Histones, poly synthetase

The enzyme catalyzing the addition of ADP-ribose units onto the histones and itself is poly(ADP-ribose) polymerase or synthetase. Poly(ADP-ribose) polymerase is a nuclear, DNA-dependent enzyme that is stimulated by DNA breaks [302]. This property of the enzyme would target its action to sites that have DNA strand breaks (regions of the genome involved in replication, repair, recombination). The enzyme is associated with chromatin areas and perichromatin regions in interphase Chinese hamster ovary cells [312]. Degradation of the ADP-ribose polymer is catalyzed by the nuclear enzyme poly(ADP-ribose) glycohydrolase and ADP-ribosyl protein lyase. 

The biological function of ADP-ribosylated nuclear proteins is not clear. Poly-(ADP-ribose) synthetase has been reported to be stimulated by histones, but histones themselves do not serve as acceptors in vitro (42). The endogenous acceptor iji vivo might involve proteins other than histones. Recently an enzyme that cleaves the ADP-ribosyl histone linkage has been purified from rat liver (43). 

Poly (adenosine 5 -diphosphate ribose) polymerase-1 (PARP-1) [also known as poly (ADP-ribose) synthetase, PARS E.C. 2.4.2.30] is a chromatin-bound enzyme, which is abundantly present in the nuclei of numerous cell types. Single strand breaks in DNA trigger the aaivation of PARP-1, which transfers ADP-ribose moieties from nicotinamide adenine dinucleotide (NAD ) to various nuclear proteins including histones and PARP (automodification domain) itself. This reaaion leads to the generation of nicotinamide, which is an inhibitor (negative feedback) of PARP-1 aaivity. Continuous or excessive activation of PARP-1 (and perhaps other, less well charaaerized members of the PARP enzyme family) produces extended... 

Recent attempts at characterization of the poly(ADP-ribose) synthetase have dealt with localization of the enzyme on chromatin, purification and requirements of synthetase for catalytic function, effects of DNA and histones on activity, and the protein substrates of the synthetase. 

Histones have been reported to be required for stimulation of synthetase activity in the presence of DNA (43, 60, 93,100,105,117,125, 148,158,159,220,221,233). As noted, however, near maximal rates of poly(ADP-ribose) formation could be obtained utilizing poly(dA) poly(dT), or "active DNA in the absence of added histones (83). Stimulation of poly ADP-ribosylation was observed when histones were added to intact (100%) or partially denatured calf thymus DNA (400%). Yoshihara suggested (83) that added histone binds to denatured DNA and masks its inhibitory action. Histones did not activate by serving as ADP-ribose acceptor in the enzyme reaction catalyzed by the purified bovine thymus poly(ADP-ribose) synthetase. It was known, however. 

In work with the bovine thymus synthetase, Tanaka et al. 212) demonstrated that the enzyme was completely dependent on histone when Mg + was omitted from the assay histone HI was ADP-ribosylated under these reaction conditions. Maximum stimulation and ADP-ribosylation occurred when the ratio of DNA to histone HI was 1 to 10 on a weight basis stimulation was lost when the amount of DNA was increased to 50% of histone HI. All other histone fractions were efiective in stimulating the reaction but none was as active as histone HI. Kawaichi et al. 109) and Ueda and co-workers 222) also observed synthesis of poly(ADP-ribosyl) histone using an apparently homogeneous preparation of rat liver poly( ADP-ribose) synthetase. As opposed to the requirement for a large excess of histone over DNA used by Tanaka et al. 212) to demonstrate modification of Hi, a ratio of DNA to histone of 1 1 (on a weight basis) appeared to be optimal. The amount of ADP-ribose incorporated into histone Hi increased linearly as the DNA to histone Hi ratio was fixed at unity and their concentrations increased from 25 to 150 jug/ml. The ADP-ribose incorporated into histone HI represented, however, only about 50% of the total poly ADP-ribosylation the remainder was polymer associated with the synthetase itself. In these studies 212), Mg + was present at a concentration of 10 mAf. All histone subfractions were tested as acceptor proteins Hi was best, followed by H2B H2A, H3, and H4 were poor acceptors. This order of effectiveness is nearly identical to that found in experiments with intact nuclei (2,28,30, 64, 76,102,103,149,162,164,178-180, 200, 215, 229). 

Kawaichi M, Ueda K, Hayaishi O (1980) Initiation of poly(ADP-ribosyl) histone synthesis by poly(ADP-ribose) synthetase. J Biol Chem 255 816-819... 

The hen liver nuclei contain both ADP-ribosyltransferase and poly(ADP-ribose) synthetase. To separate the ADP-ribosyltransferase from poly(ADP-ribose) synthetase, the 0.6 M potassium chloride extract from the nuclei was applied to a Sephadex G-200 column and eluted with the 0.1 Af Tris-buffer, pH 8.0. Each fraction was incubated with 1 vaM [adenine- H]NAD and 100 jug of whole histones, in a total volume of 0.2 ml containing 50 mAf Tris-Cl" buffer, pH 9.0, and the radioactivity in the acid-insoluble fraction was determined. The result shows the two fractions containing the enzyme activities which catalyze the incorporation of the ADP-ribose moiety from NAD to the whole histones. From the hydroxyapatite column chromatographic analysis of the products formed by the respective fraction, we found that the former fraction contains poly(ADP-ribose) synthetase and the latter fraction contains poly-... 

Poly(ADP-ribose) synthetase uses NAD as a substrate to catalyze the formation of a homopolymer of repeating ADP-ribose units [1-3]. The oligomers and polymers of ADP-ribose are synthesized in a covalent association with various proteins of which histones and [4-7] and the synthetase itself [8—10] are the best characterized. 

The formation of mono-, oligo-, and of linear or branched poly(ADP-ribose) protein conjugates by poly(ADP-ribose) synthetase has been described by various investigators. This protein modification by ADP-ribosylation has been correlated with the biochemical and biological processes of DNA replication, DNA repair, chromatin condensation, regulation of gene expression, and cellular differentiation (for reviews, see [17-19]). While the modification of chromatin constituents, especially of the histones, has been extensively studied, information on the ADP-ribosylation of other subnuclear constituents is scarce [20]. 

To determine the poly(ADP-ribose) synthetase activity in the nuclear matrix, samples were incubated as above in 10 /uM [ P]-NAD in buffer A except that PMSF was omitted. For the assay of the enzyme activity in the subnuclear fractions, 25 jug DNA ml, 25 jug histone Hi ml, and 60 jug DNase I ml were additionally included. 

Figure 1 shows the proteins contained in various subnuclear fractions. Histones were not detectable in the isolated nuclear matrix by Coomassie blue staining. But these known acceptor proteins for ADP-ribose were found in the ammonium sulfate extract. Autoradiography revealed that great amounts of the self-modified poly(ADP-ribose) synthetase - in accordance with the results of Table 1 - were released by DNase, RNase digestion of the isolated nuclei (not shown). 

At present the events triggering and the mechanism controlling muscle differentiation are not well understood they may include changes in chromatin structure, gene amplification, and specific DNA and/or protein modification. One such modification, namely, poly(ADP-ribosyl)ation of nuclear proteins has recently attracted much attention and several studies have indicated that the enzyme poly(ADP-ribose) synthetase plays a role in cellular differentiation [6-9]. This enz)mie is a nuclear chromatin-associated protein which catalyzes covalent modification of both histone and nonhistone protein acceptors (for reviews see [10-13]). The synthetase is activated by DNA strand breaks and it has been suggested that DNA fragmentation and the consequent increase in poly(ADP-ribose) activity are obligatory events for chick muscle differentiation [6]. 

Poly(ADP-ribosylation) of nuclear proteins is one of a number of post-translational events which has been demonstrated in eukaryotic cells. The reaction is catalysed by poly(ADP-ribose) synthetase which transfers ADP-ribose from NAD to a suitable acceptor protein. Histone HI and HMG proteins 1, 2, 14 and 17 have been reported as acceptors in a variety of tissues ranging from trout testis [1] to mammary carcinoma cells [2]. Poly(ADP-ribosylation) of HMG proteins is of particular interest because of the reported association of these proteins with actively transcribed genes [3]. Functionally, poly(ADP-ribosylation) has been implicated in a variety of regulatory events such as DNA synthesis [4], DNA excision repair [5, 1], gene expression [6] and cell differentiation [7]. 

Poly(ADP-ribosyl) synthetase, a nuclear-associated enzyme transfers ADP-ribose from NAD to a variety of proteins, including itself, specific histones, and a number of acidic proteins in the nucleus. Employing relatively specific inhibitors of poly(ADP-ribosyl) synthetase, it has been demonstrated that the ribosylation process is involved in differentiation of various cells, proliferation, and in DNA repair processes [1,2]. 

ADP-ribose monomers (Fig. 2). This indicates that the glycohydrolase degraded the synthetase-bound poly(ADP-ribose) up to all but the last residue of ADP-ribose. Similar results were obtained with poly(ADP-ribose) bound to histone HI, histone H2B, or nonhistone proteins. Since the glycohydrolase degraded protein-bound poly(ADP-ribose) and produced exclusively ADP-ribose monomers, the enzyme was presumed to initiate hydrolysis at the adenosine terminus. 

The nuclear enzyme, poly(ADP-ribose)synthetase, catalyzes the ADP-ribosylation of histones HI, H2a, H2b, and H3 (Ueda et al., 1975 Dixon et al., 1976). It also has been reported that a Ca, Mg -depen-dent endonuclease is ADP-ribosylated and consequently inactivated (Yoshihara et al., 1975). Poly(ADP-ribose) synthetase requires DNA for activity and is stimulated severalfold by histone (Okazaki et al., 1976). Two types of glycohydrolases degrade poly(ADP-ribose) and both are found within cell nuclei (Miyakawa et al., 1972). Poly ADP-ribosylation may inhibit (regenerating rat liver, Ehrlich carcinoma cells, HeLa S3 cells), stimulate (HeLa cells), or have no effect (lymphoid cells, hepatoma cells, human fibroblasts) on DNA replication in in vitro systems (Hayaishi and Ueda, 1977). It has been suggested that ADP-ribosylation inhibition of Ca, Mg -dependent alkaline endonuclease is the reason for NAD" -induced inhibition of DNA synthesis (Hayaishi and Ueda, 1977). It has been proposed that poly ADP-ribosylation may regulate the initiation of successive sets of replicons (Shall, 1972). An alternate idea is that poly ADP-ribosylation functions in the G2 phase to sustain continuous cell proliferation (Hayaishi and Ueda, 1977). The role of poly ADP-ribosylation is not clear, despite the fact that various nuclear proteins are ADP-ribosylated, that ADP-ribosylation patterns vary... 

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