106 ± 10 **	1.7 (+)
FAM-PARP2	gap-DNA	XRCC1	164 ± 12 *	1.2 (+)
AF-PARP2	nick-DNA	PARP1	44 ± 5 **	1.8 (+)
AF-PARP2	ds-DNA	PARP1	144 ± 16 **	1.8 (−)
AF-PARP2	nick-DNA	Cy3-PARP1			−0.04 *
AF-PARP2	ds-DNA	Cy3-PARP1			+0.08 **
FAM-PARP2	gap-DNA		≤18
AF-PARP2	nick-DNA		≤15
AF-PARP2	ds-DNA		35 ± 4

¹ Labelled PARP2 protein (40 nM) premixed with DNA (400 nM) was titrated by a protein partner. ² Model DNAs represent BER DNA intermediates: gap-DNA–DNA with a single nucleotide gap, nick-DNA–DNA with a single-strand break and an internal 5′-phosphate, ds-DNA–DNA duplex without damage. ³ EC₅₀ value is the half-maximal effective concentration of the binding partner, at which F − F₀ = (F_∞ − F₀)/2. Values are the mean (± SD) of 3–5 independent experiments. Values determined for each protein pair in the presence of DNA, which are statistically different from the respective value in the absence of DNA (Table 1), are marked p < 0.05 (*), p < 0.01 (**). Typical titration curves used to determine EC₅₀ values for complexes of FAM/AF-labelled PARP2 with DNAs are presented in Supplementary Materials, Figure S3. ⁴ Degree of increase (+) or decrease (–) in the binding affinity in the presence of DNA. ⁵ Increase (+) or decrease (–) in FRET efficiency between AF- and Cy3-labelled proteins in the presence of DNA. The respective mean E values were determined in independent measurements (n = 5–7) at three (four) sub-saturating PARP1 and Cy3-PARP1 concentrations (200–400 nM); statistically significant changes in the E value produced by DNAs are marked p < 0.05 (*), p < 0.01 (**).