Purification and characterization of poly (ADP-ribose) synthetase from human placenta

Poly(ADP-ribose) synthetase has been purified 2,000-fold to apparent homogeneity from human placenta. The purification procedure involves affinity chromatography with 3-aminobenzamide as the ligand. The purified enzyme absolutely requires DNA for the catalytic activity and catalyzes poly(ADP-ribosyl)ation of the synthetase itself (automodification) and histone H1. Mg2+ enhances both the automodification and poly(ADP-ribosyl)ation of histone H1. The enzyme is a monomeric protein with a pI of 10.0 and an apparent molecular weight of 116,000. The sedimentation coefficient and Strokes radius are 4.6 S and 5.9 nm, respectively. The frictional ratio is 1.82. Amino acid analysis and limited proteolysis with papain and alpha-chymotrypsin indicate that the human placental enzyme is very similar to the enzyme from calf thymus, although some differences are noted. Mouse antibody raised against the placental enzyme completely inhibits the activity of enzymes from human placenta and HeLa cells and cross-reacts with the enzymes from calf thymus and mouse testis. Immunoperoxidase staining with this antibody demonstrates the intranuclear localization of the enzyme in human leukemia cells. All these results indicate that molecular properties as well as antigenic determinants of poly(ADP-ribose) synthetase are highly conserved in various animal cells.     

Immunohistochemical demonstration of poly(adenosine diphosphate-ribose) synthetase in bovine tissues

The inter- and intracellular localization of poly(adenosine diphosphate-ribose)(poly(ADP-ribose] synthetase was investigated using an indirect immunofluorescence technique and a specific antibody against the enzyme purified from calf thymus. In various bovine tissues, including liver, heart, pancrease, thyroid, spleen, adrenal, and skeletal muscle, the specific immunofluorescence of poly(ADP-ribose) synthetase was localized exclusively in the nucleus. Immunostaining was inhibited by preabsorption of the antibody with purified calf thymus poly(ADP-ribose) synthetase. Nuclear immunofluorescence appeared to be more prominent in the marginal area than in the central region in most nuclei. This staining pattern is similar to that of naturally occurring poly(ADP-ribose). In bovine peripheral blood the immunofluorescence of poly(ADP-ribose) synthetase was detected in nuclei of lymphocytes, but not in granulocytes, in agreement with the finding that the enzymatic activity of poly(ADP-ribose) synthetase was barely detectable in nuclei isolated from granulocytes.     

Poly(ADP-ribosylation) of DNA topoisomerase I from calf thymus

We demonstrate that the activity of the major DNA topoisomerase I from calf thymus is severely inhibited after modification by purified poly(ADP-ribose) synthetase. Polymeric chains of poly(ADP-ribose) are covalently attached to DNA topoisomerase I. These observations with highly purified enzymes suggest that poly(ADP-ribosylation) may be a cellular mechanism for modulating DNA topoisomerase I activity in response to the state of DNA in the nucleus. Although extensive poly(ADP-ribosylation) of the Mr = 100,000 DNA topoisomerase I from calf thymus resulted in greater than 90% enzyme inhibition, exogenous poly(ADP-ribose) does not, by itself, inhibit topoisomerase activity. After modification, the apparent molecular weight of both the topoisomerase enzyme protein and of the topoisomerase enzyme activity was increased. In vitro, the extent of modification of DNA topoisomerase I could be controlled either by changing the ratio of topoisomerase to the synthetase or by varying the reaction time. More than 40 residues of ADP ribose per topoisomerase molecule could be added by the synthetase. Analysis of a poly(ADP-ribosylated) topoisomerase preparation that was about 50% inhibited revealed an average polymer chain length of 7.4, with 1-2 chains per enzyme molecule.     

Identification of minimal size requirements of DNA for activation of poly(ADP-ribose) polymerase

Poly(ADP-ribose) polymerase requires DNA as an essential enzyme activator. Using enzyme purified from lamb thymus and double-stranded deoxynucleotide oligomers of defined length, we conducted studies to identify the smallest size DNA fragment capable of successfully activating poly(ADP-ribose) polymerase. These studies revealed that a double-stranded hexadeoxynucleotide activated the enzyme 30% as effectively as highly polymerized calf thymus DNA and a double-stranded octadeoxynucleotide activated the enzyme even more effectively than calf thymus DNA. When histone H1 was also included in the reaction system, the enzyme could be activated by even smaller DNA fragments. Thus, in the presence of histone H1, a double-stranded tetradeoxynucleotide activated the enzyme 25% as effectively as calf thymus DNA, and a double-stranded hexadeoxynucleotide was equally as effective as calf thymus DNA. The time courses for activation and the stabilities of the products were identical when the enzyme was activated by a double-stranded hexadeoxynucleotide or by calf thymus DNA. Double-stranded oligodeoxynucleotides containing dephosphorylated termini were more effective activators than those containing 3'-phosphorylated termini which in turn were more effective than those containing 5'-phosphorylated termini.     

Visualization of poly(ADP-ribose) synthetase associated with polynucleosomes by immunoelectron microscopy

Antibodies showing a high specificity for poly(ADP ribose) synthetase have been purified. A fraction binding nonspecifically to histones present in antiserum and non-immune serum has been demonstrated by immunoblotting and separated by histone-Sepharose chromatography. The antibody without the nonspecific binding fraction was analyzed by Western blot with calf thymus protein extract and was found to react only with a band at 116 kDa. There was no reaction with purified topoisomerase I, this weak activity was copurified with poly(ADP-ribose) synthetase preparation. The specific IgG fraction has been used for the visualization of the interaction of poly(ADP-ribose) synthetase with chromatin by indirect gold-labelling. This immunomicroscopic study suggests that the synthetase is located in the inner part of polynucleosomes and would be associated preferentially with the core nucleosome.     

Production and characterization of monoclonal antibodies specific for the functional domains of poly(ADP-ribose) polymerase

Monoclonal antibodies were developed against poly(ADP-ribose) polymerase and analyzed for their reactivity against the NAD+- and DNA-binding fragments. Two fusions were performed to obtain hybridomas and the resulting anti-poly(ADP-ribose) polymerase antibodies were further screened by characterization of their immunoglobulin light chains. Five different hybridomas were isolated which produced different immunoglobulin light chains, all of which were specific for poly(ADP-ribose) polymerase. The specificities of these antibodies were determined by immunoblotting against the purified poly(ADP-ribose) polymerase, its autodegradation fragments, and the fragments prepared by limited proteolysis with chymotrypsin and papain. These fragments have been suggested to contain the NAD+-binding site, the DNA-binding site, and the automodification site, respectively. All the monoclonal antibodies reacted with the 116 kdalton (kDa) band corresponding to the purified enzyme. Four antibodies reacted exclusively with antigenic site(s) on the 46-kDa fragment which contains the DNA-binding site. A fifth antibody reacted exclusively with a clearly different antigenic site on the 74- and 54-kDa fragments which possess the NAD+ (substrate) binding site. The immunoreactivity with the major autodegradation products (69- and 46-kDa fragments) of the purified enzyme confirms this difference between the two groups of antibodies. The 22-kDa fragment corresponding to the auto-modification site does not show any immunoreactivity with the antibodies.     

Depression in gene expression for poly(ADP-ribose) synthetase during the interferon-gamma-induced activation process of murine macrophage tumor cells

A 2.7-kb cDNA clone coding for bovine poly(ADP-ribose) synthetase was isolated from a lambda gt11 expression library by direct immunological screening with an antiserum to the enzyme. The cDNA hybridizes to an approximately 3.8-kb bovine thymus polyadenylated RNA, which translates an immunoprecipitable 120-kDa protein with the antibody to the enzyme. The partial DNA sequence of the cDNA was determined and portions of the predicted amino acid sequence matched the sequence of 26 amino acids at the N terminal of the 41-kDa alpha-chymotryptic fragment and two cyanogen-bromide-cleaved peptides of the enzyme. A subcloned fragment from the coding region of the cDNA was used as a probe to estimate the level of mRNA for the enzyme during the interferon-gamma-induced activation process of the murine macrophage tumor P388D1 cell line. The amount of mRNA for the enzyme decreased nearly completely within 24 h after incubation in a medium containing interferon-gamma, while mRNA of the Ia antigen, one of the major histocompatibility gene products, was increased in the macrophage tumor cells by interferon-gamma as confirmed by the I-A beta cDNA as a probe. These results suggest that the gene expression for poly(ADP-ribose) synthetase is depressed during the interferon-gamma-induced activation process of macrophage tumor cells.     

Expression in E. coli of the catalytic domain of rat poly(ADP-ribose)polymerase

A 2 kilobase pair cDNA coding for the entire C-terminal catalytic domain of rat poly(ADP-ribose)polymerase has been expressed in E. coli. The overproduced 55 kDa polypeptide is active in synthesizing poly(ADP-ribose) and the 4 kDa N-terminal region of this domain is recognized by the monoclonal antibody C I,2 directed against the calf enzyme. Also, the minor alpha-chymotrypsin cleavage site found in the human catalytic domain is not present in the rat enzyme as revealed by the absence of the 40 kDa specific degradation product in the E. coli cells expressing the rat domain. The expression of this partial rat cDNA should thus permit the rapid purification and subsequent crystallization of the catalytic domain of the enzyme.     

Two proteolytic degradation products of calf-thymus poly(ADP-ribose) polymerase are efficient ADP-ribose acceptors. Implications for polymerase architecture and the automodification of the polymerase

Two polypeptides with molecular masses of 76 and 59 kDa were found to copurify with poly(ADP-ribose) polymerase from calf thymus, and to be as efficient acceptors of ADP-ribose as the polymerase itself. Analysis of their CNBr fragments by sodium dodecylsulfate/polyacrylamide gel electrophoresis revealed that the polypeptides were derived from the 112-kDa polymerase. Isolation of poly(ADP-ribose) polymerase in the absence of protease inhibitors resulted in a loss of more than 90% of the polymerase activity and an increased proportion of the 76-kDa and 59-kDa polypeptides in the final polymerase preparation. When the polymerase and the two polypeptides were separated by gel filtration or polyacrylamide gel electrophoresis in 5% acetic acid, no polymerase activity was found associated with the two fragments. Analysis of the CNBr fragments of the three polypeptides after incubation of the enzyme preparation with [32P]NAD showed that most of the fragments were radioactive, indicating multiple ADP-ribosylation sites. Several ADP-ribosylated fragments were found to be common to all three polypeptides, or to two of them.     

Inhibition of calf thymus type II DNA topoisomerase by poly(ADP-ribosylation).

The effect of poly(ADP-ribosylation) on calf thymus topoisomerase type II reactions has been investigated. Unknotting of phage P4 head DNA, and relaxation and catenation of supercoiled PM2 DNA are inhibited. We conclude that the inhibition results from poly(ADP-ribosylation) on the following grounds. Firstly, the enzyme poly(ADP-ribose) (PADPR) synthetase and NAD are required, secondly, the competitive synthetase inhibitor nicotinamide abolishes topoisomerase inhibition, and thirdly, the polymer alone is not inhibitory. The mechanism of inhibition appears to be disruption of the strand cleavage reaction. A topoisomerase-DNA complex can be formed that upon treatment with protein denaturant at low ionic strength results in strand cleavage. The amount of DNA present in such a cleavable-complex progressively decreased following pretreatment of topoisomerase type II with PADPR synthetase and increasing concentrations of NAD. Treatment of the pre-formed complex with NAD and PADPR synthetase had no effect on its salt-induced dissociation. This suggests that either poly(ADP-ribosylation) has no influence on dissociation of topoisomerase, in contrast to association, or topoisomerase is not accessible to the synthetase when bound to DNA. Similar data were obtained with calf thymus type I topoisomerase.     

In vivo ischemia and storage injury cause alterations in the activity of poly(adenosine diphosphate ribose) synthetase

The objective of this study was to determine if DNA damage caused by ischemic insult (blood depletion) causes an alteration in the activity of endogenous mouse kidney poly(ADP-ribose) synthetase. The results show that kidneys made nonviable by warm (37 degrees C) in vitro ischemia (organ storage to study the effects of blood loss at normal body temperature) and in vivo ischemia (surgical depletion of the blood supply by arterial clamping) exhibit decreased levels of enzyme activity. Kidneys made nonviable by cold (0 degrees C) storage injury (organ storage as utilized for transplantation), however, possess elevated levels of enzyme activity. The DNA isolated from ischemic kidneys was shown to have a stimulatory effect upon exogenous calf thymus poly(ADP-ribose) synthetase. Also, electron microscopy analysis of DNA from ischemic kidneys showed that cold storage injury leads to the formation of large (average size = 500 bases) single-stranded regions. The results suggest that the activities of both endogenous and exogenous poly(ADP-ribose) synthetase are related to the nature of DNA damage resulting from ischemic insult.     

Purification and characterization of poly(ADP-ribose) glycohydrolase. Different modes of action on large and small poly(ADP-ribose)

Poly(ADP-ribose) glycohydrolase was purified approximately 74,000-fold to apparent homogeneity from calf thymus with a yield of 3.2%. The enzyme was a monomeric protein of Mr = 59,000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The action of glycohydrolase on poly(ADP-ribose) was exoglycosidic in the direction of adenosine terminus----ribose terminus; radioactive ADP-ribose monomers were immediately produced from evenly labeled poly(ADP-ribose), but not from the polymer labeled selectively at the ribose terminus. The enzymatic degradation of large poly(ADP-ribose) (greater than 20 ADP-ribose residues) proceeded in a biphasic as well as bimodal manner. In the early and rapid phase, the enzyme degraded part of large polymers successively, leaving the remainder completely intact, and accumulated ADP-ribose monomers and small polymers of the size less than half of original polymers, indicating that the enzyme action was processive up to a certain extent. In the late and 20-fold slower phase, by contrast, the enzyme degraded the accumulated small polymers gradually and evenly, i.e. in a nonprocessive manner. The Km for large polymers was approximately 100-fold lower than that for small polymers. Similar rates and processivities were observed with large and small polymers bound to various proteins. These results suggested that the glycohydrolase may regulate differentially the levels of large and small poly(ADP-ribose) in the cell.     

Modulation of chromatin superstructure induced by poly(ADP-ribose) synthesis and degradation

It has been demonstrated recently by Poirier et al. (Poirier, G. G., de Murcia, G., Jongstra-Bilen, J., Niedergang, C., and Mandel, P. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 3423-3427) that poly(ADP-ribosyl)ation of pancreatic nucleosomes causes relaxation of the chromatin superstructure through H1 modification. The in vitro effect of poly(ADP-ribose) synthesis and degradation on calf thymus chromatin was investigated by the time course incorporation of ADP-ribose, electron microscopy, analytical ultracentrifugation, and autoradiography of the protein acceptors. Purified calf thymus poly(ADP-ribose) polymerase and partially purified bull testis poly(ADP-ribose) glycohydrolase were used. Degradation of ADP-ribose units on hyper(ADP-ribosyl)ated H1 by poly(ADP-ribose) glycohydrolase restores the native condensed chromatin superstructure. This reversible conformational change induced by poly(ADP-ribosyl)ation on nucleosomal arrangement could be one of the mechanisms by which the accessibility of DNA polymerases and/or excision-repair enzymes is favored, the native structure being fully restorable.     

Benzamide-DNA interactions: deductions from binding, enzyme kinetics and from X-ray structural analysis of a 9-ethyladenine-benzamide adduct

The interaction of benzamide with the isolated components of calf thymus poly(ADP-ribose) polymerase and with liver nuclei has been investigated. A benzamide-agarose affinity gel matrix was prepared by coupling o-aminobenzoic acid with Affi-Gel 10, followed by amidation. The benzamide-agarose matrix bound the DNA that is coenzymic with poly(ADP-ribose) polymerase; the matrix, however, did not bind the purified poly(ADP-ribose) polymerase protein. A highly radioactive derivative of benzamide, the 125I-labelled adduct of o-aminobenzamide and the Bolton-Hunter reagent, was prepared and its binding to liver nuclear DNA, calf thymus DNA and specific coenzymic DNA of poly(ADP-ribose) polymerase was compared. The binding of labelled benzamide to coenzymic DNA was several-fold higher than its binding to unfractionated calf thymus DNA. A DNA-related enzyme inhibitory site of benzamide was demonstrated in a reconstructed poly(ADP-ribose) polymerase system, made up from purified enzyme protein and varying concentrations of a synthetic octadeoxynucleotide that serves as coenzyme. As a model for benzamide binding to DNA, a crystalline complex of 9-ethyladenine and benzamide was prepared and its X-ray crystallographic structure was determined; this indicated a specific hydrogen bond between an amide hydrogen atom and N-3 of adenine. The benzamide also formed a hydrogen bond to another benzamide molecule. The aromatic ring of benzamide does not intercalate between ethyladenine molecules, but lies nearly perpendicular to the planes of stacking ethyladenine molecules in a manner reminiscent of the binding of ethidium bromide to polynucleotides. Thus we have identified DNA as a site of binding of benzamide; this binding is critically dependent on the nature of the DNA and is high for coenzymic DNA that is isolated with the purified enzyme as a tightly associated species. A possible model for such binding has been suggested from the structural analysis of a benzamide-ethyladenine complex.     

Properties of purified calf thymus poly(adenosine diphosphate ribose) polymerase. Comparison of the DNA-independent and the DNA-dependent enzyme.

The physicochemical properties of the purified calf thymus poly(ADP-ribose) polymerase were investigated. The enzyme purified to homogeneity was shown to contain about 10% DNA on a weight basis and its activity to be DNA independent. After removing this fragment of DNA, called the sDNA fraction, the enzyme becomes DNA dependent. The activity of this enzyme preparation was entirely dependent on, and completely restored by, added calf thymus DNA or sDNA. However, the calf thymus DNA concentration needed was a hundred times higher than that of sDNA. The properties of the two enzyme preparations, DNA independent and DNA dependent, were essentially the same. They both reacted against the specific antibody obtained with the DNA-independent poly(ADP-ribose) polymerase. The pH optimum was around 8; the activity was stimulated by Mg2+, Mn2+ and Ca2+, and inhibited by high ionic strength, p-chloromercuribenzoate, ADP-ribose, AMP and polylysine. Nicotinamide, thymidine and NADP were shown to be competitive inhibitors. The enzymatic activity was stimulated by histone H1 when the ratio of DNA to histone H1 was 2. Histones H2A, H2B, H3 and H4 had little effect on the DNA-independent enzyme activity, but were strongly inhibitory for the DNA-dependent enzyme. This inhibitory effect could be reversed by allowing the DNA-dependent enzyme to react with the sDNA fraction before adding histone subfractions. The apparent Km for NAD of the DNA-dependent poly(ADP-ribose) polymerase was shown to vary with the DNA concentration. It was minimum when the amount of sDNA was 10% of that of the enzyme. The ratio of the apparent Km for sDNA to the enzyme concentration was constant at any enzyme concentration. The minimum estimation of the number of base pairs of sDNA required for maximal activation of one enzyme molecule was 16. For calf thymus DNA, this estimation was of 640. These results suggest that the activation of the enzyme needs the formation of some complex between the protein and a specific part of the DNA. This complex was preserved in the DNA-independent enzyme preparation.     

Scl 70 autoantibodies from scleroderma patients recognize a 95 kDa protein identified as DNA topoisomerase I

Sera of patients suffering from the autoimmune disease progressive systemic sclerosis (PSS) are known to contain autoantibodies which have been reported to recognize a 70 kDa antigenic protein, designated the Scl 70 antigen. By immunoblotting of nuclear extracts from HeLa cells with sera from scleroderma patients we observed that the size of the antigen present in such cells depends on the conditions of antigen isolation. When protease inhibitors were included in the extraction buffer, a 95 kDa protein was identified instead of a 70 kDa protein. When protease inhibitors were omitted, a number of polypeptides in the size range 66 to 95 kDa was found. Furthermore, antibodies which had been affinity purified on the 95 kDa antigen, crossreacted with the 66 to 95 kDa polypeptides. These results suggest that the smaller proteins were degradation products of the 95 kDa antigen. Immunofluorescence studies on PtK-2 cells with the antibody specific for the 95 kDa protein gave staining of nuclei, nucleoli and of chromosomes and the nucleolar organizer region in mitotic cells. Since this distribution of antigens within the nucleus was reminiscent of the intranuclear distribution of DNA topoisomerase I found by others we probed purified DNA topoisomerase I from calf thymus directly with the autoantibodies from PSS patients, and also the 95 kDa antigens of HeLa cell nuclei with antibodies raised against the bovine DNA topoisomerase I. From the crossreaction pattern observed with the different antigens and antibodies we conclude that DNA topoisomerase I is one of the antigenic components against which autoantibodies are formed in scleroderma patients.     

Poly(ADP-ribosylation) of a DNA topoisomerase

A DNA topoisomerase activity, copurifying with poly(ADP-ribose) synthetase from calf thymus, is greater than 95% inhibited if extensive poly(ADP-ribosylation) is allowed to occur. The inhibited DNA topoisomerase, which has drastically different elution properties on hydroxylapatite, can be reactivated by mild alkaline treatment. These results are consistent with a poly(ADP-ribosylation) of the DNA topoisomerase and covalent attachment of the poly(ADP-ribose) moieties to the topoisomerase by alkali-labile bonds.     

Detection of poly(ADP-ribose) polymerase and its reaction product poly(ADP-ribose) by immunocytochemistry

Summary Poly(ADP-ribose) polymerase catalyses the formation of ADP-ribose polymers covalently attached to various nuclear proteins, using NAD⁺ as substrate. The activity of this enzyme is strongly stimulated upon binding to DNA single or double strand breaks. Poly(ADP-ribosyl)ation is an immediate cellular response to DNA damage and is thought to be involved in DNA repair, genetic recombination, apoptosis and other processes during which DNA strand breaks are formed. In recent years we and others have established cell culture systems with altered poly(ADP-ribose) polymerase activity. Here we describe immunocytochemistry protocols based on the use of antibodies against the DNA-binding domain of human poly(ADP-ribose) polymerase and against its reaction product poly(ADP-ribose). These protocols allow for the convenient mass screening of cell transfectants with overexpression of poly(ADP-ribose) polymerase or of a dominant-negative mutant for this enzyme, i.e. the DNA-binding domain. In addition, the immunocytochemical detection of poly(ADP-ribose) allows screening for cells with altered enzyme activity.     

Immunoquantitation and size determination of intrinsic poly(ADP-ribose) polymerase from acid precipitates. An analysis of the in vivo status in mammalian species and in lower eukaryotes

Antibodies against pig thymus poly(ADP-ribose) polymerase were obtained with enzyme-hemocyanin conjugates and used for immunoquantitation. The quick-blot procedure used allowed the determination of amounts as low as 1 ng of enzyme from whole cell trichloracetic acid precipitates. When applied to analysis of various human, rodent, and bovine cell types, surprisingly similar amounts of polymerase were found (1-5 ng of pig thymus polymerase equivalents/micrograms of DNA, 2 X 10(5) polymerase molecules/HeLa cell). Also, no significant difference was seen between normal and transformed cells. Polymerase tended to decline in several fibroblast cultures upon reaching confluency, which was not reflected by total polymerase activity. Divergence between total activity and immunogenic equivalents was also seen in alkylated cells and in rat liver treated with phenobarbital. Trichloroacetic acid-insoluble fractions dissolved in sodium dodecyl sulfate buffer could also be used to analyze, by Western blotting, the size distribution of poly(ADP-ribose) polymerase in vivo. Application to various cell types revealed that all mouse and rat cells tested had two immunogenic bands (116 and 98 kDa) of similar intensity. A highly conserved structure of poly(ADP-ribose) polymerase may be deduced from the existence of immunogenic and renaturable 116-kDa polypeptide bands even in the low eukaryotes Physarum polycephalum and Dictyostelium discoideum.     

p66 an in vivo target for poly(ADP-ribosyl)ation co-purifies with poly(ADP-ribose) polymerase

A direct immunoassay has been applied for the quantitation of poly(ADP-ribose) polymerase and its automodification in BALB/3T3 (A31) cells. As the cell population reached a high density, a 66 kDa protein (designated p66) which co-purified with the enzyme became highly poly(ADP-ribosyl)ated. Pulse-chase experiments as well as a Western blot analysis indicated that the p66 was not a degradation product of poly(ADP-ribose) polymerase.