Poly(ADP-ribosyl)ation studies in situ

We have studied the poly(ADP-ribosyl)ation of nuclear proteins in situ by examining the incorporation of [3H]NAD-derived ADP-ribose into polymers. We have devised a way to deliver [3H]NAD to cells growing in vitro, and we have determined the kinetics of uptake and incorporation into nuclear proteins using this delivery system. Incorporation into the histone fraction, known acceptors of poly(ADP-ribose), was examined and shown to be sensitive to the poly(ADP-ribose) polymerase inhibitor 3-aminobenzamide. Polyacrylamide gel electrophoresis of 3H-labeled proteins revealed radioactivity associated with known poly(ADP-ribose)-accepting proteins such as poly(ADP-ribose) polymerase and histones. These results were confirmed when we immunoreacted gel-separated proteins with anti-(ADP-ribose) generated in our laboratory.     

Poly(ADP-ribosyl)ation in mammalian ageing

Poly(ADP-ribose) polymerases (PARPs) catalyze the post-translational modification of proteins with poly(ADP-ribose). Two PARP isoforms, PARP-1 and PARP-2, display catalytic activity by contact with DNA-strand breaks and are involved in DNA base-excision repair and other repair pathways. A body of correlative data suggests a link between DNA damage-induced poly(ADP-ribosyl)ation and mammalian longevity. Recent research on PARPs and poly(ADP-ribose) yielded several candidate mechanisms through which poly(ADP-ribosyl)ation might act as a factor that limits the rate of ageing.     

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).     

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.     

Poly(ADP-ribosyl)ation during chromatin remodeling steps in rat spermiogenesis

In spermiogenesis, spermatid differentiation is marked by dramatic changes in chromatin density and composition. The extreme condensation of the spermatid nucleus is characterized by an exchange of histones to transition proteins and then to protamines as the major nuclear proteins. Alterations in DNA topology that occur in this process have been shown to require the controlled formation of DNA strand breaks. Poly(ADP-ribosyl)ation is a posttranslational modification of proteins mediated by a family of poly(ADP-ribose) polymerase (PARP) proteins, and two family members, PARP-1 and PARP-2, are activated by DNA strand breaks that are directly detected by the DNA-binding domains of these enzymes. Here, we show for the first time that poly(ADP-ribose) formation, mediated by poly(ADP-ribose) polymerases (PARP-1 and presumably PARP-2), occurs in spermatids of steps 11–14, steps that immediately precede the most pronounced phase of chromatin condensation in spermiogenesis. High levels of ADP-ribose polymer were observed in spermatid steps 12–13 in which the highest rates of chromatin nucleoprotein exchanges take place. We also detected -H2AX, indicating the presence of DNA double-strand breaks during the same steps. Thus, we hypothesize that transient ADP-ribose polymer formation may facilitate DNA strand break management during the chromatin remodeling steps of sperm cell maturation.M.L. Meyer-Ficca and H. Scherthan contributed equally to this work     

Poly(ADP-ribosyl)ation of mannose-binding lectin out of human kidney cells

Mannose-binding lectin was identified as a substrate of tankyrase 2, an enzyme that catalyzes poly(ADP-ribosyl)ation. The endogenous tankyrase 2 was isolated out of cytoplasm of human embryonic kidney cells. It was bound to a soluble complex of at least two other proteins; they were identified using specific antibodies and other approaches as keratin 1 and mannose-binding lectin. Using immunoblot analysis and radioactive labeling, we detected tankyrase-2-dependent poly(ADP-ribosyl)ation of mannose-binding lectin. In the presence of NAD(+), the complex of keratin 1 and lectin was dissociated, what was recorded during elution of its separate components out of affinity columns and by decrease of their apparent molecular masses during gel-filtration. Tankyrase 2 also inhibited the carbohydrate-binding function of the lectin. The latter effect was observed using mannose-binding lectin out of human serum, which is free from keratin 1. As a result of tankyrase-2 activity, the lectin lost its affinity to mannan-agarose. The discovery of this new biochemical mechanism justifies further analysis of its physiological and medical significance.     

Poly(ADP-ribosyl)ation of p53 Contributes to TPEN-Induced Neuronal Apoptosis

Depletion of intracellular zinc by N,N,N′,N′-tetrakis(2-pyridylmethyl) ethylenediamine (TPEN) induces p53-mediated protein synthesis-dependent apoptosis of mouse cortical neurons. Here, we examined the requirement for poly(ADP-ribose) polymerase (PARP)-1 as an upstream regulator of p53 in zinc depletion-induced neuronal apoptosis. First, we found that chemical inhibition or genetic deletion of PARP-1 markedly attenuated TPEN-induced apoptosis of cultured mouse cortical neurons. Poly(ADP-ribosyl)ation of p53 occurred starting 1 h after TPEN treatment. Suggesting the critical role of PARP-1, the TPEN-induced increase of stability and activity of p53 as well as poly(ADP-ribosyl)ation of p53 was almost completely blocked by PARP inhibition. Consistent with this, the induction of downstream proapoptotic proteins PUMA and NOXA was noticeably reduced by chemical inhibitors or genetic deletion of PARP-1. TPEN-induced cytochrome C release into the cytosol and caspase-3 activation were also blocked by inhibition of PARP-1. Taken together, these findings indicate that PARP-1 is essential for TPEN-induced neuronal apoptosis.     

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.     

Poly(ADP-ribosyl)ation of proteins and germ cell development in hyperthyroid rat testes

The effect of increased serum levels of thyroid hormone (triiodothyronine, T3) on young rat testis spermatogenesis was studied by analysing molecular and morphological parameters. Hyperthyroidism was induced by either T3-treatment or 2- and 10-day cold exposure. The poly(ADP-ribosyl)ation of proteins catalysed by poly(ADP-ribose) polymerase, which is particularly active at specific stages of rat spermatogenesis, was analysed as molecular index of DNA damage and cell stress. Poly(ADP-ribose) polymerase activity rose after both T3-treatment and 2- and 10-day cold exposure, with a trend of 10-day cold-exposed rats towards control values. In all hyperthyroid rats poly(ADP-ribose) turnover, as a contribution of both poly(ADP-ribose) polymerase and poly(ADP-ribose) glycohydrolase), was enhanced with respect to euthyroid animals. Poly(ADP-ribosyl)ation of proteins occurred with long and branched polymers suggesting an increased involvement of the modification system in DNA repair. Morphological changes of germ tissue were observed in hyperthyroid rats, mainly a high reduction of mature cells in the seminiferous tubule, and evidence of germ cell apoptosis was obtained by TUNEL method. In control animals germ cell apoptosis was within physiological levels. Conversely, in hyperthyroid rats a dramatic increase in the number of TUNEL-positive cells (some spermatogonia and numerous primary spermatocytes) was found, even though the increase was lower in 10-day than in 2-day cold-exposed animals.     

Poly(ADP-ribosyl)ation in regulation of chromatin structure and the DNA damage response

Poly(ADP-ribose) (PAR) is a post-translational modification of proteins and is synthesised by PAR polymerases (PARPs), which have long been associated with the coordination of the cellular response to DNA damage, amongst other processes. Binding of some PARPs such as PARP1 to broken DNA induces a substantial wave of PARylation, which results in significant re-structuring of the chromatin microenvironment through modification of chromatin-associated proteins and recruitment of chromatin-modifying proteins. Similarly, other DNA damage response proteins are recruited to the damaged sites via PAR-specific binding modules, and in this way, PAR mediates not only local chromatin architecture but also DNA repair. Here, we discuss the expanding role of PAR in the DNA damage response, with particular focus on chromatin regulation.     

Poly(ADP-ribosyl)ation associated changes in CTCF-chromatin binding and gene expression in breast cells

CTCF is an evolutionarily conserved and ubiquitously expressed architectural protein regulating a plethora of cellular functions via different molecular mechanisms. CTCF can undergo a number of post-translational modifications which change its properties and functions. One such modifications linked to cancer is poly(ADP-ribosyl)ation (PARylation). The highly PARylated CTCF form has an apparent molecular mass of 180?kDa (referred to as CTCF180), which can be distinguished from hypo- and non-PARylated CTCF with the apparent molecular mass of 130?kDa (referred to as CTCF130). The existing data accumulated so far have been mainly related to CTCF130. However, the properties of CTCF180 are not well understood despite its abundance in a number of primary tissues. In this study we performed ChIP-seq and RNA-seq analyses in human breast cells 226LDM, which display predominantly CTCF130 when proliferating, but CTCF180 upon cell cycle arrest. We observed that in the arrested cells the majority of sites lost CTCF, whereas fewer sites gained CTCF or remain bound (i.e. common sites). The classical CTCF binding motif was found in the lost and common, but not in the gained sites. The changes in CTCF occupancies in the lost and common sites were associated with increased chromatin densities and altered expression from the neighboring genes. Based on these results we propose a model integrating the CTCF130/180 transition with CTCF-DNA binding and gene expression changes. This study also issues an important cautionary note concerning the design and interpretation of any experiments using cells and tissues where CTCF180 may be present.     

Poly(ADP-ribosyl)ation affects stabilization of Che-1 protein in response to DNA damage

Poly(ADP-ribose) polymerase 1 (PARP-1) catalyzes a post-translational modification that plays a crucial role in coordinating the signalling cascade in response to stress stimuli. During the DNA damage response, phosphorylation by ataxia telangiectasia mutated (ATM) kinase and checkpoint kinase Chk2 induces the stabilization of Che-1 protein, which is critical for the maintenance of G2/M arrest. In this study we showed that poly(ADP-ribosyl)ation, beyond phosphorylation, is involved in the regulation of Che-1 stabilization following DNA damage. We demonstrated that Che-1 accumulation upon doxorubicin treatment is reduced after the inhibition of PARP activity in HCT116 cells and in PARP-1 knock-out or silenced cells. In accordance, impairment in Che-1 accumulation by PARP inhibition reduced Che-1 occupancy at p21 promoter and affected the expression of the corresponding gene. Epistasis experiments showed that the effect of poly(ADP-ribosyl)ation on Che-1 stabilization is independent from ATM kinase activity. Indeed we demonstrated that Che-1 protein co-immunoprecipitates with ADP-ribose polymers and that PARP-1 directly interacts with Che-1, promoting its modification in vitro and in vivo.     

Poly(ADP-ribosyl)ation accelerates DNA repair in a pathway dependent on Cockayne syndrome B protein

Activation of poly(ADP-ribose)polymerases 1 and 2 (PARP-1 and PARP-2) is one of the earliest responses of mammalian cells to DNA damage by numerous genotoxic agents. We have analysed the influence of PARP inhibition, either achieved by over-expression of the DNA binding domain of PARP-1 or by treatment with 3,4-dihydro-5-[4-(1-piperidinyl)butoxyl]-1(2H)-isoquinolinone, on the repair of single-strand breaks (SSB), pyrimidine dimers and oxidative base modifications sensitive to Fpg protein (mostly 8-hydroxyguanine) in mammalian cells at very low, non-cytotoxic levels of DNA damage. The data show that the repair rates of all three types of DNA damage are significantly lower in PARP-inhibited cells. Importantly, the retardation of the repair of base modifications is not associated with accumulation of intermediates such as SSB or abasic sites. Moreover, the influence of the PARP inhibition is not observed in cells deficient in Cockayne syndrome B protein (Csb). The results indicate that PARP activation and Csb are both involved in a novel mechanism that accelerates the global repair of various types of DNA modifications.     

Rapamycin inhibits poly(ADP-ribosyl)ation in intact cells

Rapamycin is an immunosuppressive drug, which inhibits the mammalian target of rapamycin (mTOR) kinase activity inducing changes in cell proliferation. Synthesis of poly(ADP-ribose) (PAR) is an immediate cellular response to genotoxic stress catalyzed mostly by poly(ADP-ribose) polymerase 1 (PARP-1), which is also controlled by signaling pathways. Therefore, we investigated whether rapamycin affects PAR production. Strikingly, rapamycin inhibited PAR synthesis in living fibroblasts in a dose-dependent manner as monitored by immunofluorescence. PARP-1 activity was then assayed in vitro, revealing that down-regulation of cellular PAR production by rapamycin was apparently not due to competitive PARP-1 inhibition. Further studies showed that rapamycin did not influence the cellular NAD pool and the activation of PARP-1 in extracts of pretreated fibroblasts. Collectively, our data suggest that inhibition of cellular PAR synthesis by rapamycin is mediated by formation of a detergent-sensitive complex in living cells, and that rapamycin may have a potential as therapeutic PARP inhibitor.