Poly(ADP-ribose) polymerase (PARP) is a DNA-binding enzyme that plays roles in response to DNA damage, apoptosis, and genetic stability. Recent evidence has implicated PARP in transcription of eukaryotic genes. However, the existing paradigm tying PARP function to the presence of DNA strand breaks does not provide a mechanism by which it may be recruited to gene-regulating domains in the absence of DNA damage. Here we report that PARP can bind to the DNA secondary structures (hairpins) in heteroduplex DNA in a DNA end-independent fashion and that automodification of PARP in the presence of NAD ؉ inhibited its hairpin binding activity. Atomic force microscopic images show that in vitro PARP protein has a preference for the promoter region of the PARP gene in superhelical DNA where the dyad symmetry elements likely form hairpins according to DNase probing. Using a chromatin cross-linking and immunoprecipitation assay we show that PARP protein binds to the chromosomal PARP promoter in vivo. Reporter gene assays have revealed that the transcriptional activity of the PARP promoter is 4 -5-fold greater in PARP knockout cells than in wild type fibroblasts. Reintroduction of vectors expressing full-length PARP protein or its truncated mutant (DNA-binding domain retained but lacking catalytic activity) into PARP ؊/؊ cells has conferred transcriptional down-regulation of the PARP gene promoter. These data provide support for PARP protein as a potent regulator of transcription including down-regulation of its own promoter.Poly(ADP-ribose) polymerase (PARP, 1 EC 2.4.2.30) is a chromatin-associated enzyme that catalyzes the transfer of successive units of the ADP-ribose moiety from NAD ϩ to itself and other nuclear acceptor proteins (1). PARP is a zinc fingercontaining protein, which allows enzyme binding to either double or single strand DNA breaks without any apparent sequence preference (2, 3). The catalytic activity of PARP is strictly dependent on the presence of strand breaks in DNA and is modulated by the level of automodification (4,5). Data from many studies show that PARP is involved in numerous biological functions, all of which are associated with breaking and rejoining DNA strands, and it plays a pivotal role in DNA damage repair (2, 6 -8).Recent studies have implicated PARP in transcription of eukaryotic genes (9 -16). PARP-dependent gene regulation involves poly(ADP-ribosyl)ation of transcription factors, which, in turn, prevents their binding to specific promoter sequences (10). The basal transcription factors TFIIF and TEF-1 as well as transcription factors TATA box-binding protein, YY1, SP-1, cAMP-response element-binding protein, p53, and NFB are all highly specific substrates for poly(ADP-ribosyl)ation (10,11,14,16). PARP may also interact directly with gene promoters. For instance, recombinant full-length PARP bound the DNA sequences within the MCAT1 regulatory element (11) and to the DF4 protein binding site of the Pax-6 gene neuroretinaspecific enhancer (17). Furthermore, PARP involvement in the active...