Increased ionic strength: effects on DNA fragmentation and poly(ADP-ribose) polymerase activity in HeLa nuclei

Poly(ADP-ribose) polymerase activity was measured in a crude nuclear fraction isolated from HeLa cells. It was found that the addition of ammonium sulfate or other salts to the standard incubation medium inhibited the formation of poly(ADP-ribose). Through the use of alkaline sucrose density gradients it was also noted that this same increase in ionic strength inhibited the in vitro breakdown of the HeLa DNA. Additional experiments with alkaline sucrose density gradients and deoxyribonuclease I showed that the in vitro activity of poly(ADP-ribose) polymerase is largely dependent upon DNA fragmentation but that DNA fragmentation at least in vitro is not dependent upon the formation of poly(ADP-ribose). These observations imply that this nuclear enzyme is not extremely sensitive to changes in the ionic strength of the reaction media but is affected indirectly, supposedly through changes in the endonuclease activity of the HeLa nuclei. If this proves to be true, then the addition of salt to the incubation medium for poly(ADP-ribose) polymerase could prove to be a valuable tool for the study of ADP-ribosylation reactions.     

Catalytic activities of human poly(ADP-ribose) polymerase from normal and mutagenized cells detected after sodium dodecyl sulfate-polyacrylamide gel electrophoresis

An activity gel procedure is described to identify functional polypeptides of human poly(ADP-ribose) polymerase. Purified or crude enzyme preparations from HeLa cells were electrophoresed in sodium dodecyl sulfate-polyacrylamide gels containing gapped DNA. After renaturation of the peptides in situ, the intact gel was incubated in a poly(ADP-ribose) polymerase reaction mixture containing [32P]NAD. Autoradiograms of the gels consistently exhibited a major activity band at Mr = 116,000-120,000; in many runs, three minor distinct bands at Mr = 125,000, 135,000, and 145,000 were also seen. [32P]NAD appeared to be incorporated into poly(ADP-ribose) since: (i) the activity bands were not detectable when the enzyme-inhibitor 3-aminobenzamide was added to the gel incubation mixture; and (ii) the radioactive polymer, electroeluted from the bands, was completely digested by phosphodiesterase I. Preliminary activity gel analysis of extracts of HeLa cells treated with different DNA-damaging agents revealed that the apparent activity of the Mr = 116,000 form increased by about 10-fold in cells treated with 1 mM dimethyl sulfate and 10-20-fold in cells treated with 10 microM mitomycin C. Only a small increase was obtained in cells treated with 1 mM methyl methanesulfonate, and no change in the activity band pattern was observed after 50 and 100 J/m-2 of UV irradiation.     

Effect of heparin or removal of lysine-rich histone fraction on the poly(ADP-ribose) polymerase, DNA polymerase and template activity of isolated swine aortic nuclei

Treatment with heparin or preferential removal of lysine-rich histones (LRH) stimulates endogenous DNA polymerase and template activities in swine aortic nuclei. The activities can be further enhanced in the presence of an endonuclease like DNase I. In contrast, the extraction of LRH reversibly inhibits the poly(ADP-ribose) polymerase activity of the extracted nuclei. This is due to the removal of the enzyme along with the LRH and the addition of the extract to the extracted nuclei reverses this inhibition. Heparin, on the other hand, does not inhibit the poly(ADP-ribose) polymerase unless very high concentrations are used. It appears that the removal of LRH as well as poly(ADP-ribose) polymerase exposes initiation sites for DNA polymerase.     

Purification of poly(ADP-ribose) polymerase from Ehrlich ascites tumor cells by chromatography on DNA-agarose.

Poly(ADP-ribose) polymerase with a high specific activity was obtained from Ehrlich ascites tumor cells by extraction of nuclei with 175 mM potassium phosphate, followed by chromatography on DNA-agarose. Electrophoretic analysis indicated that the preparation contained two proteins, one of which was shown to catalyze the synthesis of poly(ADP-ribose). As expected from results obtained by other workers, the synthesis was inhibited by nicotinamide and thymidine, and stimulated by DNA. Addition of histones gave inhibition of the synthesis, unless DNA was present in the reaction mixture.     

Change in activity of nuclear poly(ADP-ribose) glycohydrolase during the HeLa S3 cell cycle

The change in activity of nuclear poly(ADP-ribose) glycohydrolase during the cell cycle of HeLa S3 cells was investigated. The poly(ADP-ribose) glycohydrolase activity was solubilized from HeLa S3 cell nuclei and chromosomes only by sonication at high ionic strength. The enzyme hydrolyzed poly(ADP-ribose) exoglycosidically, producing ADP-ribose. After release from mitosis, the activity of the solubilized nuclear poly(ADP-ribose) glycohydrolase per nucleus or per unit protein, assayed with [3H]poly(ADP-ribose) (average chain length, n = 15) as substrate, was lowest in the early G1 phase and highest in the late G1 phase. The specific activity in the late G1 phase was about two times that in the early G1 phase. The high activity remained constant during the S-G2-M phase. A similar change during the cell cycle was observed after release from hydroxyurea block. These results suggest that the activity of poly(ADP-ribose) glycohydrolase doubled during the G1 phase of the cell cycle of HeLa S3 cells.     

Cytoplasmic poly(ADP-ribose) polymerase during the HeLa cell cycle.

Poly(ADP-ribose) polymerase, an enzyme that has reportedly been confined to the nucleus of eukaryotic cells, has been found in the cytoplasm of HeLa cells. The enzyme activity is stimulated more than 30-fold by the addition of both DNA and histones. These two macromolecules are absolutely necessary for maximal activity and they act in a synergistic manner. The product of the reaction was characterized as poly(ADP-ribose) by its acid insolubility, its insensitivity to hydrolysis by DNase, RNase, spleen phosphodiesterase or Pronase and by release of 5′-AMP and 2′-(5″-phosphoribosyl)-5′-AMP by incubation with snake venom phosphodiesterase. A covalent attachment between histone F1 and poly(ADP-ribose) has been established by using the cytoplasmic enzyme. The enzyme is primarily associated with ribosomes, both free ribosomes and those found in polysomes. Inhibition of protein synthesis in the intact cell reduced the level of activity in the cytoplasm. The enzyme can be removed from the ribosomes by centrifugation through sucrose gradients containing 0.6 m ammonium chloride. A relationship between this enzyme and DNA replication is suggested by the fact that the specific activity in the cytoplasm parallels the rate of DNA synthesis during the HeLa cell cycle.     

Poly(ADP-ribosyl)ation of terminal deoxynucleotidyl transferase in vitro

The activity of purified bovine thymus terminal deoxynucleotidyl transferase was markedly inhibited when the enzyme was incubated in a poly(ADP-ribose)-synthesizing system containing purified bovine thymus poly(ADP-ribose) polymerase, NAD+, Mg2+ and DNA. All of these four components were indispensable for the inhibition. The inhibitors of poly(ADP-ribose) polymerase counteracted the observed inhibition of the transferase. Under a Mg2+-depleted and acceptor-dependent ADP-ribosylating reaction condition [Tanaka, Y., Hashida, T., Yoshihara, H. and Yoshihara, K. (1979) J. Biol. Chem. 254, 12433-12438], the addition of terminal transferase to the reaction mixture stimulated the enzyme reaction in a dose-dependent manner, suggesting that the transferase is functioning as an acceptor for ADP-ribose. Electrophoretic analyses of the reaction products clearly indicated that the transferase molecule itself was oligo (ADP-ribosyl)ated. When the product was further incubated in the Mg2+-fortified reaction mixture, the activity of terminal transferase markedly decreased with increase in the apparent molecular size of the enzyme, indicating that an extensive elongation of poly(ADP-ribose) bound to the transferase is essential for the observed inhibition. Free poly(ADP-ribose) and the polymer bound to poly(ADP-ribose) polymerase were ineffective on the activity of the transferase. All of these results indicate that the observed inhibition of terminal transferase is caused by the poly(ADP-ribosyl)ation of the transferase itself.     

The effect of the x-ray irradiation of rats on the NAD-pyrophosphorylase and poly(ADP-ribose)polymerase activities of brain nuclei and on the NAD content in nerve tissue

A study was made of the influence of X-irradiation of rats with various doses on NAD-pyrophosphorylase and poly(ADP-ribose) polymerase activity of brain nuclei. It was shown that X-radiation was ineffective with regard to NAD-pyrophosphorylase activity of nuclei and increased their poly(ADP-ribose) polymerase activity. Stimulation of poly(ADP-ribose) polymerase activity of nuclei was a function of radiation dose and correlated with the decrease in the NAD content of nervous tissue. It was found that mainly nonhistone proteins were ADP-ribosylated in nuclei of both irradiated and nonirradiated rats.     

Intracellular NAD+ content and ADP-ribose polymerase activity of serum-stimulated baby hamster kidney fibroblasts

We have examined a number of events relating to ADP-ribose metabolism during serum-stimulated growth of BHK-21/C13 fibroblasts. Both the intracellular NAD+ content and the ADP-ribose polymerase activity were found to increase after serum stimulation of cells that were previously arrested by growth in low-serum medium. NAD+ content increased about two-fold, reaching a maximum of 4.2 nmol/microgram of DNA 8 hr after serum steK-21/C13 fibroblasts. Both the intracellular NAD+ content and the ADP-ribose polymerase activity were found to increase after serum stimulation of cells that were previously arrested by growth in low-serum medium. NAD+ content inreased about two-fold, reaching a maximum of 4.2 nmol/microgram of DNA 8 hr after serum step-up. The polymerase exhibited a sharp rise in activity, reaching a peak at about 5 hr after step-up; the activity declined below initial values by 10 hr, and then increased again to reach a plateau at 20 hr. We also report evidence which suggests a possible effect of ADP-ribosylation on the activity of DNA-dependent RNA polymerase I. The activity of this enzyme is diminished in isolated nuclei, and in a subsequent (NH4)2SO4 extract, when the nuclei are incubated with NAD+, the substrate for poly(ADP-ribose) polymerase. This inhibitory effect on the RNA polymerase is abolished when nuclei are incubated also with nicotinamide, a powerful inhibitor of the poly(ADP-ribose) polymerase.     

Independent induction of sister-chromatid exchanges by 3-aminobenzamide and ultraviolet radiation in HeLa cells

The effect of 3-aminobenzamide in HeLa cells was examined in several aspects. 10(-3) M 3-aminobenzamide inhibited poly(ADP-ribose) polymerase activity to more than 95% in disrupted nuclei of HeLa cells. The mode of inhibition of the enzyme was competitive inhibition with NAD at a Ki value of 2.5 microM 3-aminobenzamide. The combined treatment of HeLa cells with 3-aminobenzamide and ultraviolet radiation revealed the independent induction of SCEs by these 2 agents.     

Poly(adenosine diphosphate ribose) metabolism and regulation of myocardial cell growth by oxygen

Control of the rate of cardiac cell division by oxygen occurs most probably by altering the redox state of a control substance, e.g. NAD(+)right harpoon over left harpoonNADH. NAD(+) (and not NADH) forms poly(ADP-ribose), an inhibitor of DNA synthesis, in a reaction catalysed by poly(ADP-ribose) polymerase. Lower partial pressure of oxygen, which increases the rate of division, would shift NAD(+)-->NADH, decrease poly(ADP-ribose) synthesis, and increase DNA synthesis. Chick-embryo heart cells grown in culture in 20% O(2) (in which they divide more slowly than in 5% O(2)) did exhibit greater poly(ADP-ribose) polymerase activity (+83%, P<0.001) than when grown in 5% O(2). Reaction product was identified as poly(ADP-ribose) by its insensitivity to deoxyribonuclease, ribonuclease, NAD glycohydrolase, Pronase, trypsin and micrococcal nuclease, and by its complete digestion with snake-venom phosphodiesterase to phosphoribosyl-AMP and AMP. Isolation of these digestion products by Dowex 1 (formate form) column chromatography and paper chromatography allowed calculation of average poly(ADP-ribose) chain length, which was 15-26% greater in 20% than in 5% O(2). Thus in 20% O(2) the increase in poly(ADP-ribose) formation results from chain elongation. Formation of new chains also occurs, probably to an even greater degree than chain elongation. Additionally, poly(ADP-ribose) polymerase has very different K(m) and V(max.) values and pH optima in 20% and 5% O(2). These data suggest that poly(ADP-ribose) metabolism participates in the regulation of heart-cell division by O(2), probably by several different mechanisms.     

Poly(adenosine diphosphate ribose) polymerase activity and nicotinamide adenine dinucleotide in differentiating cardiac muscle.

Poly(ADP-ribose) polymerase activity in nuclei isolated from differentiating cardiac muscle of the rat has been characterized and its activity measured during development. Optimum enzyme activity is observed at pH 8.5. Poly(ADP-ribose) polymerase is inhibited by ATP, thymidine, nicotinamide, theophylline, 3-isobutyl-1-methylxanthine and caffeine and stimulated by actinomycin D. The activity measured under optimal assay conditions increases during differentiation of cardiac muscle and is inversely related to the rate of DNA synthesis and to the activities of DNA polymerase alpha and thymidine kinase. When DNA synthesis and the activity of DNA polymerase alpha are inhibited in cardiac muscle of the 1-day-old neonatal rat by dibutyryl cyclic AMP or isoproterenol, the specific activity of poly(ADP-ribose) polymerase measured in isolated nuclei is increased. The concentration of NAD+ in cardiac muscle increases during postnatal development. In the adult compared with the 1-day-old neonatal rat the concentration of NAD+ relative to fresh tissue weight, DNA or protein increased 1.7-fold, 5.2-fold or 1.4-fold respectively. The concentration of NAD+ in cardiac muscle of the 1-day-old neonatal rat can be increased by approx. 20% by dibutyryl cyclic AMP. These data suggest that NAD+ and poly(ADP-ribose) polymerase may be involved with the repression of DNA synthesis and cell proliferation in differentiating cardiac muscle.     

Selective probing of ADP-ribosylation reactions with oxidized 2'-deoxy-nicotinamide adenine dinucleotide

A homogeneous preparation of an arginine-specific mono(ADP-ribosyl)transferase from turkey erythrocytes effectively utilized 2'-deoxy-NAD+ for the 2'-deoxy(ADP-ribose) modification of arginine methyl ester with an apparent Km of 27.2 microM and a Vmax of 36.4 mumol min-1 (mg of protein)-1. The adduct formed was also used as a substrate by an avian erythrocyte arginine(ADP-ribose)-specific hydrolase that generated free 2'-deoxy(ADP-ribose). In contrast, 2'-deoxy-NAD+ was not a substrate in the initiation or elongation reaction catalyzed by highly purified poly(ADP-ribose) polymerase from calf thymus. However, 2'-deoxy-NAD+ was a potent noncompetitive inhibitor of NAD+ in the elongation reaction catalyzed by the polymerase, with an apparent Ki of 32 microM. These results indicate that 2'-deoxy-NAD+ may be utilized to specifically identify protein acceptors for endogenous mono(ADP-ribosyl)transferases in complex biological systems that may contain a high activity of poly(ADP-ribose) polymerase, i.e., cell nuclei preparations.     

Differential assay and biological significance of poly(ADP-ribose) polymerase activity in isolated liver nuclei

Poly(ADP-ribosyl)ation of nuclear proteins is catalyzed by poly(ADP-ribose) polymerase. This enzyme is involved in the regulation of basic cellular functions of DNA metabolism. DNA breaks induced by DNA-damaging agents trigger the activation of poly(ADP-ribose) polymerase increasing its endogenous level. This increase modifies the pattern of poly(ADP-ribosyl)ated chromatin proteins. In this paper we describe a procedure for the isolation of intact nuclei from rat liver to be used for the endogenous activity assay. Artifactual activation of the enzyme was avoided since a very low level of DNA-strand breaks occurs during the isolation of nuclei. We present a series of experiments which prove the ability of this procedure to detect increases in endogenous liver activity without modification of the total level. The application of this technique can be useful for a better understanding of the role of early changes in poly(ADP-ribose) polymerase level in physiological conditions and during exposure to DNA-damaging agents.     

Incorporation of NAD into fixed cell nuclei

Incorporation of NAD into the nuclei was determined autoradiographically in cultured HeLa cells and in cryostat sections of rat organs by incubating them with 3H-NAD after fixation with various agents. Acetone fixation was the best to render the cells permeable to NAD while preserving the cell's enzymatic activity to incorporate NAD into nuclear macromolecules. Various evidence supported that such incorporation of NAD is due mostly to the synthesis of poly(ADP-ribose) on chromatin proteins. In the sections of rat jejunum and esophagus the rate of NAD incorporation was higher in the actively proliferating cell nuclei than in the differentiated cell nuclei within the same epithelia. These results suggested that the capacity of the cells to synthesize poly(ADP-ribose) is associated with cell growth and differentiation.     

Poly(ADP-ribosylation) at successive stages of rooster spermatogenesis. Levels of polymeric ADP-ribose in vivo and poly(ADP-ribose) polymerase activity and turnover of ADP-ribosyl residues in vitro

Rooster testis cells were separated by sedimentation at unit gravity and the in vivo levels of polymeric ADP-ribose were determined both in intact cells and isolated nuclei by fluorescence methods. Poly(ADP-ribose) polymerase activity was assayed after cell permeabilization or after isolation of nuclei. The turnover of ADP-ribosyl residues was determined in isolated nuclei using benzamide. The content of poly(ADP-ribose), the poly(ADP-ribose) polymerase activity, and the turnover of ADP-ribosyl residues, decreased during the differentiation of the germinal cell line, especially at the end of spermiogenesis. Treatment of cells with 1 mM dimethyl sulfate for 1 h resulted in a marked stimulation of poly(ADP-ribose) polymerase activity in meiotic and premeiotic cells and also in round and late spermatids. The enzymatic activity was not detected and could not be induced in mature spermatozoa. These cells, however, still contained polymeric ADP-ribose with a 2% of branched form.     

The effect of Cu2+, Zn2+ cations and biogenic amines on the nuclear poly(ADP-ribose) polymerase activity in the rat brain

Study of the effects of Cu2+, Zn2+ cations and polyamines, spermine and spermidine, on the nuclear poly(ADP-ribose)polymerase activity of rat brain was carried out. It was shown that low concentrations of Cu2+ stimulate the activity of purified poly(ADP-ribose)polymerase. The poly(ADP-ribose)polymerase activity was increased 1.4-fold at 5 microM Cu2+. A further increase of Cu2+ concentration inhibited the enzymatic activity; at 50 microM Cu2+ the polymerase activity appeared to be fully inhibited. It was shown that Zn2+ inhibited only the poly(ADP-ribose)polymerase activity. Zn2+ at a concentration of 125 microM fully inhibited the enzymatic activity. Spermine and spermidine stimulated the poly(ADP-ribose)polymerase activity of brain nuclei of newborn and old rats.