Cellular regulation of ADP-ribosylation of proteins. IV. Conversion of poly(ADP-ribose) polymerase activity to NAD-glycohydrolase during retinoic acid-induced differentiation of HL60 cells.

Two enzymatic activities of the nuclear enzyme poly(ADP-ribose) polymerase or transferase (ADPRT, EC 2.4.2.30), a DNA-associating abundant nuclear protein with multiple molecular activities, have been determined in HL60 cells prior to and after their exposure to 1 microM retinoic acid, which results in the induction of differentiation to mature granulocytes in 4-5 days. The cellular concentration of immunoreactive ADPRT protein molecules in differentiated granulocytes remained unchanged compared to that in HL60 cells prior to retinoic acid addition (3.17 +/- 1.05 ng/10(5) cells), as did the apparent activity of poly(ADP-ribose) glycohydrolase of nuclei. On the other hand, the poly(ADP-ribose) synthesizing capacity of permeabilized cells or isolated nuclei decreased precipitously upon retinoic acid-induced differentiation, whereas the NAD glycohydrolase activity of nuclei significantly increased. The nuclear NAD glycohydrolase activity was identified as an ADPRT-catalyzed enzymatic activity by its unreactivity toward ethenoadenine NAD as a substrate added to nuclei or to purified ADPRT. During the decrease in in vitro poly(ADP-ribose) polymerase activity of nuclei following retinoic acid treatment, the quantity of endogenously poly(ADP-ribosylated) ADPRT significantly increased, as determined by chromatographic isolation of this modified protein by the boronate affinity technique, followed by gel electrophoresis and immunotransblot. When homogenous isolated ADPRT was first ADP-ribosylated in vitro, it lost its capacity to catalyze further polymer synthesis, whereas the NAD glycohydrolase function of the automodified enzyme was greatly augmented. Since results of in vivo and in vitro experiments coincide, it appears that in retinoic acid-induced differentiated cells (granulocytes) the autopoly(ADP-ribosylated) ADPRT performs a predominantly, if not exclusively, NAD glycohydrolase function.     

ADP-ribosylation of histones of the chicken liver nucleus at different rates of glycohydrolase hydrolysis of NAD

The rate of incorporation of nicotinamide-[adenosine-U-14C]adenine dinucleotide [( Ado-U-14C]NAD) into histones and the poly(ADPR) polymerase activity of chromatin suggest that the NAD-dependent ADP-ribosylation of histones depends on the rate of NAD hydrolysis by glycohydrolase in chicken liver nuclei. With a rise in the NAD-glycohydrolase activity after treatment of nuclei with Triton X-100 the synthesis of poly(ADP-ribose) via the poly(ADPR)polymerase reaction is augmented, as a result of which the rate of [Ado-U-14C]NAD incorporation into total histones is increased. On the contrary, the decrease of NAD-glycohydrolase hydrolysis after treatment of nuclei with SDS lowers the poly(ADPR)polymerase activity and [Ado-U-14C]NAD incorporation into histones. Under these conditions, i. e. different rates of glycohydrolase hydrolysis of NAD in the nuclei, some redistribution of [Ado U-14C]NAD incorporation into individual histones occurs.     

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.     

Herpes simplex virus 1 infection activates poly(ADP-ribose) polymerase and triggers the degradation of poly(ADP-ribose) glycohydrolase

Herpes simplex virus 1 infection triggers multiple changes in the metabolism of host cells, including a dramatic decrease in the levels of NAD(+). In addition to its role as a cofactor in reduction-oxidation reactions, NAD(+) is required for certain posttranslational modifications. Members of the poly(ADP-ribose) polymerase (PARP) family of enzymes are major consumers of NAD(+), which they utilize to form poly(ADP-ribose) (PAR) chains on protein substrates in response to DNA damage. PAR chains can subsequently be removed by the enzyme poly(ADP-ribose) glycohydrolase (PARG). We report here that the HSV-1 infection-induced drop in NAD(+) levels required viral DNA replication, was associated with an increase in protein poly(ADP-ribosyl)ation (PARylation), and was blocked by pharmacological inhibition of PARP-1/PARP-2 (PARP-1/2). Neither virus yield nor the cellular metabolic reprogramming observed during HSV-1 infection was altered by the rescue or further depletion of NAD(+) levels. Expression of the viral protein ICP0, which possesses E3 ubiquitin ligase activity, was both necessary and sufficient for the degradation of the 111-kDa PARG isoform. This work demonstrates that HSV-1 infection results in changes to NAD(+) metabolism by PARP-1/2 and PARG, and as PAR chain accumulation can induce caspase-independent apoptosis, we speculate that the decrease in PARG levels enhances the auto-PARylation-mediated inhibition of PARP, thereby avoiding premature death of the infected cell.     

Mutant cells defective in poly(ADP-ribose) synthesis due to stable alterations in enzyme activity or substrate availability

We used two different approaches to develop cell lines deficient in poly(ADP-ribose) synthesis to help determine the role of this reaction in cellular functions. One approach to this problem was to develop cell lines deficient in enzyme activity; the other approach was to develop cell lines capable of growing with such low nicotinamide adenine dinucleotide (NAD) levels so as to effectively limit substrate availability for poly(ADP-ribose) synthesis. The selection strategy for obtaining cells deficient in activity of poly(ADP-ribose) polymerase was based on the ability of this enzyme to deplete cellular NAD in response to high levels of DNA damage. Using this approach, we first obtained cell lines having 37-82% enzyme activity compared to their parental cells. We now report the development and characterization of two cell lines which were obtained from cells having 37% enzyme activity by two additional rounds of further mutagenization and selection procedures. These new cell lines contain 5-11% enzyme activity compared to the parental V79 cells. In pursuit of the second strategy, to obtain cells which limit poly(ADP-ribose) synthesis by substrate restriction, we have now isolated spontaneous mutants from V79 cells which can grow stably in the absence of free nicotinamide or any of its analogs. These cell lines maintain NAD levels in the range of 1.5-3% of that found in their parental V79 cells grown in complete medium. The pathway of NAD biosynthesis in these NAD-deficient cells is not yet known. Further characterization of these lines showed that under conditions that restricted poly(ADP-ribose) synthesis, they all had prolonged doubling times and increased frequencies of sister chromatid exchanges.     

Poly(ADP-ribosyl)ation of chromatin in an in-vitro poly(ADP-ribose)-turnover system.

This paper describes the effect of an in-vitro poly(ADP-ribose) turnover system on the poly(ADP-ribosyl)ation of chromatin. Both poly(ADP-ribose)polymerase and poly(ADP-ribose)glycohydrolase were highly purified and used in 4 different turnover systems: non-turnover, slow, medium and fast turnover. These turnover systems were designed to reflect possible turnover conditions in intact cells. The major protein acceptors for poly(ADP-ribose) are histones and the polymerase itself, a process referred to as automodification. The level of poly(ADP-ribose) modification of polymerase, histone H1 and core histones has been measured. The size of the polymer for each of the 3 groups of acceptor proteins has been determined by gel electrophoresis. After many turnover cycles at medium and fast turnover, the histones (H1 and core) become the main poly(ADP-ribose) acceptor proteins. The rate at which steady-state polymer levels are reached and the total accumulation of polymer in a given turnover system are both inversely proportional to the amount of glycohydrolase present. Furthermore, increasing amounts of glycohydrolase in the turnover systems reduces average polymer size. The polymer synthesized in the medium and fast turnover systems is degraded by glycohydrolase in a biphasic fashion and in these systems the half-life of polymer agreed with results found in intact cells. Our results show that the relative levels of polymerase and glycohydrolase activities can regulate the proportional poly(ADP-ribose) distribution on chromatin-associated acceptor proteins during steady-state turnover conditions. The patterns of modification of polymerase and histones under turnover conditions agree with in vivo observations.     

A shuttle mechanism for DNA-protein interactions. The regulation of poly(ADP-ribose) polymerase

Previously it had been shown that poly(ADP-ribose) polymerase requires DNA for its activity and that this enzyme is auto-poly(ADP-ribosyl)ated. The studies reported here indicate that this self-modification inhibits the enzyme and decreases its affinity for DNA, as shown by sucrose gradient density centrifugation. The coupling of poly(ADP-ribose) polymerase with poly(ADP-ribose) glycohydrolase reactivates the polymerase by degrading poly(ADP-ribose) and restoring the polymerase-DNA complex. The assay of polymerase in the presence of glyco-hydrolase was made possible by use of a double-label assay involving release of 14C-labelled nicotinamide and the incorporation of 3H-labelled ADP-ribose from NAD+. These results provide the basis for a shuttle mechanism in which the polymerase can be moved on and off DNA by the action of these two enzymes. Mg2+ and histone H1 appear to activate the polymerase by increasing the affinity of the polymerase for DNA.     

Poly(ADP-ribose) glycohydrolase is present in metaphase chromosomes of HeLa S3 cells

Poly(ADP-ribose) glycohydrolase was found in metaphase chromosomes of HeLa S3 cells. Adenosine diphosphate ribose and 3′, 5′-cyclic AMP inhibited the glycohydrolase activity, whereas ADP, ATP, NAD and 3′,5′-cyclic GMP did not. The hydrolytic product of poly(ADP-ribose) bound to metaphase chromosomes with this enzyme was identified as adenosine diphosphate ribose.     

Poly(ADP-ribose) turnover in quail myoblast cells: Relation between the polymer level and its catabolism by glycohydrolase

The concerted action of poly(ADP-ribose) polymerase (PARP) which synthesizes the poly(ADP-ribose) (pADPr) in response to DNA strand breaks and the catabolic enzyme poly(ADP-ribose) glycohydrolase (PARG) determine the level of polymer and the rate of its turnover. In the present study, we have shown that the quail myoblast cells have high levels of basal polymer as compared to the murine C3H10T1/2 fibroblasts. We have conducted this study to investigate how such differences influence polymer synthesis and its catabolism in the cells in response to DNA damage by alkylating agent. In quail myoblast cells, the presence of high MNNG concentration such as 200 \sgmaelig;M for 30 min induced a marginal decrease of 15% in the NAD content. For C3H10T1/2 cell line, 64 \sgmaelig;M MNNG provoked a depletion of NAD content by approximately 50%. The induction of the polymer synthesis in response to MNNG treatment was 6-fold higher in C3H10T1/2 cells than in quail myoblast cells notwithstanding the fact that 3-fold higher MNNG concentration was used for quail cells. The polymer synthesis thus induced in quail myoblast cells had a 4-5 fold longer half life than those induced in C3H10T1/2 cells. To account for the slow turnover of the polymer in the quail myoblast cells, we compared the activities of the polymer catabolizing enzyme (PARG) in the two cell types. The quail myoblast cells had about 25% less activity of PARG than the murine cells. This difference in activity is not sufficient to explain the large difference of the rate of catabolism between the two cell types implicating other cellular mechanisms in the regulation of pADPr turnover.     

Activation of cell death mediated by apoptosis-inducing factor due to the absence of poly(ADP-ribose) glycohydrolase

We previously demonstrated that the absence of poly(ADP-ribose) glycohydrolase (PARG) led to increased cell death following DNA-damaging treatments. Here, we investigated cell death pathways following UV treatment. Decreased amounts of PARG-null embryonic trophoblast stem (TS) cells were observed following doses of 10-100 J/m2 as compared to wild-type cells. In wild-type cells, caspase-cleaved poly(ADP-ribose) polymerase-1 (PARP-1) and activated caspase-3 were detected 12-24 h after UV treatment. Surprisingly, both were detected at decreased levels only after 24 h in PARG-null TS cells, indicating a decreased level and delayed presence of caspase-mediated events. Further, a time- and dose-dependent accumulation of poly(ADP-ribose) (PAR) levels after UV was observed in PARG-null TS cells and not in wild-type cells. Determination of the levels of nicotinamide adenine dinucleotide (NAD+), the substrate for PAR synthesis and a coenzyme in cellular redox reactions, demonstrated a UV dose-dependent decrease in the level of NAD+ in wild-type cells, while NAD+ levels in PARG-null TS cells remained at higher levels. This indicates no depletion of NAD+ in PARG-null TS cells following increased levels of PAR. Lastly, cell death mediated by apoptosis-inducing factor (AIF) was analyzed because of its dependence on increased PAR levels. The results demonstrate nuclear AIF translocation only in PARG-null TS cells, which demonstrates the presence of AIF-mediated cell death. Herein, we provide compelling evidence that the absence of PARG leads to decreased caspase-3 activity and the specific activation of AIF-mediated cell death. Therefore, the absence of PARG may provide a strategy for specifically inducing an alternative apoptotic pathway.     

Quantitative studies of inhibitors of ADP-ribosylation in vitro and in vivo

The ADP-ribosyl moiety of NAD+ is consumed in reactions catalyzed by three classes of enzymes: poly(ADP-ribose) polymerase, protein mono(ADP-ribosyl)transferases, and NAD+ glycohydrolases. In this study, we have evaluated the selectivity of compounds originally identified as inhibitors of poly(ADP-ribose) polymerase on members of the three classes of enzymes. The 50% inhibitory concentration (IC50) of more than 20 compounds was determined in vitro for both poly(ADP-ribose) polymerase and mono(ADP-ribosyl)transferase A in an assay containing 300 microM NAD+. Of the compounds tested, benzamide was the most potent inhibitor of poly(ADP-ribose) polymerase with an IC50 of 3.3 microM. The IC50 for benzamide for mono(ADP-ribosyl)transferase A was 4.1 mM, and similar values were observed for four additional cellular mono(ADP-ribosyl)transferases. The IC50 for NAD+ glycohydrolase for benzamide was approximately 40 mM. For seven of the best inhibitors, inhibition of poly(ADP-ribose) polymerase in intact C3H1OT1/2 cells was studied as a function of the inhibitor concentration of the culture medium, and the concentration for 50% inhibition (culture medium IC50) was determined. Culture medium IC50 values for benzamide and its derivatives were very similar to in vitro IC50 values. For other inhibitors, such as nicotinamide, 5-methyl-nicotinamide, and 5-bromodeoxyuridine, culture medium IC50 values were 3-5-fold higher than in vitro IC50 values. These results suggest that micromolar levels of the benzamides in the culture medium should allow selective inhibition of poly(ADP-ribose) metabolism in intact cells. Furthermore, comparative quantitative inhibition studies should prove useful for assigning the biological effects of these inhibitors as an effect on either poly(ADP-ribose) or mono(ADP-ribose) metabolism.     

Rapid assay of poly(ADP-ribose) glycohydrolase

We have developed a rapid, highly reproducible assay to determine poly(ADP-ribose) glycohydrolase activity which measures directly the appearance of the reaction product. We also analysed the majority of different techniques which are used to determine poly(ADP-ribose) glycohydrolase activity and found that the apparent activity can vary extensively depending on the method used. Thin-layer chromatography using PEI-F-cellulose was the only method which evaluated directly the specific release of ADP-ribose; by comparison with this method, the other procedures gave an over- or under-estimation of 2- to 10-fold of the enzymatic activity. A rapid method of affinity chromatography has also been developed to synthesize and purify in high yield poly(ADP-ribose) (35% conversion of 1 mM NAD to poly(ADP-ribose)).     

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.     

Expression of human poly(ADP-ribose) polymerase in Saccharomyces cerevisiae

The coding sequence for human poly(ADP-ribose) polymerase was expressed inducibly in Saccharomyces cerevisiae from a low-copy-number plasmid vector. Cell free extracts of induced cells had poly(ADPribose) polymerase activity when assayed under standard conditions; activity could not be detected in non-induced cell extracts. Induced cells formed poly(ADP-ribose) in vivo, and levels of these polymers increased when cells were treated with the alkylating agent N-methyl-N′-nitro-N-nitrosoguanidine (MNNG). The cytotoxicity of this agent was increased in induced cells, and in vivo labelling with [³H]adenine further decreased their viability. Increased levels of poly(ADP-ribose) found in cells treated with the alkylating agent were not accompanied by lowering of the NAD concentration.     

Decline of poly(ADP-ribosyl)ation during in vitro senescence in human diploid fibroblasts

Poly(ADP-ribose) polymerase activity was determined at various times during the in vitro life span of two human diploid fibroblast-like cell lines of different donor ages. The cell lines differed in their ability to transfer ADP-ribose, with cells from an embryonic donor exhibiting 2 to 3 times the activity found in cells obtained from a newborn donor. The activity in both cell lines decreased by 30-60% as the cells moved through their in vitro life spans. The decline could not be attributed to increases in glycohydrolase or the leakage of polymerase from older cell preparations. Enzyme activation with DNase I indicated that similar levels of enzyme were present in both cell lines at all in vitro ages. These results indicate that although poly(ADP-ribosyl)ation is inversely related to donor age as well as in vitro age the decrease is in response to other factors which change with increasing age.     

Developmental pattern of poly (ADP-ribose) synthetase and NAD glycohydrolase in the brain of the fetal and neonatal rat

Poly (ADP-ribose) synthetase and NAD glycohydrolase were examined in nuclear fractions from rat brain at sequential times during late fetal and the first two weeks of neonatal life. In whole brain, both enzymes were demonstrable at all stages of development, but followed separate patterns. Activity of the synthetase which was greatest in fetal life, fell steadily with fetal maturation from 3.90±0.06 nmol/mg DNA at 16 days, to reach a nadir of 1.36±0.09 nmol/mg DNA on the 4th postnatal day. Subsequently it underwent a non sustained neonatal rise reaching a peak of 2.46±0.07 nmol/mg DNA on the 8th day. By contrast, NAD glycohydrolase activity increased steadily throughout late fetal and during the first two weeks of neonatal life, from 12.77±0.40 nmol/mg DNA on day 16 of gestation to 25.80±.95 nmol/mg DNA on neonatal day 12. In neonatal cerebellum the activity of poly (ADP-ribose) synthetase was greater at 8 than at 4 days, could be stimulated with graded concentrations of sonicated DNA up to 100 g, but was inhibited by higher concentrations of DNA and by all concentrations of exogenous histone. In an in vitro culture system of fetal rat brain cells, the activity of poly (ADP-ribose) synthetase increased steadily over six days. Cycloheximide 10^–3 M completely inhibited the activity of this enzyme. NAD glycohydrolase activity increased progressively in vitro, and after 6 days in cycloheximide (10^–3 M), the cultures contained significantly greater levels of enzyme activity. It is suggested that changing activities of poly (ADP-ribose) synthetase and NAD glycohydrolase could both provide potential markers for brain cell differentiation in this system.     

Changes in pyridine and adenine nucleotide levels in Friend erythroleukaemia cells during growth and differentiation.

Pyridine and adenine nucleotide levels were measured in Friend erythroleukaemia cells (FELC) stimulated to growth and induced to differentiate by hexamethylene bisacetamide (HMBA) and N'-methylnicotinamide (N'-MNAM). A three- to fourfold increase in the NADP(H) was found to parallel cell growth stimulation in both the presence and absence of differentiation inducers. NAD(H) increased about twofold in control and to a minor extent in HMBA-treated FELC but did not vary significantly in N'-MNAM-treated cells. ATP was significantly higher in control cells stimulated to growth than in resting ones, but it did not vary in inducer-treated cells. These data confirm the relationship between high NADP(H) levels and cell resumption to growth; moreover they show that NAD(H) pool reduction and NAD/NADH ratio rise are associated with the process of FELC differentiation. The activities of NAD pyrophosphorylase and NAD kinase are much more enhanced in growth-stimulated FELC than in resting ones. On the other hand transition from the quiescent to the proliferative state was accompanied by a decrease in the activity of poly(ADP-ribose) polymerase. A decrease in poly(ADP-ribose) polymerase activity was also found in differentiated cells in contrast to controls.     

NAD turnover during early development of Xenopus laevis

The NAD pools of Xenopus laevis oocytes and early embryos can be radioactively labelled by microinjection of [adenine- ³H]NAD. This technique is used to study the metabolism of NAD in oocytes and during early development. The rate at which NAD is degraded in vivo has been monitored by determining the rate of transfer of adenine residues from the NAD pool into other nucleotides and polynucleotides. In oocytes, NAD turnover is extremely slow, with a half-life of about 400 h. NAD turnover increases dramatically after fertilisation, and the half-life of the compound decreases to 37 h in 5-h-old embryos and to 10 h in 40-h-old embryos. 2 mM 3-aminobenzamide, a specific inhibitor of poly(ADP-ribose) polymerase, reduces the NAD turnover rate by about 20%, whereas 5 mM isonicotinic acid hydrazide, a specific inhibitor of NAD glycohydrolase, produces no significant inhibition. This indicates that a significant fraction of the considerable NAD turnover observed involves poly(ADP-ribose) polymerase. Our results indicate that poly(ADP-ribose) polymerase is active during early development and suggest that this activity may be involved in one or more aspects of the nuclear metabolism of the embryo.     

Reconstitution of an in vitro poly(ADP-ribose) turnover system

Poly(ADP-ribose) is synthesized and degraded by poly(ADP-ribose) polymerase and glycohydrolase, respectively. We have reconstituted in vitro two turnover systems containing these two enzymes. We have measured the kinetics of NAD consumption and polymer accumulation during turnover. The combined action of the two enzymes (i.e., turnover) generates a steady state of polymer quantity. The glycohydrolase determines the time and the level at which this steady state of total polymer is reached. A major observation is that the size and calculated density of polymer bound to the total polymerase molecules is tightly regulated by the rate of polymer turnover. On the polymerase, an increase in the rate of polymer turnover does not affect the mean polymer size, but reduces the polymer density on the enzyme (i.e., the number of polymer chains per polymerase molecule). In the absence of glycohydrolase and at low histone H1 concentration (less than 1.5 micrograms/ml), poly(ADP-ribose) polymerase preferentially automodifies itself instead of modifying histone H1. In contrast, under turnover conditions, oligomer accumulation on histone H1 was greatly increased, with almost 40% of all the polymer present on H1 after 5 min of turnover. Although turnover conditions were necessary for histone H1 labelling, there was no difference between the fast and the slow turnover systems as concerns the proportion of histone H1 labelling, although the mean polymer size on histone H1 was decreased with increasing turnover rate. Due to its small size, polymer is not degraded by the glycohydrolase and accumulates on histone H1 during turnover. These data suggest that the glycohydrolase modulates the level of poly(ADP-ribosyl)action of different proteins in two ways; by degrading shorter polymers at a slower rate and probably by competing with the polymerase for polymer.