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.     

Modified nucleotide polymers as inhibitors of DNA polymerases.

We have compared the relative inhibitory activity of poly (A) with its analogues poly N6-isopentenyl adenylic acid (poly(i6 A)) and poly N6-benzyl adenylic acid (poly(bzl6A)), and of poly (U) with its analogue poly 2'-fluoro-2'-deoxyuridylic acid (poly(dUfl)), against DNA polymerase, alpha, beta and gamma and terminal deoxynucleotidyl transferase from human cells and two oncorna virus DNA polymerases. Although poly (A) and its analogues were equally inhibitory against endogenous RNA-directed DNA polymerases of murine and feline leukemia viruses, the analogues in contrast to poly (A) were strongly inhibitory against all four cellular enzymes. Poly (dUfl), on the other hand, was up to 100-fold more potent than poly (U) against both viral and cellular enzymes. Since poly (U) at 100 mug/ml and poly (dUfl) at 1 mug/ml had no effect on terminal deoxynucleotidyl transferase while inhibiting other enzymes by 80--100 per cent these polymers could be useful in the characterization and assay of terminal deoxynucleotidyl transferase. In addition, the polymers such as poly (igA) and poly (bzl5A) which were strongly inhibitory to all cellular enzymes, could be useful in cancer chemotherapy if taken up preferentially by the malignant calls due to their high pinocytic activity. The results also demonstrate potential for large variation in inhibitory activity of polyribonucleotides as related to their chemical composition.     

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-ribosyl)ation of DNA polymerase beta in vitro

DNA polymerase beta purified from bovine thymus is markedly inhibited when incubated in a reconstituted poly(ADP-ribosyl)ating reaction system. Analyses of the reaction product synthesized in this system by SDS-polyacrylamide gel electrophoresis and subsequent fluorography of the gel indicated that ADP-ribose is covalently attached to DNA polymerase beta molecule (Mr = 44,000).     

Biochemical association of poly(ADP-ribose) polymerase-1 and its apoptotic peptide fragments with DNA polymerase beta

We have characterized the biochemical association of two DNA damage-dependent enzymes, poly(ADP-ribose) polymerase-1 (PARP-1) [EC 2.4.2.30] and DNA polymerase beta (pol beta) [2.7.7.7]. We reproducibly observed that pol beta is an efficient covalent target for ADP-ribose polymers under standard conditions of enzymatically catalyzed ADP-ribosylation of betaNAD+ as a substrate. The efficiency of poly(ADP-ribosyl)ation increased as a function of the pol beta and betaNAD+ concentrations. To further characterize the molecular interactions between these two unique polymerases, we also subjected human recombinant PARP-1 to peptide-specific enzymatic degradation with either caspase-3 or caspase-7 in vitro. This proteolytic treatment, commonly referred to as 'a hallmark of apoptosis', generated the two physiologically relevant peptide fragments of PARP-1, e.g., a 24-kDa amino-terminus and an 89-kDa carboxy-terminal domain. Interestingly, co-incubation of the two peptide fragments of PARP-1 with full-length pol beta resulted in their domain-specific molecular association as determined by co-immunoprecipitation and reciprocal immunoblotting. Therefore, our data strongly suggest that, once PARP-1 is proteolyzed by either caspase-3 or caspase-7 during cell death, the specific association of its apoptotic fragments with DNA repair enzymes, such as pol beta, may serve a regulatory molecular role in the execution phase of apoptosis.     

Potentiation of DNA damage by inhibition of poly(ADP-ribosyl)ation: a test of the hypothesis for random nuclease action.

Poly(ADP-ribosyl)ation is a cellular response to DNA strand breaks by which a large array of proteins becomes covalently modified for a brief period during the lifetime of the DNA breaks. Inhibition of poly(ADP-ribose) polymerase by 3-aminobenzamide after many types of DNA damage leads to a marked increase in DNA strand breakage, repair replication, cytogenetic damage, mutagenesis, and cell killing. It has been hypothesized that poly(ADP-ribose) polymerase may modify potentially degradative endogenous nucleases that can reduce cellular viability. Thus, in the presence of DNA strand breakage, the polymer would bind these enzymes to inhibit their activity. When synthesis of the polymerase is inhibited, the enzymes would act randomly to produce nonspecific damage in the DNA. We tested this hypothesis by electroporating restriction enzymes into human cells containing the shuttle vector pHAZE. Restriction enzymes cleave at specific recognition sequences in the lacZ target gene of pHAZE, and mutations result from rejoining errors at the cleavage sites. If the hypothesis were correct, enzyme-treated cells cultured with 3-aminobenzamide to inhibit synthesis of poly(ADP-ribose) polymers would result in a significant increase in mutations outside the restriction enzyme sites. The spectrum of mutations observed after electroporation of PvuII (which produces blunt-end double-strand breaks) or PvuI (which produces cohesive-end double-strand breaks) was similar in untreated and 3-aminobenzamide-treated cells. Thus, our results do not support the hypothesis that the increase in damage observed when poly(ADP-ribosyl)ation is inhibited is due to a chaotic, nonspecific attack on DNA by endogenous cellular nucleases.     

A DNA template recognition protein: partial purification from mouse liver and stimulation of DNA polymerase alpha

A protein that specifically enhances up to 13-fold the rate of copying of poly(dT) template by DNA polymerase alpha was partially purified from chromatin of regenerating mouse liver cells. This stimulatory protein, designated herein factor D, also increases 2-3-fold the activity of polymerase alpha with heat-denatured DNA and with primed, circular single-stranded phi X174 DNA. However, factor D has no detectable effect on the copying by polymerase alpha of poly(dG), poly(dA), and poly(dC) templates. Activity of mouse DNA polymerase beta is not affected by factor D with all the tested templates. In contrast to polymerase alpha, factor D is resistant to inactivation by N-ethylmaleimide and calcium ions, but it is readily heat-inactivated at 46 degrees C and is inactivated by trypsin digestion. Partially purified factor D is not associated with detectable activities of DNA polymerase, DNA primase, deoxyribonucleotidyl terminal transferase, and endo- or exodeoxyribonuclease.     

Inhibition of poly(ADP-ribosyl)ation by overexpressing the poly(ADP-ribose) polymerase DNA-binding domain in mammalian cells

Poly(ADP-ribose) polymerase specifically recognizes DNA strand breaks by its DNA-binding domain. DNA binding activates the enzyme to catalyze the formation of poly(ADP-ribose) utilizing NAD as substrate. By a molecular genetic approach we set out to inhibit this enzyme activity in a highly specific manner, thus avoiding the inherent side effects of NAD analogs which have been used extensively as enzyme inhibitors. cDNA sequences coding for the human poly(ADP-ribose) polymerase DNA-binding domain were subcloned into eucaryotic expression plasmids and transiently transfected into monkey cells. Cells were fixed with ethanol followed by incubation with NAD. Indirect double immunofluorescence to detect both overexpressed protein and poly(ADP-ribose) in situ revealed that overexpression of the DNA-binding domain greatly inhibited poly(ADP-ribosyl)ation catalyzed by the resident enzyme during NAD postincubation. The same inhibition was observed when transfected cells were treated with N-methyl-N'-nitro-N-nitrosoguanidine to induce DNA strand breaks in vivo and subjected to trichloroacetic acid/ethanol fixation and subsequent immunofluorescence analysis, a novel method we developed for the in situ detection of polymer synthesis in intact cells. This molecular genetic approach may prove to be a selective and efficient tool to investigate possible functions of poly(ADP-ribosyl)ation in living cells.     

Regulation of DNA metabolic enzymes upon induction of preB cell development and V(D)J recombination: up-regulation of DNA polymerase delta.

Withdrawal of interleukin-7 from cultured murine preB lymphocytes induces cell differentiation including V(D)J immunoglobulin gene rearrangements and cell cycle arrest. Advanced steps of the V(D)J recombination reaction involve processing of coding ends by several largely unidentified DNA metabolic enzymes. We have analyzed expression and activity of DNA polymerases alpha, beta, delta and epsilon, proliferating cell nuclear antigen (PCNA), topoisomerases I and II, terminal deoxynucleotidyl transferase (TdT) and DNA ligases I, III and IV upon induction of preB cell differentiation. Despite the immediate arrest of cell proliferation, DNA polymerase delta protein levels remained unchanged for approximately 2 days and its activity was up-regulated several-fold, while PCNA was continuously present. Activity of DNA polymerases alpha,beta and epsilon decreased. Expression and activity of DNA ligase I were drastically reduced, while those of DNA ligases III and IV remained virtually constant. No changes in DNA topoisomerases I or II expression and activity occurred and TdT expression was moderately increased early after induction. Our results render DNA polymerase delta a likely candidate acting in DNA synthesis related to V(D)J recombination in lymphocytes.     

Structural analysis of catechin derivatives as mammalian DNA polymerase inhibitors

The inhibitory activities against DNA polymerases (pols) of catechin derivatives (i.e., flavan-3-ols) such as (+)-catechin, (-)-epicatechin, (-)-gallocatechin, (-)-epigallocatechin, (+)-catechin gallate, (-)-epicatechin gallate, (-)-gallocatechin gallate, and (-)-epigallocatechin gallate (EGCg) were investigated. Among the eight catechins, some catechins inhibited mammalian pols, with EGCg being the strongest inhibitor of pol alpha and lambda with IC(50) values of 5.1 and 3.8 microM, respectively. EGCg did not influence the activities of plant (cauliflower) pol alpha and beta or prokaryotic pols, and further had no effect on the activities of DNA metabolic enzymes such as calf terminal deoxynucleotidyl transferase, T7 RNA polymerase, and bovine deoxyribonuclease I. EGCg-induced inhibition of pol alpha and lambda was competitive with respect to the DNA template-primer and non-competitive with respect to the dNTP (2'-deoxyribonucleotide 5'-triphosphate) substrate. Tea catechins also suppressed TPA (12-O-tetradecanoylphorbol-13-acetate)-induced inflammation, and the tendency of the pol inhibitory activity was the same as that of anti-inflammation. EGCg at 250 microg was the strongest suppressor of inflammation (65.6% inhibition) among the compounds tested. The relationship between the structure of tea catechins and the inhibition of mammalian pols and inflammation was discussed.     

Lesion bypass by human DNA polymerase mu reveals a template-dependent, sequence-independent nucleotidyl transferase activity

DNA polymerase mu (pol mu), which is related to terminal deoxynucleotidyl transferase and DNA polymerase beta, is thought to be involved in non-homologous end joining and V(D)J recombination. Pol mu is induced by ionizing radiation and exhibits low fidelity. Analysis of translesion replication by purified human pol mu revealed that it bypasses a synthetic abasic site with high efficiency, using primarily a misalignment mechanism. It can also replicate across two tandem abasic sites, using the same mechanism. Pol mu extends primers whose 3'-terminal nucleotides are located opposite the abasic site. Most remarkably, this extension occurs via a mode of nucleotidyl transferase activity, which does not depend on the sequence of the template. This is not due to simple terminal nucleotidyl transferase activity, because pol mu is unable to add dNTPs to an oligo(dT)29 primer or to a blunt end duplex oligonucleotide under standard conditions. Thus, pol mu is a dual mode DNA-synthesizing enzyme, which can act as either a classical DNA polymerase or as a non-canonical, template-dependent, but sequence-independent nucleotidyl transferase. To our knowledge, this is the first report on a DNA-synthesizing enzyme with such properties. These activities may be required for its function in non-homologous end joining in the processing of DNA ends prior to ligation.     

Relationship between poly(ADP-ribose) polymerase activity and DNA synthesis in cultured hepatocytes

Previous studies have demonstrated that an increase in poly(ADP-ribose) polymerase activity could be closely related to DNA replication during liver regeneration and to DNA repair synthesis in different experimental systems. This relationship was further investigated by studying the time course of endogenous and total poly(ADP-ribose) polymerase activity in cultured rat hepatocytes stimulated by epidermal growth factor. This mitogen has been shown to stimulate DNA synthesis in liver cells both in vivo and in vitro. A 6-fold increase in endogenous activity was observed early after epidermal growth factor addition, just before DNA synthesis. A subsequent 4-fold increment in total enzyme activity, concomitant with DNA synthesis, was detected. Orotic acid, which has recently shown mitoinhibitory effect, abolished the epidermal-growth-factor-induced increase in endogenous and total poly(ADP-ribose) polymerase activity, as well as DNA synthesis. On the contrary, 3-aminobenzamide inhibitor of poly(ADP-ribose) polymerase completely suppressed the endogenous activity but only partially modified the increase in total catalytic level and the overall pattern of thymidine incorporation. Taken together, these data indicate that, in cultured hepatocytes, the induction of DNA synthesis is supported by an increased poly(ADP-ribose) polymerase activity.     

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.