Figure 2. Poly(ADP-ribosyl)ation and inhibition of recombinant DNAS1L3 by PARP-1 in vitro.

Figure 2

Poly(ADP-ribosyl)ation and inhibition of recombinant DNAS1L3 by PARP-1 in vitro. A, poly(ADP-ribosyl)ation of DNAS1L3 by PARP-1. The indicated combinations of recombinant human PARP-1, [32P]NAD (lanes 1-5) or nonradioactive NAD (lane 6), recombinant DNAS1L3, and 3-AB were incubated with high molecular weight (lanes 1-4 and 6) or activated (lane 5) DNA. The reaction products were then analyzed by SDS-PAGE and autoradiography. The positions of PARP-1, PARP-1 fragments, and DNAS1L3 are indicated. B, effect of poly(ADP-ribosyl)ation on DNAS1L3 activity. The indicated combinations of recombinant human PARP-1, nonradioactive NAD, recombinant DNAS1L3, and 3-AB were incubated for 30 min at room temperature in a reaction mixture containing high molecular weight DNA. The mixtures were then placed on ice, and portions were subsequently incubated for 30 min at 37°C with phage DNA in a reaction containing 25 mM Tris-HCl (pH 7.4), 5 mM MgCl2, and 2.5 mM CaCl2. DNA integrity was then analyzed by electrophoresis through a 1.5% agarose gel. The leftmost lane contains 1-kb DNA size markers. Reprinted with permission from: Yakovlev et al. J Biol Chem. 2000;275(28):21302-21308. © 2000 The American Society for Biochemistry and Molecular Biology, Inc.

From: Genome Degradation by DNAS1L3 Endonuclease: A Key PARP-1-Regulated Event in Apoptosis

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