The Sequence-specific DNA Binding of NF-κB Is Reversibly Regulated by the Automodification Reaction of Poly (ADP-ribose) Polymerase 1

Abstract

Recent studies suggest that the synthesis of protein-bound ADP-ribose polymers catalyzed by poly(ADP-ribose) polymerase-1 (PARP-1) regulates eucaryotic gene expression, including the NF-κB-dependent pathway. Here, we report the molecular mechanism by which PARP-1 activates the sequence-specific binding of NF-κB to its oligodeoxynucleotide. We co-incubated pure recombinant human PARP-1 and the p50 subunit of NF-κB (NF-κB-p50) in the presence or absence of βNAD+ in vitro. Electrophoretic mobility shift assays showed that, when PARP-1 was present, NF-κB-p50 DNA binding was dependent on the presence of βNAD +. DNA binding by NF-κB-p50 was not efficient in the absence of βNAD+. In fact, the binding was not efficient in the presence of 3-aminobenzamide (3-AB) either. Thus, we conclude that NF-κB-p50 DNA binding is protein-poly(ADP-ribosyl)ation dependent. Co-immunoprecipitation and immunoblot analysis revealed that PARP-1 physically interacts with NF-κB-p50 with high specificity in the absence of βNAD+. Because NF-κB-p50 was not an efficient covalent target for poly(ADP-ribosyl)ation, our results are consistent with the conclusion that the auto-poly(ADP-ribosyl)ation reaction catalyzed by PARP-1 facilitates the binding of NF-κB-p50 to its DNA by inhibiting the specific protein·protein interactions between NF-κB-p50 and PARP-1. We also report the activation of NF-κB DNA binding by the automodification reaction of PARP-1 in cultured HeLa cells following exposure to H2O2. In these experiments, preincubation of HeLa cells with 3-AB, prior to oxidative damage, strongly inhibited NF-κB activation in vivo as well.