Research Paper Volume 4, Issue 10 pp 674—685

Figure 3. PARP-1 inhibits NEIL1 incision activity. (A) NEIL1 (38 nM; 33 ng) was incubated with buffer, NAD+ with or without PARP-1 (180 nM; 400 ng), or with PAR and reacted with a 5'- ³²P-labeled oligonucleotide duplex containing a 5OHU lesion for 15 min at 37°C. The cleavage products were analyzed on a 20% denaturing gel containing 7 M urea. Percent incision was calculated by normalizing the amount of cleaved substrate (bottom band) to the amount of uncleaved product (top band). Data was then normalized to the amount of incision activity of NEIL1 alone (100%). (B) Incision assays were performed as in (A) in the presence of the indicated concentrations of PARP-1 with (activated PARP-1) or without NAD+ (PARP-1) and quantified. (C) NEIL1 (33 ng) incision assays were performed in the presence of 400 ng of the indicated PARP-1 domains or full-length PARP-1 in the presence of NAD+ (10 μM). (D) PARP-1 inhibits NEIL1 activity at both 5 nM and 20 nM concentrations of DNA substrate. The histograms in (A) and (B) represent the mean + SEM from three, (C) from five, and (D) from four independent experiments.**p≤0.01 and ***p≤0.001 compared to the incision activity of NEIL1 alone using one-way ANOVA and Tukey's post-hoc test. (C) NEIL1 was incubated with buffer or PARP-1 with or without NAD+ and incubated with a non-radiolabeled oligonucleotide containing the 5OHU lesion as above. Samples were separated by SDS-PAGE and probed with anti-PAR, anti-PARP-1 and anti-NEIL1 antibodies.