The enzymatic mechanism of poly(ADP-ribose) polymerase (PARP-1) has been analyzed in two in vitro systems: (a) in solution and (b) when the acceptor histones were attached to a solid surface. In system (a), it was established that the coenzymatic function of dsDNAs was sequence-independent. However, it is apparent from the calculated specificity constants that the AT homopolymer is by far the most effective coenzyme and randomly damaged DNA is the poorest. Rates of auto(poly-ADP-ribosylation) with dsDNAs as coenzymes were nearly linear for 20 min, in contrast to rates with dcDNA, which showed product [(ADPR)n] inhibition. An allosteric activation of auto(poly-ADP-ribosylation) by physiologic cellular components, Mg2+, Ca2+, and polyamines, was demonstrated, with spermine as the most powerful activator. On a molar basis, histones H(1) and H(3) were the most effective PARP-1 activators, and their action was abolished by acetylation of lysine end groups. It was shown in system (b) that oligo(ADP-ribosyl) transfer to histone H(1) is 1% of that of auto(poly-ADP-ribosylation) of PARP-1, and this trans(ADP-ribosylation) is selectively regulated by putrescine (activator). Physiologic cellular concentrations of ATP inhibit PARP-1 auto(poly-ADP-ribosylation) but less so the transfer of oligo(ADP-ribose) to histones, indicating that PARP-1 auto(ADP-ribosylation) activity is dormant in bioenergetically intact cells, allowing only trans(ADP-ribosylation) to take place. The inhibitory mechanism of ATP on PARP-1 consists of a noncompetitive interaction with the NAD site and competition with the coenzymic DNA binding site. A novel regulation of PARP-1 activity and its chromatin-related functions by cellular bioenergetics is proposed that occurs in functional cells not exposed to catastrophic DNA damage.

The enzymatic transfer of ADP-ribose from NAD to histone H 1 (defined as trans-poly(ADP-ribosylation)) or to PARP I (defined as auto-poly(ADP-ribosylation)) was studied with respect to the nature of the DNA required as a coenzyme. Linear double-stranded DNA (dsDNA) containing the MCAT core motif was compared with DNA containing random nicks (discontinuous or dcDNA). The dsDNAs activated trans-poly(ADP-ribosylation) about 5 times more effectively than dcDNA as measured by V max . Activation of auto-poly(ADP-ribosylation) by dcDNA was 10 times greater than by dsDNA. The affinity of PARP I toward dcDNA or dsDNA in the auto-poly(ADP-ribosylation) was at least 100-fold lower than in trans-poly(ADP-ribosylation) (K a ‫؍‬ 1400 versus 3-15, respectively). Mg 2؉ inhibited trans-poly(ADP-ribosylation) and so did dcDNA at concentrations required to maximally activate auto-poly(ADP-ribosylation). Mg 2؉ activated auto-poly(ADP-ribosylation) of PARP I. These results for the first time demonstrate that physiologically occurring dsDNAs can serve as coenzymes for PARP I and catalyze preferentially trans-poly(ADPribosylation), thereby opening the possibility to study the physiologic function of PARP I.The activity of poly(ADP-ribose) polymerase (PARP I, 1 EC 2.4.2.30) was originally considered to represent a novel form of covalent protein modification analogous to phosphorylation, acetylation, etc. (1). Immunochemical analysis of normal adult rat tissues for poly(ADP-ribose) (pADPR) showed that 99% of covalently bound polymer was found in the non-histone fraction of nuclear proteins, with histone H 1 being the major acceptor among the remaining 1% (2). The actual quantities of protein-bound pADPR in normal tissues, however, are small (200 -250 ng/g, dry weight) as compared with the large abundance of PARP I protein itself (0.5 ϫ 10 6 copies/cell (cf. Ref. 4). This difference suggests that PARP I activity in vivo is highly regulated. The secondary structure of pADPR (3) predicts that binding of modified proteins to other proteins or to DNA will be altered by poly(ADP-ribosylation). Furthermore, unmodified PARP I avidly binds to a large number of nuclear proteins (4), an association that is completely blocked by added histone H 1 . In addition, an assortment of transcription factors was shown to bind to PARP I (cf. Ref. 5), and a transcriptional regulatory role of PARP I in muscle differentiation has been identified (6). The MCAT element derived from this work (cf. Ref. 6) served as a basic DNA sequence for the construction of dsDNAs studied in the present experiment. Recently the topology of the direct binding of PARP I to Topo 1 has been demonstrated in detail (7). The binding of PARP I to histone H 1 induces conformational changes in the latter coinciding with new phosphorylation sites for Cdc2-kinase (8).Although the enzymatic transfer of ADP-ribose from NAD to PARP I and to several nuclear proteins has been well established (1), it has remained unclear what factors determine protein auto-or heteromodification by poly(ADP-ribos...

The enzymatic transfer of ADP-ribose from NAD to histone H(1) [defined as trans(oligo-ADP-ribosylation)] or to PARP-1 [defined as auto(poly-ADP-ribosylation)] requires binding of coenzymic DNA. The preceding paper [Kun, E., et al. (2004) Biochemistry 43, 210-216] shows that oligonucleotides of dsDNA can serve as coenzymic DNA for PARP-1 trans- or auto-modification activity. Results of DNA-protein binding (EMSA) experiments reported here demonstrate that short DNA oligonucleotides containing the 5'-TGTTG-3' nucleotide sequence motif preferentially bind to cloned PARP-1 in vitro. The same nucleotide sequence motif is responsible for striated myocyte-selective transcription of a contractile protein gene encoding cardiac troponin T (cTnT). Results of experiments reported here demonstrate that mutation of this motif also abolishes the differentiation-dependent activation of the transfected cTnT promoter in myoblasts cultured in vitro, indicating that nucleotide sequence-dependent binding of PARP-1 to promoter DNA of the cTnT gene is also necessary for differentiation-dependent activation. Thus, PARP-1 has two types of dsDNA binding activity: (1) nucleotide sequence-dependent binding, analyzed here with EMSA experiments, and (2) coenzymic binding, measured catalytically, which does not depend on the nucleotide sequence of the dsDNA. We hypothesize that the well-known association of PARP-1 with chromatin can be attributed to its stable binding to chromosomal dsDNA, some portion of which is likely to be nucleotide sequence-dependent binding. According to this hypothesis, the distribution of this protein-modifying enzyme in chromatin may be targeted to specific genomic loci and vary according to cell type and developmental stage.

Human fibroblasts were subjected to nutritionally induced GI block, followed by release and subsequent entry into S phase, and exposed to nontoxic concentrations of carcinogens in early S phase. Cell transformation occurred as determined by early morphologic cell alterations, anchorage-independent colony formation, cell invasiveness, and augmentation of Ab 376 human malignancy-specific cell-surface antigenic determinant. Methylazoxymethanol acetate was the most potent transforming agent at doses that were negative in toxicity tests. Benzamide (10 FM intracellular concentration), a specific inhibitor of poly(ADPribose) polymerase, prevented transformation in a cell cycle-specific manner, maximal prevention coinciding with early S phase, also characteristic of maximal susceptibility to transformation. Neither an interference of carcinogen deoxyguanosine nucleoside adduct formation nor a chemical reaction between benzamide and carcinogens was detected. Methylazoxymethanol acetate at transforming but nontoxic dose partially inhibited poly(ADP-ribosyl)ation to about the same extent as benzamide. However, simultaneous exposure of cells to both agents in early S phase, resulting in the prevention of transformation, augmented poly(ADPribosyl)ation above the controls. Enzymatic activities ran parallel with the formation of DNA-associating polymer-nonhistone protein adducts that are assumed to regulate the physiological function of chromatin at the structural level.A significant increase in poly(ADP-ribosyl)ation of nonhistone proteins was found in an early precancerous state of hamster liver (1) and in regenerating rat liver (2), suggesting that cellular regeneration may be a common reason for the accelerated enzymatic rates. Cellular regeneration is known to augment the number of cells in S phase and this has been correlated with the facilitation of neoplastic transformation in organs (3) and in cultured C3H/101/3 CL8 mouse fibroblasts (4). Poly(ADP-ribosyl)ation also exhibits a cell cycle-dependent oscillation, a maximum coinciding with S phase after a release of G1 block (5). These observations tend to suggest an as yet undefined connection between (poly ADP-ribosyl)ation, cell cycle, and neoplastic transformation.Ontogenic development (6), the effect of developmental hormones (7-9), and differentiation (10) also coincide with changes in rates of poly(ADP-ribosyl)ation of mainly nonhistone proteins (11, 12) that are thought to regulate selective gene expression (13). Identification of poly(ADP-ribose) as a unique nucleic acid (14, 15) that is covalently bound to presumably DNA-associated proteins may provide a molecular model of chromatin regulation. Ionic environment-dependent helical polymer chains of poly(ADP-ribose) (15) may act as crosslinking agents between regulatory proteins and, depending on their nature and localization, could alter chromatin conformations, expressed as karyological changes that accompany the cell cycle in normal and malignant cells (16,17). The poly(ADP-ribosyl)ation-dependent var...

The molecular interactions of poly(ADP-ribose) polymerase I (PARP I) and topoisomerase I (Topo I) have been determined by the analysis of physical binding of the two proteins and some of their polypeptide components and by the effect of PARP I on the enzymatic catalysis of Topo I. Direct association of Topo I and PARP I as well as the binding of two Topo I polypeptides to PARP I are demonstrated. The effect of PARP I on the`global' Topo I reaction (scission and religation), and the activation of Topo I by the 36 kDa polypeptide of PARP I and catalytic modifications by poly(ADP-ribosyl)ation are also shown. The covalent binding of Topo I to circular DNA is activated by PARP I similar to the degree of activation of thè global' Topo I reaction, whereas the religation of DNA is unaffected by PARP I. The geometry of PARP I^Topo I interaction compared to automodified PARP I was reconstructed from direct binding assays between glutathione S-transferase fusion polypeptides of Topo I and PARP I demonstrating highly selective binding, which was correlated with amino acid sequences and with the`C clamp' model derived from X-ray crystallography. ß 2001 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.