Modulation of chromatin structure by poly(ADP-ribosyl)ation

Poly(ADP-ribose) polymerase is a nuclear enzyme that is highly conserved in eucaryotes. Its activity is totally dependent on the presence of DNA containing single or double stranded breaks. We have shown that this activation results in a decondensation of chromatin superstructure in vitro, which is caused mainly by hyper(ADP-ribosy)ation of histone H1. In core particles, the modification of histone H2B leads to a partial dissociation of DNA from core histones. The conformational change of native chromatin by poly(ADP-ribosyl)ation is reversible upon degradation of the histone H1-bound poly(ADP-ribose) by poly(ADP-ribose) glycohydrolase. We propose that cuts produced in vivo on DNA during DNA repair activate poly(ADP-ribose) polymerase, which then synthesizes poly(ADP-ribose) on histone H1, in particular, and contributes to the opening of the 25-nm chromatin fiber, resulting in the increased accessibility of DNA to excision repair enzymes. This mechanism is fast and reversible.     

The effect of poly(ADP-ribosyl)ation on native and H1-depleted chromatin. A role of poly(ADP-ribosyl)ation on core nucleosome structure

The effect of poly(ADP-ribosyl)ation on native and H1-depleted chromatin was analyzed by gel electrophoresis, electron microscopy, and velocity sedimentation. In parallel, the interaction of automodified poly(ADP-ribose) polymerase with native and H1-depleted chromatin was analyzed. In H1-depleted chromatin histone H2B becomes the major poly(ADP-ribose) histone acceptor protein, whereas in native chromatin histone H1 was the major histone acceptor. Poly(ADP-ribosyl)ation of H1-depleted chromatin prevented the recondensation of polynucleosomes reconstituted with exogenous histone H1. This is probably due to the presence of modified poly(ADP-ribose) polymerase and hyper(ADP-ribosyl)ated histone H2B. Indeed, about 40% of the modified enzyme remained associated with H1-depleted chromatin, while less than 1% of the modified enzyme was bound to native chromatin. The influence of poly(ADP-ribosyl)ation on the chromatin conformation was also studied at the level of nucleosome in using monoclonal and polyclonal antibodies specific for individual histones and synthetic peptides of histones. In native chromatin incubated in the presence of Mg2+ there was a drop in the accessibility of histone epitopes to monoclonal and polyclonal antibodies whereas upon poly(ADP-ribosyl)ation their accessibility was found to remain even in the presence of Mg2+. In poly(ADP-ribosyl)ated H1-depleted chromatin an increased accessibility of some histone tails to antibodies was observed.     

Poly(ADP-ribose) accessibility to poly(ADP-ribose) glycohydrolase activity on poly(ADP-ribosyl)ated nucleosomal proteins

Hydrolysis of protein-bound 32P-labelled poly(ADP-ribose) by poly(ADP-ribose) glycohydrolase shows that there is differential accessibility of poly(ADP-ribosyl)ated proteins in chromatin to poly(ADP-ribose) glycohydrolase. The rapid hydrolysis of hyper(ADP-ribosyl)ated forms of histone H1 indicates the absence of an H1 dimer complex of histone molecules. When the pattern of hydrolysis of poly(ADP-ribosyl)ated histones was analyzed it was found that poly(ADP-ribose) attached to histone H2B is more resistant than the polymer attached to histone H1 or H2A or protein A24. Polymer hydrolysis of the acceptors, which had been labelled at high substrate concentrations (greater than or equal to 10 microM), indicate that the only high molecular weight acceptor protein is poly(ADP-ribose) polymerase and that little processing of the enzyme occurs. Finally, electron microscopic evidence shows that hyper(ADP-ribosyl)ated poly(ADP-ribose) polymerase, which is dissociated from its DNA-enzyme complex, binds again to DNA after poly(ADP-ribose) glycohydrolase action.     

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.     

Importance of poly(ADP-ribose) glycohydrolase in the control of poly(ADP-ribose) metabolism.

Poly(ADP-ribosyl)ation is a posttranslational modification that alters the functions of the acceptor proteins and is catalyzed by the poly(ADP-ribose) polymerase (PARP) family of enzymes. Following DNA damage, activated poly(ADP-ribose) polymerase-1 (PARP-1) catalyzes the elongation and branching of poly(ADP-ribose) (pADPr) covalently attached to nuclear target proteins. Although the biological role of poly(ADP-ribosyl)ation has not yet been defined, it has been implicated in many important cellular processes such as DNA repair and replication, modulation of chromatin structure, and apoptosis. The transient nature and modulation of poly(ADP-ribosyl)ation depend on the activity of a unique cytoplasmic enzyme called poly(ADP-ribose) glycohydrolase which hydrolyzes pADPr bound to acceptor proteins in free ADP-ribose residues. While the PARP homologues have been recently reviewed, there are relatively scarce data about PARG in the literature. Here we summarize the latest advances in the PARG field, addressing the question of its putative nucleo-cytoplasmic shuttling that could enable the tight regulation of pADPr metabolism. This would contribute to the elucidation of the biological significance of poly(ADP-ribosyl)ation.     

Poly(ADP-ribose) polymerases: managing genome stability

The importance of poly(ADP-ribose) metabolism in the maintenance of genomic integrity following genotoxic stress has long been firmly established. Poly(ADP-ribose) polymerase-1 (PARP-1) and its catabolic counterpart, poly(ADP-ribose) glycohydrolase (PARG) play major roles in the modulation of cell responses to genotoxic stress. Recent discoveries of a number of other enzymes with poly(ADP-ribose) polymerase activity have established poly(ADP-ribosyl)ation as a general biological mechanism in higher eukaryotic cells that not only promotes cellular recovery from genotoxic stress and eliminates severely damaged cells from the organism, but also ensures accurate transmission of genetic information during cell division. Additionally, emerging data suggest the involvement of poly(ADP-ribosyl)ation in the regulation of intracellular trafficking, memory formation and other cellular functions. In this brief review on PARP and PARG enzymes, emphasis is placed on PARP-1, the best understood member of the PARP family and on the relationship of poly(ADP-ribosyl)ation to cancer and other diseases of aging.     

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.     

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.     

In vitro induction of H1-H1 histone cross-linking by adenosine diphosphate-ribose polymers

It is well-known that H1-H1 interactions are very important for the induction of 30 nm chromatin fiber and that, among all posttranslational modifications, poly(ADP-ribosyl)ation is one of those capable of modifying chromatin structure, mainly through H1 histone. As this protein can undergo both covalent and noncovalent modifications by poly(ADP-ribosyl)ation, our aim was to investigate whether and how ADP-ribose polymers, by themselves, are able to affect the formation of H1-H1 oligomers, which are normally present in a condensed chromatin structure. The results obtained in our in vitro experimental system indicate that ADP-ribose polymers are involved in chromatin decondensation. This conclusion was reached as the result of two different observations: (a) H1 histone molecules can be hosted in clusters on ADP-ribose polymers, as shown by their ability to be chemically cross-linked, and (b) H1 histone has a higher affinity for ADP-ribose polymers than for DNA; ADP-ribose polymers compete, in fact, with DNA for H1 histone binding.     

Purification and characterization of an (ADP-ribose)n glycohydrolase from human erythrocytes

An (ADP-ribose)n glycohydrolase from human erythrocytes was purified approximately 13,000-fold and characterized. On sodium dodecyl sulfate/polyacrylamide gel the purified enzyme appeared homogeneous and had an estimated relative molecular mass (Mr) of 59,000. Amino acid analysis showed that the enzyme had a relatively high content of acidic amino acid residues and low content of basic amino acid residues. Isoelectrofocusing showed that the enzyme was an acidic protein with pI value of 5.9. The mode of hydrolysis of (ADP-ribose)n by this enzyme was exoglycosidic, yielding ADP-ribose as the final product. The Km value for (ADP-ribose)n (average chain length, n = 15) was 5.8 microM and the maximal velocity of its hydrolysis was 21 mumol.min-1.mg protein-1. The optimum pH for enzyme activity was 7.4 KCl was more inhibitory than NaCl. The enzyme activity was inhibited by ADP-ribose and cAMP but not the dibutyryl-derivative (Bt2-cAMP), cGMP or AMP. These physical and catalytic properties are similar to those of cytosolic (ADP-ribose)n glycohydrolase II, but not to those of nuclear (ADP-ribose)n glycohydrolase I purified from guinea pig liver [Tanuma, S., Kawashima, K. & Endo, H. (1986) J. Biol. Chem. 261, 965-969]. Thus, human erythrocytes contain (ADP-ribose)n glycohydrolase II. The kinetics of degradation of poly(ADP-ribose) bound to histone H1 by purified erythrocyte (ADP-ribose)n glycohydrolase was essentially the same as that of the corresponding free poly(ADP-ribose). In contrast, the glycohydrolase showed appreciable activity of free oligo(ADP-ribose), much less activity on the corresponding oligo(ADP-ribose) bound to histone H1. The enzyme had more activity on oligo(ADP-ribose) bound to mitochondrial and cytosolic free mRNA ribonucleoprotein particle (mRNP) proteins than on oligo(ADP-ribose) bound to histone H1. It did not degrade mono(ADP-ribosyl)-stimulatory guanine-nucleotide-binding protein (Gs) and -inhibitory guanine-nucleotide-binding protein (Gi) prepared with cholera and pertussis toxins, respectively. These results suggest that cytosolic (ADP-ribose)n glycohydrolase II may be involved in extranuclear de(ADP-ribosyl)n-ation, but not in membrane de-mono(ADP-ribosyl)ation.     

Oligo(3'-deoxy ADP-ribosyl)ation of the nuclear matrix lamins from rat liver utilizing 3'-deoxyNAD as a substrate

It has previously been shown that the levels of poly(ADP-ribose)polymerase and polymers of ADP-ribose that co-purify with the nuclear matrix in regenerating liver fluctuate with the levels of in vivo DNA replication [(1988) FEBS Lett. 236, 362-366]. We have now electrophoretically identified lamins A and C, and poly(ADP-ribose)polymerase as the main protein targets for poly(ADP-ribosyl)ation in isolated nuclear matrices from adult rat liver. The identification of these protein acceptors was facilitated by the utilization of 32P-radiolabeled 3'-deoxyNAD as a substrate for nuclear matrix extracts in the presence of exogenously added DNA-dependent poly(ADP-ribose)polymerase from calf thymus. The extent of protein modification was time- and substrate concentration-dependent. These results are consistent with the hypothesis that the poly(ADP-ribose) modification of the lamins A and C and poly(ADP-ribose)polymerase are important to modulate chromatin-nuclear matrix interactions in rat liver.     

Purification and characterization of poly (ADP-ribose) synthetase from human placenta

Poly(ADP-ribose) synthetase has been purified 2,000-fold to apparent homogeneity from human placenta. The purification procedure involves affinity chromatography with 3-aminobenzamide as the ligand. The purified enzyme absolutely requires DNA for the catalytic activity and catalyzes poly(ADP-ribosyl)ation of the synthetase itself (automodification) and histone H1. Mg2+ enhances both the automodification and poly(ADP-ribosyl)ation of histone H1. The enzyme is a monomeric protein with a pI of 10.0 and an apparent molecular weight of 116,000. The sedimentation coefficient and Strokes radius are 4.6 S and 5.9 nm, respectively. The frictional ratio is 1.82. Amino acid analysis and limited proteolysis with papain and alpha-chymotrypsin indicate that the human placental enzyme is very similar to the enzyme from calf thymus, although some differences are noted. Mouse antibody raised against the placental enzyme completely inhibits the activity of enzymes from human placenta and HeLa cells and cross-reacts with the enzymes from calf thymus and mouse testis. Immunoperoxidase staining with this antibody demonstrates the intranuclear localization of the enzyme in human leukemia cells. All these results indicate that molecular properties as well as antigenic determinants of poly(ADP-ribose) synthetase are highly conserved in various animal cells.     

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.     

ADP-ribosylation of nuclear proteins in rat ventral prostate during ageing

Poly(ADPR)polymerase activity and poly(ADP-ribosyl)ation of nuclear proteins have been investigated in ventral prostate nuclei of different aged rats (14, 28, 60, 180, 360 day old animals), by reverse-phase HPLC and acetic acid-urea polyacrylamide gel electrophoresis. The major ADP-ribose acceptor proteins were identified as histone H1 and H2b. It is concluded that concomitant with major changes to chromatin organization, poly(ADP-ribosyl)ation reaction is progressively inhibited during aging of rat ventral prostate. These results support the hypothesis that prostatic dysfunction in senescent animals is related to a failure of DNA repair mechanisms and deregulated template activity.     

Poly(ADP-ribosyl)ation-dependent Transient Chromatin Decondensation and Histone Displacement following Laser Microirradiation

Chromatin undergoes a rapid ATP-dependent, ATM and H2AX-independent decondensation when DNA damage is introduced by laser microirradiation. Although the detailed mechanism of this decondensation remains to be determined, the kinetics of decondensation are similar to the kinetics of poly(ADP-ribosyl)ation. We used laser microirradiation to introduce DNA strand breaks into living cells expressing a photoactivatable GFP-tagged histone H2B. We find that poly(ADP-ribosyl)ation mediated primarily by poly(ADP-ribose) polymerase 1 (PARP1) is responsible for the rapid decondensation of chromatin at sites of DNA damage. This decondensation of chromatin correlates temporally with the displacement of histones, which is sensitive to PARP inhibition and is transient in nature. Contrary to the predictions of the histone shuttle hypothesis, we did not find that histone H1 accumulated on poly(ADP-ribose) (PAR) in vivo. Rather, histone H1, and to a lessor extent, histones H2A and H2B were rapidly depleted from the sites of PAR accumulation. However, histone H1 returns to chromatin and the chromatin recondenses. Thus, the PARP-dependent relaxation of chromatin closely correlates with histone displacement.     

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.     

In vitro poly(ADP-ribosyl)ated histones H1a and H1t modulate rat testis chromatin condensation differently

Rat testis H1 proteins were poly(ADP-ribosyl)ated in vitro. The modifying product, poly(ADP-ribose), was found covalently bound to each histone variant at various extents and exhibited distinct structural features (linear and short, rather than branched and long chains). Interest was focused on the somatic H1a, particularly abundant in the testis, as compared with other tissues, and the testis-specific H1t, which appears only at the pachytene spermatocyte stage of germ cell development. These H1s were modified with poly(ADP-ribose) by means of two in vitro experimental approaches. In the first system, each variant was incubated with purified rat testis poly(ADP-ribose)polymerase in the presence of [(32)P] NAD. In parallel, poly(ADP-ribosyl)ated H1s were also prepared following incubation of intact rat testis nuclei with [(32)P] NAD. In both experiments, the poly(ADP-ribosyl)ated proteins were purified from the native forms by means of phenyl boronic agarose chromatography. The results from both analyses were in agreement and showed qualitative differences with regard to the poly(ADP-ribose) covalently associated with H1a and H1t. Comparison of the bound polymers clearly indicated that the oligomers associated with H1a were within 10-12 units long, whereas longer chains (相似文献