DNA maintenance following bleomycin-induced strand breaks does not require poly(ADP-ribosyl)ation activation in Drosophila S2 cells

BackgroundPoly-ADP ribosylation (PARylation) is a post translational modification, catalyzed by Poly(ADP-ribose)polymerase (PARP) family. In Drosophila, PARP-I (human PARP-1 ortholog) is considered to be the only enzymatically active isoform. PARylation is involved in various cellular processes such as DNA repair in case of base excision and strand-breaks.ObservationsStrand-breaks (SSB and DSB) are detrimental to cell viability and, in Drosophila, that has a unique PARP family organization, little is known on PARP involvement in the control of strand-breaks repair process. In our study, strands-breaks (SSB and DSB) are chemically induced in S2 Drosophila cells using bleomycin. These breaks are efficiently repaired in S2 cells. During the bleomycin treatment, changes in PARylation levels are only detectable in a few cells, and an increase in PARP-I and PARP-II mRNAs is only observed during the recovery period. These results differ strongly from those obtained with Human cells, where PARylation is strongly activating when DNA breaks are generated. Finally, in PARP knock-down cells, DNA stability is altered but no change in strand-breaks repair can be observed.ConclusionsPARP responses in DNA strands-breaks context are functional in Drosophila model as demonstrated by PARP-I and PARP-II mRNA increases. However, no modification of the global PARylation profile is observed during strand-breaks generation, only changes at cellular levels are detectable. Taking together, these results demonstrate that PARylation process in Drosophila is tightly regulated in the context of strands-breaks repair and that PARP is essential during the maintenance of DNA integrity but dispensable in the DNA repair process.     

Histone shuttling by poly ADP-ribosylation

The enzymes poly(ADP-ribose)polymerase and poly(ADP-ribose) glycohydrolase may cooperate to drive a histone shuttle mechanism in chromatin. The mechanism is triggered by binding of the N-terminal zinc-finger domain of the polymerase to DNA strand breaks, which activates the catalytic activities residing in the C-terminal domain. The polymerase converts into a protein carrying multiple ADP-ribose polymers which displace histones from DNA by specifically targeting the histone tails responsible for DNA condensation. As a result, the domains surrounding DNA strand breaks become accessible to other proteins. Poly(ADP0ribose) glycohydrolase attacks ADP-ribose polymers in a specific order and thereby releases histones for reassociation with DNA. Increasing evidence from different model systems suggests that histone shuttling participates in DNA repairin vivo as a catalyst for nucleosomal unfolding.     

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.     

Growth-phase-dependent response to DNA damage in poly(ADP-ribose) polymerase deficient cell lines: basis for a new hypothesis describing the role of poly(ADP-ribose) polymerase in DNA replication and repair

We have studied the role of poly(ADP-ribose) polymerase in the repair of DNA damage induced by x-ray and N-methyl N-nitro-N-nitrosoguanidine (MNNG) by using V79 chinese hamster cells, and two derivative mutant cell lines, ADPRT54 and ADPRT351, that are deficient in poly(ADP-ribose) polymerase activity. Under exponentially growing conditions these mutant cell lines are hypersensitive to x-irradiation and MNNG compared to their parental V79 cells which could be interpreted to suggest that poly(ADP-ribose) polymerase is involved in the repair of DNA damage. However, the level of DNA strand breaks induced by x-irradiation and MNNG and their rates of repair are similar in all the cell lines, thus suggesting that it may not be the difference in strand break formation or in its rate of repair that is contributing to the enhanced cell killing in exponentially growing poly(ADP-ribose) polymerase deficient cell lines. In contrast, under growth-arrested conditions, all three cell lines become similarly sensitive to both x-irradiation and MNNG, thus suggesting that poly(ADP-ribose) polymerase may not be involved in the repair of DNA damage in growth-arrested cells. These paradoxical results could be interpreted to suggest that poly(ADP-ribose) polymerase is involved in DNA repair in a cell-cycle-dependent fashion, however, it is functionally active throughout the cell cycle. To resolve this dilemma and explain these results and those obtained by many others, we propose that the normal function of poly(ADP-ribose) polymerase is to prevent DNA recombination processes and facilitate DNA ligation.     

Characterization of human poly(ADP-ribose) polymerase with autoantibodies

The addition of poly(ADP-ribose) chains to nuclear proteins has been reported to affect DNA repair and DNA synthesis in mammalian cells. The enzyme that mediates this reaction, poly(ADP-ribose) polymerase, requires DNA for catalytic activity and is activated by DNA with strand breaks. Because the catalytic activity of poly(ADP-ribose) polymerase does not necessarily reflect enzyme quantity, little is known about the total cellular poly(ADP-ribose) polymerase content and the rate of its synthesis and degradation. In the present experiments, specific human autoantibodies to poly(ADP-ribose) polymerase and a sensitive immunoblotting technique were used to determine the cellular content of poly(ADP-ribose) polymerase in human lymphocytes. Resting peripheral blood lymphocytes contained 0.5 X 10(6) enzyme copies per cell. After stimulation of the cells by phytohemagglutinin, the poly(ADP-ribose) polymerase content increased before DNA synthesis. During balanced growth, the T lymphoblastoid cell line CEM contained approximately 2 X 10(6) poly(ADP-ribose) polymerase molecules per cell. This value did not vary by more than 2-fold during the cell growth cycle. Similarly, mRNA encoding poly(ADP-ribose) polymerase was detectable throughout S phase. Poly(ADP-ribose) polymerase turned over at a rate equivalent to the average of total cellular proteins. Neither the cellular content nor the turnover rate of poly(ADP-ribose) polymerase changed after the introduction of DNA strand breaks by gamma irradiation. These results show that in lymphoblasts poly(ADP-ribose) polymerase is an abundant nuclear protein that turns over relatively slowly and suggest that most of the enzyme may exist in a catalytically inactive state.     

Temporal Patterns of Poly(ADP-Ribose) Polymerase Activation in the Cortex Following Experimental Brain Injury in the Rat

The activation of poly(ADP-ribose) polymerase, a DNA base excision repair enzyme, is indicative of DNA damage. This enzyme also undergoes site-specific proteolysis during apoptosis. Because both DNA fragmentation and apoptosis are known to occur following experimental brain injury, we investigated the effect of lateral fluid percussion brain injury on poly(ADP-ribose) polymerase activity and cleavage. Male Sprague-Dawley rats (n = 52) were anesthetized, subjected to fluid percussion brain injury of moderate severity (2.5-2.8 atm), and killed at 30 min, 2 h, 6 h, 24 h, 3 days, or 7 days postinjury. Genomic DNA from injured cortex at 24 h, but not at 30 min, was both fragmented and able to stimulate exogenous poly(ADP-ribose) polymerase. Endogenous poly(ADP-ribose) polymerase activity, however, was enhanced in the injured cortex at 30 min but subsequently returned to baseline levels. Slight fragmentation of poly(ADP-ribose) polymerase was detected in the injured cortex in the first 3 days following injury, but significant cleavage was detected at 7 days postinjury. Taken together, these data suggest that poly(ADP-ribose) polymerase-mediated DNA repair is initiated in the acute posttraumatic period but that subsequent poly(ADP-ribose) polymerase activation does not occur, possibly owing to delayed apoptosis-associated proteolysis, which may impair the repair of damaged DNA.     

TcPARP: A DNA damage-dependent poly(ADP-ribose) polymerase from Trypanosoma cruzi

Poly(ADP-ribose) polymerase (PARP) is a nuclear enzyme present in most eukaryotes and has been involved in processes such as DNA repair and gene expression. The poly(ADP-ribose) polymer (PAR) is mainly catabolised by poly(ADP-ribose) glycohydrolase. Here, we describe the cloning and characterisation of a PARP from Trypanosoma cruzi (TcPARP). The recombinant enzyme (Mr=65) required DNA for catalytic activity and it was strongly enhanced by nicked DNA. Histones purified from T. cruzi increased TcPARP activity and the covalent attachment of [32P]ADP-ribose moieties to histones was demonstrated. TcPARP required no magnesium or any other metal ion cofactor for its activity. The enzyme was inhibited by 3-aminobenzamide, nicotinamide, theophylline and thymidine but not by menadione. We demonstrated an automodification reaction of TcPARP, and that the removal of attached PAR from this protein resulted in an increase of its activity. The enzyme was expressed in all parasite stages (amastigotes, epimastigotes and trypomastigotes). When T. cruzi epimastigotes were exposed to DNA-damaging agents such as hydrogen peroxide or beta-lapachone, PAR drastically increased in the nucleus, thus confirming PAR synthesis in vivo and suggesting a physiological role for PARP in trypanosomatid DNA repair signalling.     

Isolation of Chinese hamster ovary cells with reduced poly(ADP-ribose) polymerase activity

The biological function of poly(ADP-ribose) polymerase in DNA repair, cell-cycle regulation and cellular differentiation has yet to be defined. Isolation of cells which are deficient in poly(ADP-ribose) synthesis would greatly facilitate the determination of the biological role of this enzyme. A method is described for isolating Chinese hamster ovary (CHO) cells deficient in the poly(ADP-ribose) polymerase activity by direct screening of colonies for enzyme activity. Colonies with decreased production of poly(ADP-ribose) are recovered from nylon replicas for further analysis. Using this method we have isolated a series of CHO cells which have 50% or less poly(ADP-ribose) polymerase activity. These mutants have normal generation times and are 20% more sensitive to the effects of DNA (m)ethylating agents than the parental cell. However, these mutants display normal sensitivity to gamma-rays.     

Poly(ADP-ribose)polymerase: a novel finger protein.

By Energy Dispersive X-ray fluorescence we have determined that calf thymus poly(ADP-ribose) polymerase binds two zinc ions per enzyme molecule. Using 65Zn (II) for detection of zinc binding proteins and polypeptides on western blots, we found that the zinc binding sites are localized in a 29 kd N-terminal fragment which is included in the DNA binding domain. Metal depletion and restoration experiments proved that zinc is essential for the binding of this fragment to DNA as tested by Southwestern assay. These results correlate with the existence of two putative zinc finger motifs present in the N-terminal part of the human enzyme. Poly(ADP-ribose)polymerase fingers could be involved in the recognition of DNA strand breaks and therefore in enzyme activation.     

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.     

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) in the cellular response to DNA damage

Poly(ADP-ribose) polymerase is a chromatin-bound enzyme which, on activation by DNA strand breaks, catalyzes the successive transfer of ADP-ribose units from NAD to nuclear proteins. Poly(ADP-ribose) synthesis is stimulated by DNA strand breaks, and the polymer may alter the structure and/or function of chromosomal proteins to facilitate the DNA repair process. Electronmicroscopic studies show that poly(ADP-ribose) unwinds the tightly packed nucleosomal structure of isolated chromatin. Recent studies also show that the presence of poly(ADP-ribose) enhances the activity of DNA ligase. This may increase the capacity of the cell to complete DNA repair. Inhibitors of poly(ADP-ribose) polymerase or deficiencies of the substrate, NAD, lead to retardation of the DNA repair process. When DNA strand breaks are extensive or when breaks fail to be repaired, the stimulus for activation of poly(ADP-ribose) persists and the activated enzyme is capable of totally consuming cellular pools of NAD. Depletion of NAD and consequent lowering of cellular ATP pools, due to activation of poly(ADP-ribose) polymerase, may account for rapid cell death before DNA repair takes place and before the genetic effects of DNA damage become manifest.     

Identification of ADPR-transferase activity in the rat tapeworm, Hymenolepis diminuta

1. The nuclear fraction of the rat tapeworm Hymenolepis diminuta (Cestoda) contains the enzyme adenosine diphosphoribosyl transferase (ADPR-transferase). 2. The enzyme catalyzes the postsynthetic modification of some nuclear proteins by the covalent attachment of the (ADP-ribose) moiety of NAD to such proteins. 3. The reaction is dependent on DNA which contains strand-breaks, and chain lengths equivalent to (ADP-ribose) is estimated. 4. The formation of polynucleotide products was competitively inhibited by 3-acetamidobezamide, with a Km of 125 microM. 5. The catalytic properties of ADPR-transferase in Hymenolepis diminuta are similar to those in T. brucei.     

Poly(ADP-ribosylated) histones in chromatin replication

Poly(ADP-ribosylation) of histones and several other nuclear proteins seem to participate in nuclear processes involving DNA strand breaks like repair, replication, or recombination. This is suggested from the fact that the enzyme poly(ADP-ribose) polymerase responsible for this modification is activated by DNA strand breaks produced in these nuclear processes. In this article I provide three lines of evidence supporting the idea that histone poly(ADP-ribosylation) is involved in chromatin replication. First, cellular lysates from rapidly dividing mouse or human cells in culture synthesize a significant number of oligo- in addition to mono(ADP-ribosylated) histones. Blocking the cells by treatment of cultures with 5 mM butyrate for 24 h or by serum or nutrient depletion results in the synthesis of only mono- but not of oligo(ADP-ribosylated) histones under the same conditions. Thus, the presence of oligo(ADP-ribosylated) histones is related to cell proliferation. Second, cellular lysates or nuclei isolated under mild conditions in the presence of spermine and spermidine and devoid of DNA strand breaks mainly synthesize mono(ADP-ribosylated) histones; introduction of a small number of cuts by DNase I or micrococcal nuclease results in a dramatic increase in the length of poly(ADP-ribose) attached to histones presumably by activation of poly(ADP-ribose) polymerase. Free ends of DNA that could stimulate poly(ADP-ribosylation) of histones are present at the replication fork. Third, putatively acetylated species of histone H4 are more frequently ADP-ribosylated than nonacetylated H4; the number of ADP-ribose groups on histone H4 was found to be equal or exceed by one the number of acetyl groups on this molecule. Since one recognized role of tetraacetylated H4 is its participation in the assembly of new nucleosomes, oligo(ADP-ribosylation) of H4 (and by extension of other histones) may function in new nucleosome formation. Based on these results I propose that poly(ADP-ribosylated) histones are employed for the assembly of histone complexes and their deposition on DNA during replication. Modified histones arise at the replication fork by activation of poly(ADP-ribose) polymerase by unligated Okazaki fragments.     

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.     

Inhibition of DNA polymerase alpha, DNA polymerase beta, terminal deoxynucleotidyl transferase, and DNA ligase II by poly(ADP-ribosyl)ation reaction in vitro

Incubation of DNA polymerase alpha, DNA polymerase beta, terminal deoxynucleotidyl transferase, or DNA ligase II in a reconstituted poly(ADP-ribosyl)ating enzyme system markedly suppressed the activity of these enzymes. Components required for poly(ADP-ribose) synthesis including poly(ADP-ribose) polymerase, NAD+, DNA, and Mg2+ were all essential for the observed suppression. Purified poly(ADP-ribose) itself, however, was slightly inhibitory to all of these enzymes. Furthermore, the suppressed activities of DNA polymerase alpha, DNA polymerase beta, and terminal deoxynucleotidyl transferase were largely restored (3 to 4-fold stimulation was observed) by a mild alkaline treatment, a procedure known to hydrolyze alkaline-labile ester linkage between poly(ADP-ribose) and an acceptor protein. All of these results strongly suggest that the four nuclear enzymes were inhibited as a result of poly(ADP-ribosyl)ation of either the enzyme molecule itself or some regulatory proteins of these enzymes.     

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.