Resistance of ADP-ribosylated histones and HMG proteins to proteases.

Calf thymus histones (individually isolated or mixtures) and high mobility group proteins were ADP-ribosylated in vitro using [32P]NAD+ and immobilized purified poly(ADP-ribose) polymerase. The modified histones were then subjected to V8 protease or alpha-chymotrypsin digestion and the resulting peptides were separated by electrophoresis on acetic acid-urea-Triton gels. It was found that in vitro ADP-ribosylated histones were much more resistant to proteases than unmodified histones. A similar approach was applied to histones modified by the endogenous poly(ADP-ribose) polymerase in permeabilized NS-1 mouse myeloma cells in culture. In this case, the proteases could not discriminate between modified and unmodified histones and putative mono(ADP-ribosyl)ated peptides appeared in a digestion frame corresponding to that of bulk peptides. These differences are most probably due to the specificity or number of ADP-ribose groups added to the histones by the endogenous or exogenous poly(ADP-ribose) polymerase. Thus, depending on the size of poly(ADP-ribose) attached to nuclear proteins, these modified proteins might display different degrees of resistance to proteolysis.     

Variations in ADP-ribosylation of nuclear scaffold proteins during the HeLa cell cycle

Cell cycle variations in ADP-ribosylation of nuclear scaffold proteins were determined. Nuclei of synchronized cells were isolated and labeled with [32P]NAD before nuclear scaffolds were obtained by digestion of DNA with DNase I and extraction of proteins with 2M NaCl. Autoradiograms revealed the three groups of "lamins" and a species identified as poly (ADP-ribose) polymerase to be the primary ADP-ribosylated proteins. The patterns of modification of nuclear scaffold proteins displayed similar features through the cell cycle. Radioactivity in the lamins increased from 20% in early-S phase to 40% in G1 phase of the next cell cycle.     

ATP for the DNA ligation step in base excision repair is generated from poly(ADP-ribose)

In mammalian cells, the base excision repair (BER) pathway is the main route to counteract the mutagenic effects of DNA lesions. DNA nicks induce, among others, DNA polymerase activities and the synthesis of poly(ADP-ribose). It is shown here that poly(ADP-ribose) serves as an energy source for the final and rate-limiting step of BER, DNA ligation. This conclusion was drawn from experiments in which the fate of [(32)P]poly(ADP-ribose) or [(32)P]NAD added to HeLa nuclear extracts was systematically followed. ATP was synthesized from poly(ADP-ribose) in a pathway that strictly depended on nick-induced DNA synthesis. NAD was used for the synthesis of poly(ADP-ribose), which, in turn, was converted to ATP by pyrophosphorylytic cleavage utilizing the pyrophosphate generated from dNTPs during DNA synthesis. The adenylyl moiety was then preferentially used to adenylate DNA ligase III, from which it was transferred to the 5'-phosphoryl end of the nicked DNA. Finally, ligation to the 3'-OH end resulted in the release of AMP. When using NAD, but not poly(ADP-ribose), in the presence of 3-aminobenzamide, the entire process was blocked, confirming poly(ADP-ribosyl)ation to be the essential initial step. Thus, poly(ADP-ribose) polymerase-1, DNA polymerase beta, and ligase III interact with x-ray repair cross-complementing protein-1 within the BER complex, which ensures that ATP is generated and specifically used for DNA ligation.     

Cell cycle variations in ADP-ribosylation of HeLa nuclear proteins

Changes in ADP-ribosylation of nuclear proteins during the HeLa cell cycle were determined. Portions of synchronized cultures were withdrawn at intervals and cells were permeabilized by resuspension in hypotonic buffer containing detergents. Nuclear proteins were radioactively labeled by incubating samples with [32P]NAD. Modified species were resolved using one-dimensional and two-dimensional polyacrylamide gel electrophoresis. Measurements of the incorporation of [32P]NAD by permeabilized cells showed that ADP-ribosylation is a significant modification throughout the cell cycle. A twofold increase was detected during S phase. Autoradiograms of one-dimensional sodium dodecyl sulfate-polyacrylamide gels revealed that many nuclear nonhistones are modified, though the major acceptors of 32P were the histones and a 116,000-Da species (poly(ADP-ribose) polymerase). The same modified proteins were present through the cell cycle, but densitometry of autoradiograms demonstrated a general increase in the level of incorporation in S phase. Autoradiograms of two-dimensional gels of nuclear proteins labeled with [32P]NAD were consistent with these results. Although nonhistones of isolated metaphase chromosomes show a substantial reduction in ADP-ribosylation, histone modification is essentially unchanged in metaphase.     

Decrease in ADP-ribosylation of HeLa non-histone proteins from interphase to metaphase

Variations for non-histones in the ADP-ribosylating activities of interphase and metaphase cells were investigated. 32P-Labeled nicotinamide adenine dinucleotide ([32P]NAD), the specific precursor for the modification, was used to radioactively label proteins. Permeabilized interphase and mitotic cells, as well as isolated nuclei and chromosomes, were incubated with the label. One-dimensional and two-dimensional gels of the proteins of total nuclei and chromatin labeled with [32P]NAD showed more than 100 modified species. Changing the labeling conditions resulted in generally similar patterns of modified proteins, though the overall levels of incorporation and the distributions of label among species were significantly affected. A less complex pattern was found for nuclear scaffolds. The major ADP-ribosylated proteins included the lamins and poly(ADP-ribose) polymerase. Inhibitors of ADP-ribosylation were effective in preventing the incorporation of label by most non-histones. Snake venom phosphodiesterase readily removed protein-bound 32P radioactivity. A fundamentally different distribution of label from that of interphase nuclei and chromatin was found for metaphase chromosome non-histones. Instead of 100 or more species, the only major acceptor of label was poly(ADP-ribose) polymerase. This profound change during mitosis may indicate a structural role for ADP-ribosylation of non-histone proteins.     

A 3-aminobenzamide-resistant labeled protein in [32P]NAD+-labeled cells

A series of proteins are covalently labeled when human lymphocytes are incubated with [32P]NAD+. The majority of this labeling is effectively inhibited when the lymphocytes are coincubated with 3-aminobenzamide, a potent inhibitor of poly(ADP-ribose) polymerase. However, labeling of a 72 000 molecular weight protein was resistant to the inhibitory effect of 3-aminobenzamide. Labeling of this protein from [32P]NAD+ was shown to be Mg2+-dependent. The 72 000 molecular weight protein could also be labeled on incubation with [alpha-32P]ATP, [gamma-32P]ATP and [32P]orthophosphate, but not from [3H]NAD+ or [14C]NAD+. In the present study, we show that the 72 000 molecular weight protein is not ADP-ribosylated but rather, phosphorylated on incubation with [32P]NAD+. This phosphorylation appears to occur via an Mg2+-dependent conversion of NAD+ to AMP with the eventual utilization of the alpha-phosphate for phosphorylation of the 72 000 molecular weight protein.     

Cholera toxin affects nuclear ADP-ribosylation in GH1 cells

Incubation of GH1 cells with cholera toxin for 24 h inhibits [32P]ADP-ribose incorporation into histones and non-histone nuclear proteins by more than 50%. The toxin produces a generalized decrease of incorporation into all protein acceptors and into the poly(ADP-ribosyl)ated components excised from chromatin after micrococcal nuclease digestion. The cellular levels of NAD were also decreased (40 to 80%) after treatment with cholera toxin. The inhibition of poly(ADP-ribosyl)ation is preceded by an increase of [32P]ADP-ribose incorporation, since incubation with the toxin for 3 h caused an increase instead of a decrease of incorporation. Incubation with dibutyryl cyclic AMP for 24 h also inhibited nuclear poly(ADP-ribosyl)ation, thus showing that the effect of cholera toxin might be mediated by cyclic AMP.     

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-ribose) in the cellular response to DNA damage

Poly(ADP-ribose) polymerase is a chromatin-bound enzyme which, on activation by DNA strand breaks, catalyzes the successive transfer of ADP-ribose units from NAD to nuclear proteins. Poly(ADP-ribose) synthesis is stimulated by DNA strand breaks, and the polymer may alter the structure and/or function of chromosomal proteins to facilitate the DNA repair process. Electronmicroscopic studies show that poly(ADP-ribose) unwinds the tightly packed nucleosomal structure of isolated chromatin. Recent studies also show that the presence of poly(ADP-ribose) enhances the activity of DNA ligase. This may increase the capacity of the cell to complete DNA repair. Inhibitors of poly(ADP-ribose) polymerase or deficiencies of the substrate, NAD, lead to retardation of the DNA repair process. When DNA strand breaks are extensive or when breaks fail to be repaired, the stimulus for activation of poly(ADP-ribose) persists and the activated enzyme is capable of totally consuming cellular pools of NAD. Depletion of NAD and consequent lowering of cellular ATP pools, due to activation of poly(ADP-ribose) polymerase, may account for rapid cell death before DNA repair takes place and before the genetic effects of DNA damage become manifest.     

Strategy for selection of cell variants deficient in poly(ADP-ribose) polymerase

A selection strategy to obtain cells deficient in poly(ADP-ribose) polymerase was developed based on the fact that treatment with high levels of N-methyl-N'-nitro-N-nitrosoguanidine results in sufficient activation of poly(ADP-ribose) polymerase to cause NAD and ATP depletion leading to cessation of all energy-dependent processes and rapid cell death. In contrast, cells with low levels of poly(ADP-ribose) polymerase should not consume their NAD and might therefore be more likely to survive the DNA damage. Using this approach, we have cloned a number of cell lines containing 37-82% enzyme activity. The apparent decrease in poly(ADP-ribose) polymerase activity is not due to increases in NAD glycohydrolase, poly(ADP-ribose) glycohydrolase, or phosphodiesterase activities. Further characterization of the poly(ADP-ribose) polymerase-deficient cells indicates that they have prolonged generation times and increased rates of spontaneous sister chromatid exchanges.     

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.     

Mutant cells defective in poly(ADP-ribose) synthesis due to stable alterations in enzyme activity or substrate availability

We used two different approaches to develop cell lines deficient in poly(ADP-ribose) synthesis to help determine the role of this reaction in cellular functions. One approach to this problem was to develop cell lines deficient in enzyme activity; the other approach was to develop cell lines capable of growing with such low nicotinamide adenine dinucleotide (NAD) levels so as to effectively limit substrate availability for poly(ADP-ribose) synthesis. The selection strategy for obtaining cells deficient in activity of poly(ADP-ribose) polymerase was based on the ability of this enzyme to deplete cellular NAD in response to high levels of DNA damage. Using this approach, we first obtained cell lines having 37-82% enzyme activity compared to their parental cells. We now report the development and characterization of two cell lines which were obtained from cells having 37% enzyme activity by two additional rounds of further mutagenization and selection procedures. These new cell lines contain 5-11% enzyme activity compared to the parental V79 cells. In pursuit of the second strategy, to obtain cells which limit poly(ADP-ribose) synthesis by substrate restriction, we have now isolated spontaneous mutants from V79 cells which can grow stably in the absence of free nicotinamide or any of its analogs. These cell lines maintain NAD levels in the range of 1.5-3% of that found in their parental V79 cells grown in complete medium. The pathway of NAD biosynthesis in these NAD-deficient cells is not yet known. Further characterization of these lines showed that under conditions that restricted poly(ADP-ribose) synthesis, they all had prolonged doubling times and increased frequencies of sister chromatid exchanges.     

Participation of Poly(ADP-ribose)-polymerase of nuclear matrix in DNA repair

The poly(ADP-ribose)-polymerase activity of brain and liver cell nuclei is changed during X-irradiation of rats. In the nuclear matrix, poly(ADP-ribose)-polymerase activity increases at a low dose of irradiation (1.7 Gy) and decreases at a high dose (6.7 Gy). A significant part of the activity of nuclear NMN-adenylyltransferase, a key enzyme for biosynthesis of NAD (the substrate of poly(ADP-ribose)-polymerase), has been found in the nuclear matrix. An interrelation between ADP-ribosylation taking place on the matrix level and eukaryotic cell DNA repair is suggested.     

Two proteolytic degradation products of calf-thymus poly(ADP-ribose) polymerase are efficient ADP-ribose acceptors. Implications for polymerase architecture and the automodification of the polymerase

Two polypeptides with molecular masses of 76 and 59 kDa were found to copurify with poly(ADP-ribose) polymerase from calf thymus, and to be as efficient acceptors of ADP-ribose as the polymerase itself. Analysis of their CNBr fragments by sodium dodecylsulfate/polyacrylamide gel electrophoresis revealed that the polypeptides were derived from the 112-kDa polymerase. Isolation of poly(ADP-ribose) polymerase in the absence of protease inhibitors resulted in a loss of more than 90% of the polymerase activity and an increased proportion of the 76-kDa and 59-kDa polypeptides in the final polymerase preparation. When the polymerase and the two polypeptides were separated by gel filtration or polyacrylamide gel electrophoresis in 5% acetic acid, no polymerase activity was found associated with the two fragments. Analysis of the CNBr fragments of the three polypeptides after incubation of the enzyme preparation with [32P]NAD showed that most of the fragments were radioactive, indicating multiple ADP-ribosylation sites. Several ADP-ribosylated fragments were found to be common to all three polypeptides, or to two of them.     

Poly(adenosine diphosphate ribose) metabolism and regulation of myocardial cell growth by oxygen

Control of the rate of cardiac cell division by oxygen occurs most probably by altering the redox state of a control substance, e.g. NAD(+)right harpoon over left harpoonNADH. NAD(+) (and not NADH) forms poly(ADP-ribose), an inhibitor of DNA synthesis, in a reaction catalysed by poly(ADP-ribose) polymerase. Lower partial pressure of oxygen, which increases the rate of division, would shift NAD(+)-->NADH, decrease poly(ADP-ribose) synthesis, and increase DNA synthesis. Chick-embryo heart cells grown in culture in 20% O(2) (in which they divide more slowly than in 5% O(2)) did exhibit greater poly(ADP-ribose) polymerase activity (+83%, P<0.001) than when grown in 5% O(2). Reaction product was identified as poly(ADP-ribose) by its insensitivity to deoxyribonuclease, ribonuclease, NAD glycohydrolase, Pronase, trypsin and micrococcal nuclease, and by its complete digestion with snake-venom phosphodiesterase to phosphoribosyl-AMP and AMP. Isolation of these digestion products by Dowex 1 (formate form) column chromatography and paper chromatography allowed calculation of average poly(ADP-ribose) chain length, which was 15-26% greater in 20% than in 5% O(2). Thus in 20% O(2) the increase in poly(ADP-ribose) formation results from chain elongation. Formation of new chains also occurs, probably to an even greater degree than chain elongation. Additionally, poly(ADP-ribose) polymerase has very different K(m) and V(max.) values and pH optima in 20% and 5% O(2). These data suggest that poly(ADP-ribose) metabolism participates in the regulation of heart-cell division by O(2), probably by several different mechanisms.     

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.     

Direct radioactive labelling of poly(ADP-ribose) in developingXenopus laevis embryos

It has not previously been possible to label the nuclear protein modification poly(ADP-ribose) directly from NAD because of the impermeability of the cell membrane. We have overcome this important problem by micro-injection of radioactively labelled NAD into Xenopus laevis early embryos. The polymer was identified and then quantified by its insensitivity to DNAase, RNAase, and spleen phosphodiesterase and by the chromatographic mobility of the products of digestion with snake-venom phosphodiesterase. The quantity of poly(ADP-ribose) present after 25 h of development (129 ng/mg DNA) is lower than that found in fully differentiated tissue.     

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.