Research Paper Volume 4, Issue 10 pp 674—685

Figure 2. Mapping the interacting regions of PARP-1 and NEIL1. (A) Schematic representation of GST-NEIL1 fusion proteins. (B) Purified GST-NEIL1 fusion proteins were analyzed by SDS-PAGE followed by staining with Coomassie Colloidal Blue. (C) PARP-1 binds to the C-terminal 289–390 aa of NEIL1. PARP-1 (250 ng) was incubated with either GST, GST tagged full-length NEIL1 or indicated NEIL1 fragments (1 μg) in an in-vitro binding assay. GST precipitations were probed with anti-PARP-1 antibodies and stained with Ponceau S. ~5% of purified PARP-1 bound to both GST-NEIL and NEIL 289–390aa. (D) NEIL1 binds to the BRCT domain of PARP-1. The DNA binding (41 kDa), BRCT (15 kDa) and catalytic (39 kDa) domains of PARP-1 (1 μg) were incubated with 1 μg GST-NEIL1 (69 kDa) either in the presence (+) or absence (−) of ethidium bromide. GST-NEIL1 precipitations were immunoblotted with anti-His (for the DNA binding and catalytic domains) and anti-BRCT (for BRCT domain) antibodies and then stained with Coomassie to reveal the amount of GST-NEIL1 in the precipitations. The different PARP-1 domains and GST-NEIL1 are indicated by arrows. ~4% of purified BRCT domain bound to GST-NEIL1. (E) A schematic of the different PARP-1 proteins is shown.