Poly(ADP-ribosylation) of DNA topoisomerase I from calf thymus

We demonstrate that the activity of the major DNA topoisomerase I from calf thymus is severely inhibited after modification by purified poly(ADP-ribose) synthetase. Polymeric chains of poly(ADP-ribose) are covalently attached to DNA topoisomerase I. These observations with highly purified enzymes suggest that poly(ADP-ribosylation) may be a cellular mechanism for modulating DNA topoisomerase I activity in response to the state of DNA in the nucleus. Although extensive poly(ADP-ribosylation) of the Mr = 100,000 DNA topoisomerase I from calf thymus resulted in greater than 90% enzyme inhibition, exogenous poly(ADP-ribose) does not, by itself, inhibit topoisomerase activity. After modification, the apparent molecular weight of both the topoisomerase enzyme protein and of the topoisomerase enzyme activity was increased. In vitro, the extent of modification of DNA topoisomerase I could be controlled either by changing the ratio of topoisomerase to the synthetase or by varying the reaction time. More than 40 residues of ADP ribose per topoisomerase molecule could be added by the synthetase. Analysis of a poly(ADP-ribosylated) topoisomerase preparation that was about 50% inhibited revealed an average polymer chain length of 7.4, with 1-2 chains per enzyme molecule.     

DNA topoisomerase I from calf thymus is inhibited in vitro by poly(ADP-ribosylation)

A slight DNA topoisomerase I activity was detected in highly purified poly(ADP-Rib)polymerase prepared from calf thymus. This copurified activity was found to be suppressed under conditions where the poly(ADP-ribosylation) reaction occurs in the presence of NAD. Purified topoisomerase I from calf thymus was shown to be ADP-ribosylated by poly(ADP-Rib) polymerase purified from the same tissue. Poly(ADP-ribosylation) of topoisomerase I produces an inhibition of the enzymatic activity in parallel to the extent of ADP-ribosylation. The fact that a slight poly(ADP-Rib) polymerase activity was also found to copurify with a topoisomerase I preparation and that topoisomerase I activity can be modified by ADP-ribosylation, may suggest a spatial and functional correlation of these two enzymes in chromatin.     

Poly(ADP-ribosylated) histones in chromatin replication

Poly(ADP-ribosylation) of histones and several other nuclear proteins seem to participate in nuclear processes involving DNA strand breaks like repair, replication, or recombination. This is suggested from the fact that the enzyme poly(ADP-ribose) polymerase responsible for this modification is activated by DNA strand breaks produced in these nuclear processes. In this article I provide three lines of evidence supporting the idea that histone poly(ADP-ribosylation) is involved in chromatin replication. First, cellular lysates from rapidly dividing mouse or human cells in culture synthesize a significant number of oligo- in addition to mono(ADP-ribosylated) histones. Blocking the cells by treatment of cultures with 5 mM butyrate for 24 h or by serum or nutrient depletion results in the synthesis of only mono- but not of oligo(ADP-ribosylated) histones under the same conditions. Thus, the presence of oligo(ADP-ribosylated) histones is related to cell proliferation. Second, cellular lysates or nuclei isolated under mild conditions in the presence of spermine and spermidine and devoid of DNA strand breaks mainly synthesize mono(ADP-ribosylated) histones; introduction of a small number of cuts by DNase I or micrococcal nuclease results in a dramatic increase in the length of poly(ADP-ribose) attached to histones presumably by activation of poly(ADP-ribose) polymerase. Free ends of DNA that could stimulate poly(ADP-ribosylation) of histones are present at the replication fork. Third, putatively acetylated species of histone H4 are more frequently ADP-ribosylated than nonacetylated H4; the number of ADP-ribose groups on histone H4 was found to be equal or exceed by one the number of acetyl groups on this molecule. Since one recognized role of tetraacetylated H4 is its participation in the assembly of new nucleosomes, oligo(ADP-ribosylation) of H4 (and by extension of other histones) may function in new nucleosome formation. Based on these results I propose that poly(ADP-ribosylated) histones are employed for the assembly of histone complexes and their deposition on DNA during replication. Modified histones arise at the replication fork by activation of poly(ADP-ribose) polymerase by unligated Okazaki fragments.     

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.     

Regulation of glutamate dehydrogenase by reversible ADP-ribosylation in mitochondria

Mitochondrial ADP-ribosylation leads to modification of two proteins of approximately 26 and 53 kDA: The nature of these proteins and, hence, the physiological consequences of their modification have remained unknown. Here, a 55 kDa protein, glutamate dehydrogenase (GDH), was established as a specific acceptor for enzymatic, cysteine-specific ADP-ribosylation in mitochondria. The modified protein was isolated from the mitochondrial preparation and identified as GDH by N-terminal sequencing and mass spectrometric analyses of tryptic digests. Incubation of human hepatoma cells with [14C]adenine demonstrated the occurrence of the modification in vivo. Purified GDH was ADP-ribosylated in a cysteine residue in the presence of the mitochondrial activity that transferred the ADP-ribose from NAD+ onto the acceptor site. ADP- ribosylation of GDH led to substantial inhibition of its catalytic activity. The stoichiometry between incorporated ADP-ribose and GDH subunits suggests that modification of one subunit per catalytically active homohexamer causes the inactivation of the enzyme. Isolated, ADP-ribosylated GDH was reactivated by an Mg2+-dependent mitochondrial ADP-ribosylcysteine hydrolase. GDH, a highly regulated enzyme, is the first mitochondrial protein identified whose activity may be modulated by ADP-ribosylation.     

Increase in histone poly (ADP-ribosylation) in mitogen-activated lymphoid cells

Poly (ADP-ribosylated) histones appear to be intermediates in nuclear processes that involve DNA strand breaks. We have studied histone ADP-ribosylation in cellular lysates from activated human lymphoid cells in culture. Modified histones differing in the number of ADP-ribose groups gave separate bands upon two-dimensional gel electrophoresis. Cellular lysates from control cells contained histones modified with 1 to 15 ADP-ribose groups. Stimulation of the cells during culture with phytohemagglutinin (PHA) or a phorbol ester (TPA) as well as combinations of these two reagents led to a significant increase in the upper limit number of ADP-ribose groups attached to histones in the presence of divalent metal ions. Hyper (ADP-ribosylated) H2B carrying at least 32 ADP-ribose groups gave a distinctly characteristic pattern on two-dimensional gels showing that highly ordered enzymatic steps are followed for its synthesis. Moreover, it was found that PHA and/or TPA induces branching of the poly (ADP-ribose) on H2B. The increase in histone poly (ADP-ribosylation) following lymphocyte activation was less dramatic during incubation of cellular lysates in the absence of divalent metal ions. The increased histone modification observed in this study may result from an increase in cell proliferation during activation of lymphoid cells. The finding that the number of ADP-ribose groups on H4 equals or exceeds by one the number of acetyl groups suggests that the two modifications may share common functions.     

In vitro poly(ADP-ribosyl)ation of seminal ribonuclease

The site of in vitro ADP-ribosylation of seminal ribonuclease was determined. Seminal enzyme was found to be a good receptor of [14C]ADP-ribose residues under the reaction conditions used. The recovery of [14C]ADP-ribosylated RNase was about 65% after purification. After tryptic digestion of modified enzyme, a fraction containing [14C]ADP-ribosylated peptides was separated from the others by ion-exchange chromatography on M82 resin. Radioactive peptides were then purified by affinity chromatography on anti-poly(ADP-ribose)IgG-Sepharose. High performance liquid chromatography of a mixture obtained after pronase digestion of purified ADP-ribosylated peptides revealed only one radioactive peptide whose amino acid composition corresponded to a peptide that has equimolar quantities of aspartic acid, serine, and glycine. Carboxypeptidase Y digestion of this peptide showed that its amino acid sequence was Asp-Ser-Gly. Only position 14-16 of seminal RNase corresponded to this sequence. The chemical stability of the ADP-ribose/enzyme linkage indicated that aspartic acid 14 is the modification site in seminal RNase.     

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.     

Time course of polynucleosome relaxation and ADP-ribosylation. Correlation between relaxation and histone H1 hyper-ADP-ribosylation

Isolated rat pancreatic polynucleosomes were poly(ADP-ribosylated) with purified calf thymus poly(ADP-ribose) polymerase. A time course study was performed using an NAD concentration of 200 microM and changes in nucleosomal structure were investigated by means of electron microscopy visualization and sedimentation velocity determinations. In parallel, analyses of histone H1 poly(ADP-ribosylation) and determinations of DNA polymerase alpha activity on ADP-ribosylated polynucleosomes were done at different time intervals. A direct kinetic correlation between ADP-ribose incorporation, polynucleosome relaxation amd histone H1 hyper-ADP-ribosylation was established. In addition, DNA polymerase alpha activity was highly stimulated on ADP-ribosylated polynucleosomes as compared to control ones, suggesting increased accessibility of DNA to enzymatic action. Because of the strong evidence implicating histone H1 in the maintenance of higher-ordered chromatin structures, the present study may provide a basis for the interpretation of the involvement of the histone H1 ADP-ribosylation reaction in DNA rearrangements during DNA repair, replication or gene expression.     

Adenosine diphosphate ribosylation of chicken-erythrocyte histones H1, H5 and high-mobility-group proteins by purified calf-thymus poly(adenosinediphosphate-ribose) polymerase

Poly(ADP-ribosylation) of histones H1, H5 and non-histone chromosomal high-mobility-group proteins HMG 1, 2, 14 and 17 from chicken erythrocytes by purified calf thymus poly(ADP-ribose) polymerase was studied using acid/urea/Triton gel electrophoresis and autoradiography. With histone H1, besides ADP-ribosylated H1 supporting short chains of polymer, the appearance of H1 'dimer' was observed and this reaction was dependent on NAD concentration and incubation time. In addition, highly modified and/or aggregated species of histone H1 were observed. Histone H5 was slightly ADP-ribosylated at low NAD concentrations. At higher NAD concentrations or after longer incubations the formation of H5 'dimer' and of more modified forms of H5 could be observed. HMG 1 and HMG 2 were found to be ADP-ribosylated, the reaction being dependent on NAD concentration and time. Here again some discrete intermediates appeared. HMG 14 and HMG 17 were only slightly ADP-ribosylated under our experimental conditions. These results indicate that the purified DNA-independent poly(ADP-ribose) polymerase can catalyse the formation of H1 'dimer' as in nuclei and nucleosomes and that H5 and HMG proteins can also be ADP-ribosylated and produce well-defined higher complexes. These modifications of nuclear proteins may provide a means of localized conformational changes of the chromatin structure in vivo.     

The ADP-ribosylation of Sulfolobus solfataricus Sso7 modulates protein/DNA interactions in vitro

The 7 kDa Sso7 is a basic protein particularly abundant in Sulfolobus solfataricus and is involved in DNA assembly. This protein undergoes in vitro ADP-ribosylation by an endogenous poly(ADP-ribose) polymerase-like enzyme. The circular dichroism spectrum of purified ADP-ribosylated Sso7 shows that this modification stabilizes the prevalent protein β-conformation, as suggested by shifting of negative ellipticity minimum to 220 nm. Moreover, a short ADP-ribose chain (up to 6-mers) bound to Sso7 is able to reduce drastically the thermoprotective and DNA condensing ability of the protein, suggesting a possible regulatory role of ADP-ribosylation in sulfolobal DNA organization.     

Activation of NUDT5, an ADP-ribose pyrophosphatase, by nitric oxide-mediated ADP-ribosylation

The ADP-ribose (ADPR) pyrophosphatase (ADPRase) NUDT5, a member of a superfamily of Nudix hydrolases, hydrolyzes ADP-ribose (ADPR) to AMP and ribose 5'-phosphate. Nitric oxide (NO) enhances nonenzymatic ADP-ribosylation of proteins such as beta-actin and glyceraldehydes 3-phosphate dehydrogenase in the presence of free ADPR, suggesting a possibility that NUDT5 could also be ADP-ribosylated by its substrate, ADPR. Here, we show that NO stimulates nonenzymatic ADP-ribosylation of NUDT5 using ADP-ribose and consequently activates its ADPRase activity. We found that ADPRase activity in J774 macrophage cells is increased by the treatment with SNP, an exogenous NO generator or TNF-alpha/IFN-gamma, endogenous NO inducers. Anti-NUDT5 antibody pulled down most of the ADPRase activity increased by NO, indicating that the ADPRase regulated by NO is NUDT5. Using recombinant human NUDT5, we also demonstrated that the increase of ADPRase activity is mediated via ADP-ribosylation at cysteine residue(s) in the presence of reductant. This result suggests that NO activates NUDT5 through ADP-ribosylation at cysteine residues of the enzyme in macrophages.     

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.     

Poly(ADP-ribose) reactivates stalled DNA topoisomerase I and Induces DNA strand break resealing

Regulating the topological state of DNA is a vital function of the enzyme DNA topoisomerase I. However, when acting on damaged DNA, topoisomerase I may get trapped in a covalent complex with nicked DNA (stalled topoisomerase I), that, if unrepaired, may lead to genomic instability or cell death. Here we show that ADP-ribose polymers target specific domains of topoisomerase I and reprogram the enzyme to remove itself from cleaved DNA and close the resulting gap. Two members of the poly(ADP-ribose) polymerase family, PARP-1 and 2, act as poly(ADP-ribose) carriers to stalled topoisomerase I sites and induce efficient repair of enzyme-associated DNA strand breaks. Thus, by counteracting topoisomerase I-induced DNA damage, PARP-1 and PARP-2 act as positive regulators of genomic stability in eukaryotic cells.     

The effect of gamma-irradiation on the poly(ADP-ribosylation) of nuclear proteins in rat thymocytes

Differences in the spectra of modified nuclear proteins of thymocytes of control and irradiated rats were investigated using antibodies specific for poly(ADP-ribose) and incorporation of a label from 14C-NAD in vitro. Two classes of modified proteins were identified differing in the rate of the polymer metabolism and the degree of poly(ADP-ribosylation). No postirradiation changes were detected in poly(ADP-ribosylation) of the nuclear sap proteins and chromatin. A pronounced increase in modification of proteins with the molecular mass of 72 and 83 kD and a sharp decrease in poly(ADP-ribosylation) of a protein group with the molecular mass of 47 to 65 kD were detected within the nuclear matrix by the second hour following irradiation. A study was made of the localization of modified proteins in polydeoxynucleotide fractions of different sizes (mononucleosomes and their oligomers).     

Poly(ADP-ribosylation) of atypical CS histone variants is required for the progression of S phase in early embryos of sea urchins.

The patterns of poly(ADP-ribosylation) in vivo of CS (cleavage stage) histone variants were compared in sea urchin zygotes at the entrance and the exit of S1 and S2 in the initial developmental cell cycles. This post-translational modification was detected by Western immunoblots with rabbit sera anti-poly(ADP-ribose) that was principally reactive against ADP-ribose polymers and slightly against ADP-ribose oligomers. The effect of 3 aminobenzamide (3-ABA), an inhibitor of the poly(ADP-ribose) synthetase, on S phase progression was determined in vivo by measuring the incorporation of 3H thymidine into DNA. The results obtained indicate that the CS histone variants are poly(ADP-ribosylated) in a cell cycle dependent manner. A significantly positive reaction of several CS variants with sera anti-poly(ADP-ribose) was found at the entrance into S phase, which decreases after its completion. The incubation of zygotes in 3-ABA inhibited the poly(ADP-ribosylation) of CS variants and prevented both the progression of the first S phase and the first cleavage division. These observations suggest that the poly(ADP-ribosylation) of atypical CS histone variants is relevant for initiation of sea urchin development and is required for embryonic DNA replication.