Control of histone H1 dimer-poly(ADP-ribose) complex formation by poly(ADP-ribose) glycohydrolase

In nuclei incubated in vitro with [³H]NAD to promote poly(ADP-ribose) synthesis, about 6% of the polymer synthesized is differentially extracted into cold 5% PCA along with the H1 histone. Polyacrylamide gel electrophoresis of the extracts revealed large differences in the mobility of the incorporated radioactivity depending on the source of the nuclei used. With rat mammary tumors, the radioactivity co-migrated with the H1 histone on both acid-urea and SDS-urea gels. In contrast, the labeled polymer from HBL-100 mammary cell nuclei co-electrophoresed with a minor protein component which moved more slowly than H1. With lactating mammary glands, an intermediate profile was seen. The difference in mobility on the gels was found to be due to differences in the chain lengths of the poly(ADP-ribose) attached in the H1 protein. The difference in chain length produced was inversely related to the level of poly(ADP-ribose) degrading activity in the various nuclear preparations.     

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.     

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.     

ADP-ribosylation of rye histones

Chromatin from etiolated rye seedlings synthesized protein-bound, acid-insoluble material from [3H]NAD, presumably poly(ADP-ribose). [3H]ADP-ribosylated histone fractions were isolated from crude chromatin and characterized by gel electrophoresis and exclusion chromatography. It was found that histone H2B was the main acceptor, that H2A and H1 were modified to a lesser extent, and that H3 and H4 were only slightly modified. The average chain length on purified histones was 2.5 units of polymer.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Catalytic activities of human poly(ADP-ribose) polymerase from normal and mutagenized cells detected after sodium dodecyl sulfate-polyacrylamide gel electrophoresis

An activity gel procedure is described to identify functional polypeptides of human poly(ADP-ribose) polymerase. Purified or crude enzyme preparations from HeLa cells were electrophoresed in sodium dodecyl sulfate-polyacrylamide gels containing gapped DNA. After renaturation of the peptides in situ, the intact gel was incubated in a poly(ADP-ribose) polymerase reaction mixture containing [32P]NAD. Autoradiograms of the gels consistently exhibited a major activity band at Mr = 116,000-120,000; in many runs, three minor distinct bands at Mr = 125,000, 135,000, and 145,000 were also seen. [32P]NAD appeared to be incorporated into poly(ADP-ribose) since: (i) the activity bands were not detectable when the enzyme-inhibitor 3-aminobenzamide was added to the gel incubation mixture; and (ii) the radioactive polymer, electroeluted from the bands, was completely digested by phosphodiesterase I. Preliminary activity gel analysis of extracts of HeLa cells treated with different DNA-damaging agents revealed that the apparent activity of the Mr = 116,000 form increased by about 10-fold in cells treated with 1 mM dimethyl sulfate and 10-20-fold in cells treated with 10 microM mitomycin C. Only a small increase was obtained in cells treated with 1 mM methyl methanesulfonate, and no change in the activity band pattern was observed after 50 and 100 J/m-2 of UV irradiation.     

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.     

The effect of poly(ADP-ribose) on interactions of DNA with histones H1, H3 and H4

The competition between poly(ADP-ribose) and DNA for binding of the histones H1, H3 and H4 was studied, using a membrane filter-binding test. Poly(ADP-ribose) differently affected the interaction between DNA and the individual histones. While poly(ADP-ribose) effectively competed with DNA for binding of histone H4, it equally competed with DNA for binding of histone H3 and only inefficiently competed with DNA for binding of histone H1. Moreover, preformed complexes were correspondingly affected by the addition of competing polynucleotides, thereby also indicating the reversibility of complex formation. The competition capacity of DNA for histone H4 binding did not depend on DNA size. Competition experiments with poly(A) also indicated that poly(ADP-ribose) preferentially affected DNA-histone H4 interaction. The significance of the differing binding properties is discussed with regard to the possible molecular function of poly(ADP-ribose), especially with regard to its potential effect on nucleosome structure.     

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.     

Large-scale preparation and characterization of poly(ADP-ribose) and defined length polymers

Poly(ADP-ribose) (pADPr) is a large, structurally complex polymer of repeating ADP-ribose units. It is biosynthesized from NAD(+) by poly(ADP-ribose) polymerases (PARPs) and degraded to ADP-ribose by poly(ADP-ribose) glycohydrolase. pADPr is involved in many cellular processes and exerts biological function through covalent modification and noncovalent binding to specific proteins. Very little is known about molecular recognition and structure-activity relationships for noncovalent interaction between pADPr and its binding proteins, in part because of lack of access to the polymer on a large scale and to units of defined lengths. We prepared polydisperse pADPr from PARP1 and tankyrase 1 at the hundreds of milligram scale by optimizing enzymatic synthesis and scaling up chromatographic purification methods. We developed and calibrated an anion exchange chromatography method to assign pADPr size and scaled it up to purify defined length polymers on the milligram scale. Furthermore, we present a pADPr profiling method to characterize the polydispersity of pADPr produced by PARPs under different reaction conditions and find that substrate proteins affect the pADPr size distribution. These methods will facilitate structural and biochemical studies of pADPr and its binding proteins.     

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   

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 and properties of poly(adenosine diphosphate ribose) synthetase.

Poly(ADP-ribose) synthetase has been purified approximately 5000-fold from rat liver nuclei. The activity of the purified enzyme is absolutely dependent upon the presence of native or synthetic DNA, and the further addition of histone(s) stimulates the activity 3- to 5-fold. When the ADP-ribosylated material synthesized in the absence or presence of various histones is analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the major product in all cases migrates between histones H1 and H3-H2B with the same RF value of 0.58 relative to the marker dye. No ADP-ribose was found to co-electrophorese with any of thehistones. The addition of histones does not affect the chain number of the poly(ADP-ribose) synthesized but does result in an increase in the average chain length of the polymer. In the presence of histones, the Km for NAD+ decreases from 80 micron to 25 micron and the Vmax doubles. These results indicate that, in the purified poly(ADP-ribose) synthetase system, histones are not ADP-robosylated but act as allosteric activators.