Properties of the complex between histone H1 and poly(ADP-ribose synthesised in HeLa cell nuclei

Preparations of H1 histone from HeLa cell nuclei incubated with [3H]NAD to permit poly(ADP-ribose) synthesis were electrophoresed on polyacrylamide gels. The incorporated radioactivity migrated as a sharply defined peak in association with a protein band which moved more slowly than H1, the major protein component. The following observations indicate that this complex is composed of two molecules of H1 and a single chain of poly(ADP-ribose) with one detectable covalent linkage of polymer to protein. 1. The [14C]arginine/[3H]lysine ratio is identical in H1 histone and in the protein moiety of the complex. 2. Protein is displaced from H1 histone to the complex during poly(ADP-ribose) synthesis. At least 90% of the protein in the complex (stainable protein and labelled protein) is derived from H1. 3. Sedimentation rate studies indicate a molecular weight of the complex about twice that of H1 histone. 4. The average chain length of the polymer is 15 ADP-ribose units and there are 7--8 ADP-ribose units for each molecule of H1 histone in the 'complex'. 5. Poly(ADP-ribose) glycohydrolase, which hydrolyses the polymer exoglycosidically from the AMP terminus, degrades the complex producing ADP-ribose and mono-ADP-ribosylated H1 histone which co-electrophoreses with unmodified H1. Although only one covalent linkage between protein and polymer has been detected, the 'complex' does not dissociate when electrophoresed on dodecylsulfate gels. Nor can the noncovalently linked H1 histone of the complex readily exchange with free H1. Complex formation does not occur when purified poly(ADP-ribose) and H1 are mixed.     

Rapid determination of chain length pattern in poly(ADP-ribose) samples

(³H)poly(ADP-ribose) synthesized from nuclei by incubation with (³H)NAD was released from protein by alkaline treatment and electrophoresed in dodecyl sulfate gels. Individual polymers up to at least 33 units were completely separated according to their chain length. Size distribution was visualized by fluorography of the gels, and quantified by radioactivity determination of sliced gels The method could be applied to crude nuclear extracts. It showed that nuclei of Ehrlich ascites tumor cells produced a poly(ADP-ribose) pattern distinctly different from that of rat liver nuclei.     

3'-Deoxy-NAD+ as a substrate for poly(ADP-ribose)polymerase and the reaction mechanism of poly(ADP-ribose) elongation

Adenyl-32P-Labeled 3'-deoxy-NAD+ was utilized as a substrate by pure DNA-dependent poly(ADP-ribose)polymerase (EC 2.4.2.30) from calf thymus in the automodification reaction with an apparent Km of 20 microM and a Vmax of 80 nmol/min/mg of protein. Analysis by lithium lauryl sulfate-polyacrylamide gel electrophoresis revealed a single 32P-labeled protein of 116-kDa which comigrated with automodified enzyme. Addition of increasing amounts of histone H1 up to a concentration of 15 micrograms/ml stimulated the synthesis of protein-bound polymers of 3'-deoxy-ADP-ribose. However, the average polymer size was equal to 2 in the presence and 4 in the absence of histone H1, respectively. The synthesis of protein-bound oligomers of 3'-deoxy-ADP-ribose was inhibited by the polymerase inhibitors benzamide, nicotinamide, thymidine, and NaCl. A pulse labeling of polymer synthesis with 40 microM [32P]3'-deoxy-NAD+ either in the presence or absence of 15 micrograms/ml of histone H1, followed by a chase with 1 mM [3H]NAD+, was used to determine the mechanism of poly(ADP-ribose) elongation. Following enzyme digestion of these polymers with phosphodiesterase, it was found that 52 and 24% of the total 32P radiolabel was associated with the 3'-deoxy-AMP termini of the polymers synthesized in the pulse reactions, in the presence or absence of histone H1, respectively. In contrast, less than 10% of the total radioactivity was associated with 3'-deoxy-AMP in the product of the chase reactions. These results are consistent with the conclusion that the initially attached residue of 3'-deoxy-ADP-ribose to either the polymerase or histone H1, is elongated by the "protein-distal" addition of ADP-ribose residues to the AMP terminus of the growing polymer chain.     

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.     

Initiation of poly(ADP-ribosyl) histone synthesis by poly(ADP-ribose) synthetase

Initiation of poly(ADP-ribosyl) histone synthesis was achieved in vitro using an apparently homogeneous preparation of poly(ADP-ribose) synthetase. When poly(ADP-ribose) was synthesized in the presence of DNA and increase amounts of histone H1, increasing portions (up to about 55%) of the product were found associated with the histone, judging from solubility in 5% HClO4 and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Most of the polymers were directly attached to the histone protein and not produced by elongation from pre-existing ADP-ribose; the cohesive end of poly(ADP-ribose), isolated as ribose 5-phosphate with snake venom phosphodiesterase digestion, was labeled almost quantitatively with [ribose (NMN)-14C]NAD. The poly(ADP-ribose) . histone linkage was labile in mild alkali and neutral NH2OH, suggesting that the same bond, probably ester, was formed in this system as in crude chromatin or isolated nuclei. Elongation of a histone-bound monomer into a polymer by this enzyme was previously demonstrated (Ueda, K., Kawaichi, M., Okayama, H., and Hayaishi, O. (1979) J. Biol. Chem. 254, 679-687), but initiation of ADP-ribose chains on histone has never been shown with a purified enzyme. This appeared to be due to the low concentrations of histone so far used. These findings indicated that a single enzyme catalyzes two different types of reaction, i.e. an attachment of ADP-ribose to histone and its elongation into a polymer.     

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.     

Effect of DNA on poly (ADP-ribose) glycohydrolase and the degradation of histone H1-poly (ADP-ribose) complex from HeLa cell nuclei.

A poly(ADP-ribose)-H1 histone complex has been isolated from HeLa cell nuclei incubated with NAD. The rate of poly(ADP-ribose) glycohydrolase catalyzed hydrolysis of the polymer in the complex is only 1/9 that of free poly(ADP-ribose), indicating that the polymer is in a protected environment within the complex. Comparison of the rate of hydrolysis of free poly(ADP-ribose) in the presence or absence of H1 to that in the complex synthesized de novo indicates a specific mode of packaging of the complex. This is further indicated by the fact that alkaline dissociation of the complex followed by neutralization markedly exposes the associated poly(ADP-ribose) to the glycohydrolase. The complex also partially unfolds when it binds to DNA as evidenced by a 2-fold increase in the rate of glycolytic cleavage of poly(ADP-ribose). This effect of DNA is not due to a stimulation of the glycohydrolase per se since hydrolysis of free polymer by the enzyme is strongly inhibited by DNA, especially single-stranded DNA. Inhibition of glycohydrolase by DNA results from the binding of the enzyme to DNA and conditions which decrease this binding (increased ionic strength or addition of histone H1 which competes for DNA binding) relieve the DNA inhibition.     

Regulation of poly(ADP-ribose) polymerase. Histone-specific adaptations of reaction products

The post-translational poly ADP-ribosylation of proteins by the nuclear enzyme poly(ADP-ribose) polymerase (EC 2.4.2.30) involves a complex pattern of ADP-ribose polymers. We have determined how this enzyme produces the various polymer size patterns responsible for altered protein function. The results show that histone H1 and core histones are potent regulators of both the numbers and sizes of ADP-ribose polymers. Each histone induced the polymerase to synthesize a specific polymer size pattern. Various other basic and/or DNA binding proteins as well as other known stimulators of poly(ADP-ribose) polymerase (spermine, MgCl2, nicked DNA) were ineffective as polymer size modulators. Testing specific proteolytic fragments of histone H1, the polymer number and polymer size modulating activity could be mapped to specific polypeptide domains. The results suggest that histones specifically regulate the polymer termination reaction of poly(ADP-ribose) polymerase.     

Properties of poly(ADP-ribose) synthetase from rat pancreas and poly(ADP-ribosylation) of basic nuclear proteins.

The isolated nuclei of rat pancreas contain an enzyme system that will incorporate 3H-labeled NAD into an acid-insoluble product, which is shown to be poly(ADP-ribose). The enzyme has an optimum pH of 7.8 and the optimum temperature is between 20 and 30 degrees C. Optimum Mg2+ concentration is 8 mM and dithiothreitol also stimulates the enzyme at a concentration of 8 mM. Under standard conditions, the Km value for the reaction is 0.25 mM and an inhibition by the substrate is observed at high substrate concentrations. It has also been found that only one basic nuclear protein, that is, histone H1, is modified by the synthetase. An average chain length of 5.0 is found in the nuclei and of 4.5 on histone H1. Radioautographic studies show that poly(ADP-ribose) is closely associated with chromatin.     

Cytoplasmic poly(ADP-ribose) polymerase and poly(ADP-ribose) glycohydrolase in AEV-transformed chicken erythroblasts

Poly(ADP-ribose) polymerase and poly(ADP-ribose) glycohydrolase activities were both investigated in chicken erythroblasts transformed by Avian Erythroblastosis Virus. Respectively 21% and 58% of these activities were found to be present in the post-mitochondrial supernatant (PMS). Fractionation of the PMS on sucrose gradients and poly(A⁺) mRNA detection by hybridization to [³H] poly(U) show that cytoplasmic poly(ADP-ribose) polymerase is exclusively localized in free mRNP. The glycohydrolase activity sedimented mostly in the 6 S region but 1/3 of the activity was in the free mRNP zone. Seven poly(ADP-ribose) protein acceptors were identified in the PMS in the Mr 21000–120000 range. The Mr 120000 protein corresponds to automodified poly(ADP-ribose) polymerase. A Mr 21000 protein acceptor is abundant in PMS and a Mr 34000 is exclusively associated with ribosomes and ribosomal subunits. The existence of both poly(ADP-ribose) polymerase and glycohydrolase activities in free mRNP argues in favour of a role of poly(ADP-ribosylation) in mRNP metabolism. A possible involvement of this post translational modification in the mechanisms of repression-derepression of mRNA is discussed.Abbreviations ADP-ribose adenosine (5) diphospho(5)--D ribose - poly(ADP-ribose) polymer of ADP-ribose - mRNP messenger ribonucleoprotein particles - PMSF phenylmethylsulfonyl fluoride - LDS lithium dodecyl sulfate - TCA trichloroacetic acid     

Chromosomal protein poly(ADP-ribosyl)ation in pancreatic nucleosomes

When pancreatic chromatin fragments were prepared and resolved in the presence of 80 mM NaCl, endogenous poly(ADP-ribose) polymerase activity was found to be maximal in nucleosome periodicities of four to five units and did not respond to any further increases in nucleosomal architecture. Furthermore, in nucleosome complexities spanning 1 through 14 and over unit lengths, polyacrylamide gel electrophoresis on acid-urea and acid-urea-Triton gels has shown pancreatic histone H1 to be the only actively ADP-ribosylated histone species. The extent of ADP-ribosylation of histone H1 was also demonstrated to retard the protein's mobility in acid-urea, acid-urea-Triton, and lithium dodecyl sulfate polyacrylamide gels and to consist of at least 12 distinct ADP-ribosylated species extractable in all nucleosome complexities studied. Finally, extraction and subsequent electrophoresis of total chromosomal proteins in the presence of lithium dodecyl sulfate also evidenced heavy ADP-ribosylation at the level of nonhistone chromosomal proteins of the high mobility group comigrating in the core histone region, as well as in the topmost region of the gels where poly(ADP-ribose) polymerase was found to form a poly(ADP-ribosyl)ated aggregate.     

Hyper(ADP-ribosyl)ation of histone H1

Nucleosomal chains of various repeat unit lengths were generated by a mild micrococcal nuclease digestion of purified pancreatic nuclei. Maximal nucleosome associated poly(ADP-ribose) polymerase activity was recovered in trimeric to tetrameric chromatin fragments, after which the enzyme activity gradually decreased and stabilized towards oligomeric periodicities of 11 to 16 nucleosomes. Electrophoresis of [32P]ADP-ribosylated histones on first-dimension acid-urea or acid-urea-Triton gels and on second-dimension acid--urea--cetyltriammonium bromide gels revealed that, of all histones, only histone H1 could be significantly poly(ADP-ribosyl)ated while only minimal modification could be recovered with histone H1(0). Furthermore, the extent of ADP-ribosylation present on pancreatic histone H1 is shown to proportionally retard this protein's electrophoretic mobility in all gel systems and to consist of a distinct series of at least 12 modification intermediates which can be evidenced, in nuclei or nucleosomes, and fully recovered along with histone H1 upon its selective extraction with 5% perchloric acid. The generation of these increasingly ADP-ribosylated forms of histone H1 is also demonstrated to be time dependent and the more complex ADP-ribosylated forms of this histone are favored at high NAD+ concentrations. Moreover, the electrophoretic mobilities of all intermediates are unaffected by the presence of the nonionic detergent Triton X-100.     

Theophylline reduces poly(ADP-ribose) synthetase from chick embryo liver nuclei

The effect of theophylline on poly(ADP-ribosyl)ation was investigated. The poly(ADP-ribose) synthetase activity in vitro was markedly reduced in the liver nuclei prepared from theophylline-treated chick embryo. This reduction was not due to the enzyme inhibition by theophylline contamination in the nuclear fraction. The hydroxyapatite column chromatographic analysis of [³H]adenosine-labelled poly(ADP-ribose) molecules formed in vivo revealed that the in vivo formation of poly(ADP-ribose) molecules was also decreased by theophylline administration. The theophylline-induced reduction of poly(ADP-ribose) synthesis was not due to either low NAD levels or to a decrease in the chain length of the poly(ADP-ribose) molecule, rather this reduction was derived from a decrease in the number of poly(ADP-ribose) molecules. Possible mechanisms related to reduction of poly(ADP-ribose) synthesis in vivo are discussed.     

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.     

Purification of non-histone acceptor proteins for ADP-ribose from mouse testis nuclei.

Acceptor proteins for poly(ADP-ribose) have been purified from mouse testis nuclei. Nuclear proteins were labelled in vitro with [14C]ribose and [3H]adenine, extracted with 5% (v/v) HClO4 and 0.25 M-HCl and separated by ion-exchange chromatography. Non-histone proteins were found to be the major acceptors in both the 5% (w/v)-HClO4-soluble and 5%-HClO4-insoluble HCl-extractable fractions. Of the two groups of non-histone proteins associated with chromatin, the LMG (low-mobility-group) proteins were preferentially ADP-ribosylated. HMG (high-mobility group) proteins were labelled to lower specific radioactivity. Six LMG proteins were purified to approx. 90% homogeneity and were identified from their mobility on polyacrylamide gels at pH 2.9 and from their amino acid composition. The average length of the poly(ADP-ribose) chain was estimated to be four to six repeating ADP-ribose units. It is suggested that ADP-ribosylation of LMG proteins, a long-neglected group of chromatin-associated proteins, is important during spermatogenesis for the production of spermatozoa with intact and competent DNA.     

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.     

The isolation and identification of a novel intermediate in ubiquinone-6 biosynthesis by Saccharomyces cerevisiae.

The reaction product obtained from HeLa cell nuclei incubated with [³H]NAD was specifically hydrolyzed with snake venom phosphodiesterase. Analysis of the hydrolyzed product revealed that it is a homopolymer consisting of 4–5 repetition of ADP-ribose units. The [³H]poly ADP-ribosylated histone fraction was anslyzed by urea-acetic acid polyacrylamide gel electrophoresis. The radioactive peak was clearly separated from the stained histone H1 band, while a slight overlap was observed. When chromatographed on a SP-Sephadex C-50 column, more than 90% of the radioactivity of [³H]poly(ADP-ribose) was eluted in accordance with histones but not with nonhistone contaminants. On a sodium dodecyl sulfate polyacrylamide gel electrophoresis, a major radioactive peak appeared at a position very close to the histone Hl band, which disappeared by the treatment with alkali prior to electrophoresis. A selective extraction of histone Hl with 5% perchloric acid showed that histone Hl contained about 85% of the radioactivity incorporated into whole histones.     

Noncovalent interactions of poly(adenosine diphosphate ribose) with histones.

Covalent linkage of ADP-ribose polymers to proteins is generally considered essential for the posttranslational modification of protein function by poly(ADP-ribosyl)ation. Here we demonstrate an alternative way by which ADP-ribose polymers may modify protein function. Using a highly stringent binding assay in combination with DNA sequencing gels, we found that ADP-ribose polymers bind noncovalently to a specific group of chromatin proteins, i.e., histones H1, H2A, H2B, H3, and H4 and protamine. This binding resisted strong acids, chaotropes, detergents, and high salt concentrations but was readily reversible by DNA. When the interactions of variously sized linear and branched polymer molecules with individual histone species were tested, the hierarchies of binding were branched polymers greater than long, linear polymers greater than short, linear polymers and H1 greater than H2A greater than H2B = H3 greater than H4. For histone H1, the target of polymer binding was the carboxy-terminal domain, which is also the domain most effective in inducing higher order structure of chromatin. Thus, noncovalent interactions may be involved in the modification of histone functions in chromatin.     

Cytological detection of poly (ADP-ribose) polymerase

An attempt was made to demonstrate poly (ADP-ribose) polymerase cytologically. In vitro incorporation from the nucleotide, [³H]NAD was detected in frozen sections of onion embryo and meristematic tissue by autoradiography. In meristematic tissue, there was a correlation between the number of cells displaying intensein vitro incorporation from [³H]NAD and cytological DNA polymerase activity. Performed enzymes effecting a distinct incorporation from [³H]NAd were localized in the nuclei of all tissues of the ungerminated seed except the endosperm. Evidence for poly (ADP-ribose) polymerase has been obtained for the first time from higher plant cells and localized cytologically.