(A) Representative image from the filter binding assay monitoring the incorporation of 32P-NAD into PARP1 (autoPARylation) in the presence of Nuc165. Different time points (0, 20, 40, 60, 80, 100, 120, and 150 s) are represented in the vertical direction and different concentrations of Nuc165 (0.5–1000 nM by factors of 2 in concentration) are represented in the horizontal direction. (B) Representative data from monitoring the incorporation of 32P-NAD into PARP1 (autoPARylation) in the presence of Nuc165 (different concentrations indicated in nM). Good linearity of rates is observed at all concentrations of nucleosome up to 150 s. (C) Representative curve determining the Km for NAD+ in the presence of p18mer, Nuc165, or Nuc165 in the presence of HPF1. The Km values for p18mer, Nuc165, and Nuc165 in the presence of HPF1 are 38 ± 9 µM (n = 8), 39 ± 10 µM (n = 3), and 54 ± 8 µM (n = 3), respectively. The kcat values for p18mer, Nuc165, and Nuc165 in the presence of HPF1 are 4.4 ± 1.9 s−1, 2.4 ± 0.8 s−1, and 4.6 ± 1.0 s−1, respectively. (D) Representative activation curves for p165mer, Nuc165, p621mer, and LE-Tri demonstrating that nucleosomes lead to greater maximal autoPARylation activity. Indicated error bars are from triplicate assay points. Derived apparent values for kcat for these and other activators of PARP1 and their replicates are shown in Table 1.