©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Identification of Domains of Poly(ADP-ribose) Polymerase for Protein Binding and Self-association (*)

(Received for publication, October 7, 1994; and in revised form, December 1, 1994)

Kalman G. Buki (§) Pal I. Bauer (§) Alaeddin Hakam Ernest Kun (¶)

From the Laboratory for Environmental Toxicology and Chemistry and the Octamer Research Foundation, Romberg Tiburon Centers, San Francisco State University, Tiburon, California 94920

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Cellular proteins extracted from normal and cancer cells bind polymerizing ADP-ribose transferase (pADPRT) on nitrocellulose membrane transblots. Histones at 1 mg/ml concentration completely prevent the binding of pADPRT to cellular proteins, indicating that the binding of histones to pADPRT sites competitively blocks the association of pADPRT to proteins other than histones. The direct binding of pADPRT to histones is shown by cross-linking with glutaraldehyde. The COOH-terminal basic histone H1 tail binds to the basic polypeptide domain of pADPRT. The basic domain present in the NH(2)-terminal part of core histones is the probable common structural feature of all core histones that accounts for their binding to pADPRT. Two polypeptide domains of pADPRT were identified, by way of CNBr fragments, to bind histones. These two domains are located within the 64-kDa fragment of pADPRT and are contiguous with the polypeptide domains that were shown to participate in self-association of pADPRT, ending at the 606th amino acid residue. The polypeptide domains of pADPRT which participate in DNA binding are thus shown to associate also with other proteins. Intact pADPRT binds to both the zinc-free or zinc-reconstituted basic polypeptide fragments of pADPRT. Histones activate auto-poly(ADP)-ribosylation of pADPRT by increasing the number of short oligomers on pADPRT. This reaction is also dependent in a biphasic manner on the concentration of pADPRT. Histones in solution are only marginally poly(ADP)-ribosylated but are good polymer acceptors when incorporated into artificial nucleosome structures.


INTRODUCTION

Poly(ADP-ribose) polymerase (ADP-ribosyltransferase, polymerizing, pADPRT, (^1)EC 2.4.2.30) is a highly abundant nuclear protein of higher eukaryotes (1, 2) that exhibits at least two catalytic functions: ADPR polymerizing and NAD glycohydrolase activities, which are also regulated by the state of differentiation of particular cell types(3) . Assuming the same molecular activity in vitro and in vivo, pADPRT activity in maximally stimulated cells appears to account for only one percent of the molecular activity of this enzyme(1, 2) , approximating the magnitude detectable in the isolated homogeneous protein in the absence of coenzymic DNA(4) , an activity present even in the 56-kDa polypeptide fragment of pADPRT (5) that contains no DNA recognition sites(6) . These observations suggest that the enzymatic activity of pADPRT is highly regulated in physiologically operating cells. Immunochemical estimation of native and automodified pADPRT in intact cells in culture (AA-2 and MT-2 cells) indicated that only 4% (for AA-2) or 20% (for MT-2) of pADPRT was auto-ADP-ribosylated, thus a significant portion of this protein is available for macromolecular associations(7) . Indeed recent results (8) show that in vitro pADPRT significantly stimulates DNA polymerase alpha but not beta. Microinjection of the 46-kDa DNA-binding polypeptide fragment of pADPRT into human fibroblasts inhibits MNNG-induced unscheduled DNA synthesis(56) , further supporting the contention that protein-protein or protein-DNA interactions involving pADPRT may have biological consequences. Binding of pADPRT to the DNA primer of human immunodeficiency virus reverse transcriptase was also shown to inhibit this enzyme(9) . A potentially important cellular function of pADPRT in NAD-dependent repair of damaged DNA in vitro has been also traced to the release of the binding of pADPRT from DNA termini by auto-poly(ADP)-ribosylation of the enzyme protein(10, 11) . Self-association (dimerization) of pADPRT has been shown to be essential for auto-poly(ADP)-ribosylation of the enzyme protein(12, 13) . Furthermore, binding to and trans-ADP-ribosylation (14) to an assortment of highly significant enzymes that act on DNA, such as the calcium/magnesium-dependent endonuclease(15) , DNA polymerase alpha and beta, terminal deoxynucleotidyl transferase, DNA ligase II(16, 17) , topoisomerase I(18, 19) , and topoisomerase II (20) are also of critical cell biochemical significance, because trans-ADP-ribosylation to these enzymes produces a down-regulation of their catalytic activities. As we show here, the binding of pADPRT to a large number of proteins, extracted from 3T3, CHO, and PC12 cells, occurs readily in protein transblots on nitrocellulose membranes (see Fig. 1), emphasizing the significant protein-protein associative capacity of pADPRT. We also show that histones by apparent competition can completely inhibit the binding of pADPRT to cellular proteins, suggesting that pADPRT-histone association and the association of pADPRT with other cellular proteins may occur at the same pADPRT site. It follows that a more detailed analysis of pADPRT-histone association may shed light on the general pADPRT-protein binding sites of pADPRT. Clarification of pADPRT-protein binding in terms of identification of binding domains on pADPRT may have cell biological importance since inhibitors of pADPRT which do not inhibit poly(ADP-ribose) catabolism eventually convert cellular pADPRT to a protein- and DNA-binding cellular component that can regulate complex cellular processes(5, 7, 10, 11, 21) . As a beginning to this highly involved series of pADPRT-protein interactions we first determined histone and self-association sites of pADPRT by nitrocellulose transblot binding and protein cross-linking methods. The study of binding kinetics of proteins in solution is subject to further experimental work.


Figure 1: The binding of pADPRT to cellular proteins, their inhibition by histone H1 and 0.5 M NaCl. Panel A, electrophoretically separated cellular proteins on transblots were stained with Ponceau S (see ``Materials and Methods''). Lane 1, 3T3 cells; lane 2, CHO cells; lane 3, PC12 cells. Panel B, autoradiography identifying the binding of I-labeled pADPRT (10 µg, 4 times 10^5 cpm) to the cellular proteins on transblots, as described in Panel A. Lane 1, 3T3 cells; lane 2, CHO cells; lane 3, PC12 cells. Panel C, the binding experiment, as shown in Panel B, was performed in the presence of 1 mg/ml solution of histone H1. Panel D, the binding assay shown in Panel B was performed in the presence of 0.5 M NaCl. Panel E, immunochemical assay for pADPRT antibody-reactive proteins shown in Panel A. The same amounts of cellular proteins were employed in experiments shown in Panels A-E, hence the faint immunopositive pADPRT bands shown in Panel E, lanes 1 and 2, indicate apparently smaller concentrations of pADPRT in 3T3 and CHO cells.



In and of itself, pADPRT-histone binding is a significant biochemical problem. By far the most abundant nuclear proteins that are trans-ADP-ribosylated by pADPRT are histones (22) and we have demonstrated the in vivo occurrence of poly(ADP)-ribosylated histones by specific antibodies directed against the polymer(23) . Besides serving as ADPR acceptors (24) under certain conditions histones also activate rates of poly(ADP)-ribosylation(25) . In stimulated thymocytes histone H3 is selectively poly(ADP)-ribosylated (26) . Apart from the binding of the zinc fingers of pADPRT to DNA termini(27) , molecular details of protein-protein associations of pADPRT are unknown. We report here the identification of binding domains on pADPRT for histones which probably represent general protein binding sites including a self-association site of the pADPRT enzyme protein, and provide evidence for a regulatory effect of these sites on enzymatic activity.


MATERIALS AND METHODS

Electrophoretically 98% homogeneous pADPRT and coenzymic DNA were isolated from calf thymus as reported elsewhere(28, 29) . Unavoidable traces of lower mass bands were peptides of pADPRT produced by proteases as identified by immunotransblots(28) . The isolated protein did not contain covalently bound poly(A)DP-ribose as determined by the absence of binding to phenylboronate affinity columns(7) . The commercial sources of reagents were as follows: CNBr-activated Sepharose 4B and the Mono S column from Pharmacia Biotech Inc., Affi-Gel 10 from Bio-Rad, [P]NAD and the I-labeled Bolton-Hunter reagent from ICN (Irvine, CA), ZnCl(2) from DuPont-NEN), and Centricon concentrators from Amicon (Danvers, MA). Histones were obtained from Sigma. All other chemicals used were of the highest purity available.

Binding of pADPRT to Cellular Proteins

3T3, CHO, and PC12 cells were grown and harvested by scraping into PBS at midconfluent state. Cells were washed twice with PBS to remove medium and finally were resuspended into PBS. Aliquots of 2 times 10^5 cells were withdrawn and mixed with electrophoretic sample buffer, boiled for 5 min, and then loaded onto 10% SDS-PAGE gels made according to Laemmli (30) . After electrophoresis, proteins were transblotted onto nitrocellulose membranes and renatured, and the aspecific binding sites on the membrane were blocked with bovine albumin. Membrane-bound proteins were probed with I-labeled pADPRT (10 µg, 4 times 10^5 cpm) either in the absence or in the presence of 1 mg/ml histone H1 for 30 min at 23 °C.

Isolation of Polypeptides of pADPRT Obtained by Proteases or CNBr Cleavage

Partial digestion of pADPRT with chymotrypsin (31) was done as follows. pADPRT (2 mg/ml in 50 mM Tris-HCl, 200 mM NaCl, 20 mM 2-mercaptoethanol, pH 8.0) was digested with 3.3 µg/ml chymotrypsin at 25 °C for 30 min, then the reaction was stopped with 1 mM PMSF and the polypeptides isolated by ion exchange chromatography on the Mono S column as reported(6) . The basic NH(2)-terminal 64-kDa polypeptide eluted at 0.45 M NaCl from the cation exchanger column. The COOH-terminal 56-kDa polypeptide, which did not bind to the cation exchanger, was isolated on a benzamide-Affi-Gel 10 affinity column as described earlier(6) .

Cyanogen bromide (CNBr) fragments of pADPRT were prepared by the following technique. Homogeneous pADPRT (200 µg) was precipitated with 20% trichloroacetic acid, and pelleted by centrifugation, washed with 70% ethanol, and dried. The dried protein was dissolved in 100 µl of 88% formic acid, 300 µl of 0.1 M HCl and 20 µl of CNBr (100 mg/ml in ethanol) were added, and the mixture was allowed to stand for 48 h at room temperature. Then the solvent was evaporated by freeze-drying and the residue dissolved in 400 µl of ``renaturation buffer''(32) . Digestion of pADPRT with plasmin was carried out as described earlier(6) .

Digestion of Histone H1 with Chymotrypsin(33, 34

Histone H1 (70 µg) was digested with 0.12 µg of chymotrypsin for 25 min at 25 °C in 45 µl 0.1 M Tris-HCl (pH 8.0). The reaction was stopped by addition of PMSF (1 mM final concentration).

Labeling of pADPRT or Its Fragments by I

The polypeptides to be labeled with I were dissolved in 25-50 µl of 150 mM phosphate (pH 8.2) and pipetted into Eppendorf centrifuge tubes, which contained 2 µl of I-labeled Bolton-Hunter reagent (1888 Ci/mmol, 33 µCi in benzene-dimethylformamide) previously evaporated to dryness by a stream of N(2). After mixing by aspiration into the tip of a micropipette several times, the iodination reaction was allowed to proceed at 6 °C for 1 h. The unreacted Bolton-Hunter reagent was quenched with 5 µl of 1 M Tris-HCl (pH 9.0) and the reaction mixture gel-filtered through an 1.5 ml Sephadex G25 (fine) column equilibrated with 0.1 M sodium phosphate buffer (pH 7.5) containing 0.1% gelatin. The exclusion volume contained the iodinated polypeptides. The incorporation of I was in the range of 3-5%. The I-labeled protein was used on the same day of its preparation for binding experiments.

Labeling of pADPRT by [P]ADPR

This was carried out by incubating the protein with 25 nM of [P]-NAD as described earlier(14) .

Labeling of DNA

Sonicated calf thymus DNA, average length 250 bp, was used for labeling. The DNA (50 µg) was dissolved in 100 µl of Klenow buffer containing 50 µM dNTPs and 10 µCi of [P]TTP and then enzymatically end-labeled with Klenow fragment of DNA Pol I as described elsewhere(35) . The labeled DNA was purified by phenol extraction and ethanol precipitation.

Electrophoretic Techniques

pADPRT and its chymotryptic fragments were separated by 10% SDS-PAGE (30) and histones or CNBr fragments of pADPRT by a 17.5% acrylaminde-SDS system as previously described(36) . Electroblotting was carried out in 10 mM CAPS-NaOH (pH 11.0) buffer containing 15% methanol for 90 min using nitrocellulose membranes with 0.45-µm pore size. When amino acid sequencing was performed, PVDF membranes (ProBlot membrane, Applied Biosystems, Foster City, CA) were used and the excised pieces containing the transblots of peptides were sequenced using the model 470-A gas-phase sequencer and an on-line 120A phenylthiohydantoin horomone analyzer (Applied Biosystems, Foster City, CA) according to a published method(37) .

Preparation of Histone-Sepharose and pADPRT-Sepharose Affinity Matrices

Affinity matrices were prepared from CNBr-activated Sepharose 4B and histones or purified bovine pADPRT, according to the manufacturer's protocol. The amount of proteins bound to the matrix was determined in 0.1-ml aliquots of the settled resin by a published method(38) . On the average 0.9-1.2 mg of histones and 2 mg of pADPRT were covalently bound per 1 ml of packed bed of the gel matrix.

Cross-linking of pADPRT with Histone 2B with Glutaraldehyde (12

Histone 2B was labeled with I using the Bolton-Hunter reagent. The unbound labeling was removed by gel filtration. pADPRT (3.2 µg) and I-labeled histone 2B (3 µg; 5 times 10^4 cpm) were incubated either alone or in combination with 8 mM of glutaraldehyde in a 40-µl volume reaction mixture containing 50 mM triethanolamine-HCl buffer (pH 8.0) for 10 min at 23 °C . After incubation 10 µl of 5 times sample buffer (made according to Laemmli) was added and loaded onto 5-20% SDS-PAGE gradient gels without prior boiling. In addition, 1/50th of each sample was loaded onto another gel for immunological characterization. After electrophoresis part of the samples were stained with Coomassie blue, destained, dried and autoradiographed while the other gel was developed as a Western blot.

Binding of Labeled pADPRT and Its Polypeptides and Labeled Histones to Transblotted Peptides

Membranes containing electroblotted peptides were soaked in ``renaturation buffer,'' 50 mM Tris-HCl (pH 8.0), 100 mM NaCl, 1 mM dithiothreitol, and 0.3% Tween 20 (32) for 1 h then blocked by incubation with 2% defatted powdered milk (dissolved in PBS containing 0.1 mM PMSF) for 2-6 h at 25 °C(37) . The membranes were then washed with a ``low salt buffer'' (50 mM Tris-HCl (pH 7.5), 10 mM 2-mercaptoethanol, 0.1 mM PMSF, 0.05% bovine serum ablumin, and 0.05% Tween 20). The incubation with labeled polypeptides (2-5 times 10^5cpm, 2-5 µg) was performed in the same buffer (15-25 ml) as the one used for washing for 30 min at 25 °C. Then the membranes were washed once with the low salt buffer and twice with the low salt buffer containing 50 mM NaCl. Finally they were dried and exposed overnight to x-ray film. Polypeptides of histone H1 obtained by chymotrypsin and pepsin digestion were isolated as reported (33, 34, 39) .

Binding of Labeled DNA to Transblotted Peptides

Polypeptides transblotted to membranes were renatured (32) and blocked with defatted milk, then washed once with 0.1 M potassium phosphate buffer (pH 7.6) containing 1 mM EDTA, and incubated in the same buffer (15 ml) with [P]DNA (5-10 times 10^4 cpm, 2-4 µg) for 20 min at 25 °C. After four washings with the same buffer the membranes were dried and exposed to x-ray films overnight.

Incorporation of Zn into Transblotted Zinc Finger-containing Peptides

Transblotted peptides were renatured (32) in the presence of 5 µCi of ZnCl(2) in 10 ml of renaturation buffer for 1 h at 25 °C, then the membranes washed with renaturation buffer until no radioactivity over background was found in the washings. The membranes were exposed to x-ray films to locate Zn-containing bands.

Binding of the Polypeptide Fragments of pADPRT to Histone-Sepharose and pADPRT-Sepharose Affinity Column

A histone-Sepharose column (1-ml bed volume) was loaded with 50 µg of CNBr fragments dissolved in 0.4 ml of 50 mM Tris-HCl, (pH 8.0) containing 10 mM 2-mercaptoethanol and allowed to bind for 30 min at 25 °C. The column was then stepwise eluted with 2-ml aliquots of the above buffer containing increments of NaCl: 50, 100, 200, 400, and 1000 mM. The eluted fractions were concentrated on Centricon 3, and one-third of the total amount of concentrates applied onto 17.5% SDS-PAGE. After separation, the gel was stained with Coomassie Blue or transblotted onto PVDF membranes for sequencing.

pADPRT-Sepharose columns (1-ml bed volume) were loaded with either 50 µg of histone H1 or its chymotryptic polypeptides. Chromatography was carried out exactly as described above.

Poly(ADP-ribose) Polymerase Assay

The assays were carried out by published techniques (6) with modifications as given in the figure legends.

Nucleosome Reconstruction(40

Coenzymic DNA (600 µg) and an equal amount of core histones were dissolved in 1.5 ml of TE buffer (10 mM Tris-HCl, 0.2 mM EDTA, pH 7.4) containing 0.8 M NaCl. This solution was dialyzed (at 4 °C) stepwise against TE buffer that contained progressively lower concentrations of NaCl (0.8, 0.6, 0.4, and 0.2 M NaCl). Each dialysis step lasted 90 min, except the final dialysis against TE buffer containing no added NaCl was extended overnight. The DNA content was adjusted to 200 µg/ml by dilution.

Poly(ADP-ribose) Chain Length Analysis

This analysis was carried out according to Althaus and co-workers (41) using DNA sequencing type gels.


RESULTS

The binding of pADPRT to cellular proteins extracted from three cell types is shown in Fig. 1. Among the numerous protein fractions separated by gel electrophoresis (Fig. 1, Panel A) upon transblot to nitrocellulose membranes approximately 16-20 major bands adsorbed I-labeled pADPRT to varying extents. (Fig. 1, Panel B). When the binding experiment was repeated in the presence of 1 mg/ml of histone H1 added to the binding mixture, pADPRT exhibited no association with the transblotted cellular proteins (Fig. 1, Panel C). Histone H1 could be replaced by any other histone (not shown). A similar inhibition of binding occurred in the presence of 0.5 M NaCl, a salt concentration usually required to extract pADPRT from nuclei(28) . It was also found that the assay for pADPRT binding to other polypeptides or the binding of pADPRT-derived peptides to histones was limited by the size of the polypeptide, which was 6-10 kDa, and no association by this technique could be detected if the polypeptide was of a smaller size. This size limitation puts some constraints on binding experiments employing the transblot technique, as will be shown below. When the presence of pADPRT in extracts of 2 times 10^5 cells was probed with pADPRT-specific polyclonal antibody(28) , faint bands were seen in 3TC and CHO cells (Fig. 1, Panel E, lanes 1 and 2) and stronger bands, including a proteolytic degradation product of pADPRT, were detectable in the extract of the more malignant PC12 cells (Fig. 1, Panel E, lane 3).

Since histones prohibited the association of pADPRT with other cellular proteins, implying that histones effectively competed at protein binding sites of pADPRT with other proteins, we reasoned that determination of histone-binding polypeptide domains of pADPRT may identify general protein association sites. The prerequisite for this assumption was a direct demonstration of the association of histones with pADPRT, which was carried out by the glutaraldehyde cross-linking technique (12) as shown in Fig. 2. In Fig. 2, Panel A, Coomassie Blue-stained protein bands identify pADPRT and histone 2B separately but not in the cross-linked complex (lanes 4 and 6) because of the limited resolving power of SDS-PAGE. In the autoradiography shown in Panel B of Fig. 2, unlabeled pADPRT (lane 7) gave no signal, whereas I-labeled histone 2B (lane 8) and glutaraldehyde cross-linked histone 2B (lane 9) clearly separated from the pADPRT-I-labeled-histone 2B cross-linked product, which is shown in lane 10. The pADPRT component of the cross-linked pADPRT-histone 2B complex was identified by immunoblot (Fig. 2, Panel C). Lane 11 is pADPRT, lane 12 is glutaraldehyde cross-linked pADPRT, and lane 14 is the adduct of pADPRT with glutaraldehyde cross-linked I-labeled histone 2B, containing also some glutaraldehyde cross-linked dimers of pADPRT. Lane 13 contains histone H2B which does not react with anti-pADPRT serum.


Figure 2: Cross-linking with glutaraldehyde of histone 2B with pADPRT. Panel A, Coomassie Blue-stained proteins. Lane 1, molecular weight standards; lane 2, pADPRT and degradation products; lane 3, I-labeled histone 2B; lane 4, glutaraldehyde-cross-linked pADPRT; lane 5, glutaraldehyde-cross-linked I-labeled histone H2B; lane 6, glutaraldehyde-cross-linked I-labeled histone H2B with pADPRT. Panel B, autoradiography of labeled proteins given in Panel A. Lane 7, unlabeled pADPRT; lane 8, I-labeled histone H2B; lane 9, same as lane 8 except cross-linked with glutaraldehyde; lane 10, glutaraldehyde cross-linked pADPRT with I-labeled histone H2B. Panel C, identification of pADPRT component in Panel B by immunoblot. Lane 11, pADPRT; lane 12, cross-linked pADPRT; lane 13, histone H2B; lane 14, cross-linked pADPRT with I-labeled histone H2B.



The Binding of P-ADP-ribosylated pADPRT to Histone H1 and to Its Chymotryptic Peptide Fragments

Proteolytic fragments of histone H1 (33, 34, 39) fulfilled physical requirements for binding assays, and as shown in Fig. 3, the COOH-terminal basic histone tail of H1 readily binds to pADPRT, implying that this lysine-rich polypeptide domain is the probable common denominator that provides binding of all types of histones to pADPRT. The pADPRT binding lysine-rich histone domain is in the carboxyl-terminal of histone H1, but in core histones this polypeptide is part of the amino-terminal (42, 43) . Fig. 3A illustrates the electrophoretic separation of histone H1 (lane 1) and its chymotryptic fragments (lane 2) stained by Ponceau red, whereas the binding of labeled pADPRT to histone H1 is shown in lane 3, and to chymotryptic fragments of histone H1 in lane 4. The binding of histone H1 and its chymotryptic COOH-terminal polypeptide to the pADPRT affinity column is demonstrated in Fig. 3B which is a Coomassie Blue-stained gel (for details see legend of Fig. 3B). Since histone H1 (Fig. 3B, lane 12) and its COOH-terminal tail (lane 5) both elute with 200 mM NaCl, their affinity to the pADPRT column appears to be identical. A weak binding of the NH(2)-terminal fragment is also apparent (Fig. 3B, lane 4). However, this weak association does not show up in the nitrocellulose binding assay (Fig. 3A, lanes 3 and 4) that is carried out under more stringent conditions, therefore the main pADPRT-binding COOH-terminal tail of histone H1 is the significant pADPRT-binding histone polypeptide. The NH(2)-terminal CNBr fragment of histone H4, as predicted, binds to pADPRT in the same manner as the COOH-terminal chymotryptic fragment of H1 (not shown). According to published results (41) the pADPRT-binding histone fragments bind also free poly(ADP-ribose).


Figure 3: Binding of pADPRT to histone H1 and to its chymotryptic fragments. A, the binding of [P]ADP-ribosylated pADPRT to histone H1 and to its chymotryptic polypeptide fragments. 10 µg of histone H1 (lanes 1 and 3) and 10 µg of chymotrypsin-digested histone H1 (lanes 2 and 4) were separated by SDS-PAGE, transblotted onto nitrocellulose membranes, and probed with [P]ADP-ribosylated pADPRT (3 µg, 10^5 cpm; obtained by incubating pADPRT with 25 nM [P]NAD for 10 min followed by gel filteration on a Sephadex G75 column) and then processed (see ``Materials and Methods''). Lanes 1 and 2 are Ponceau Red-stained polypeptides and lanes 3 and 4 are autoradiograms. The doublets in lanes 2 and 4 (17 and 18 kDa) are the COOH-terminal basic fragments of histone H1. B, the binding of histone H1 and its chymotryptic fragments to pADPRT-Sepharose affinity columns. The pADPRT-Sepharose columns were loaded either with chymotryptic fragments of 50 µg of histone H1 (lanes 1-7) or with 50 µg of histone H1 (lanes 8-14). The columns were then eluted with NaCl solutions with increasing ionic strength, and aliquots from each fraction were loaded onto SDS-PAGE gels, electrophoresed, and Coomassie Blue-stained. Lane 1 shows polypeptides of histone H1 obtained by chymotryptic digestion; lanes 2 and 9 are flow-through fractions with buffer only; lanes 3 and 10 are eluates with 50 mM NaCl; lanes 4 and 11 are eluates with 100 mM NaCl; lanes 5 and 12 are eluates with 200 mM NaCl. Lanes 6 and 13 are eluates with 400 mM NaCl and lanes 7 and 14 are eluates with 1000 mM NaCl, indicating that there are no further absorbed polypeptides present in the adsorbed form.



The Binding of I-Labeled Histones to Chymotryptic Fragments of pADPRT

The binding of polypeptides derived from pADPRT by chymotryptic digestion to a mixture of radioiodinated core histones was assayed on nitrocellulose membranes. The separation of chymotryptic polypeptides of pADPRT, mainly representing molecular masses of 64, 56, and 42 kDa, are shown (Coomassie Blue-stained) in Fig. 4, lane 1, coinciding with published results(31) . When the chymotryptic polypeptides of pADPRT were transblotted onto nitrocellulose membranes and incubated with radioiodinated core histones, only the basic polypeptide of pADPRT (64 kDa) indicated histone binding (Fig. 4, lane 2).


Figure 4: Binding of labeled histones to transblotted chymotryptic fragments of pADPRT. The digestion of pADPRT, separation of polypeptides, electroblotting onto a nitrocellulose membrane, and the procedure for binding I-labeled histones are described under ``Materials and Methods.'' Lane 1, Coomassie Blue-stained peptides, lane 2, autoradiography of bound I-labeled histones to transblotted peptides of pADPRT.



The correctness of these conclusions was tested also by ``opposite'' labeling, i.e. radioiodination of polypeptides of pADPRT. The same results as shown in Fig. 4were obtained, i.e. only the basic half of pADPRT bound to histones, regardless which protein species was labeled with I. Fig. 5, lane 1, shows the Coomassie Blue-stained histones (mixed histones; 8 µg/lane). Lane 2 illustrates that the 56-kDa polypeptide (labeled with I) of pADPRT (the catalytic domain) did not bind to transblotted histones at all. The binding of the labeled basic 64-kDa polypeptide to transblotted histones is apparent from lane 3 together with lane 4, a positive control where the binding of labeled intact pADPRT protein to histones is shown. The intact protein bound to transblotted histones in the same manner as the 64-kDa basic polypeptide (compare lanes 3 and 4).


Figure 5: Binding of I-labeled pADPRT and its N- and COOH-terminal fragments to transblotted histones. Four µg of mixed histones were electrophoresed (36) then transblotted onto nitrocellulose membranes and probed with I-labeled pADPRT. Lane 1 shows Coomassie Blue-stained histones, all the other lanes are autoradiograms. The absence of binding of radiolabeled 56-kDa polypeptide of pADPRT to histones is shown in lane 2. Lanes 3 and 4 illustrate the histone binding of the labeled 64-kDa polypeptide and of the entire pADPRT protein as a control.



Electrophoretic Separation of CNBr Fragments of pADPRT Eluted from a Histone-Sepharose Column

A more detailed localization of histone binding sites on pADPRT was achieved by percolating CNBr-generated peptide fragments of pADPRT through a histone-Sepharose affinity matrix and identifying the bound fragments. From the known amino acid sequence of bovine pADPRT (44) it is predictable that CNBr fragments of pADPRT in the region between zinc finger II and the catalytic domain yield peptides of 8-18 kDa size, which are large enough to bind to pADPRT. In contrast, CNBr fragmentation of the zinc finger region produces some very short peptides which would not bind to pADPRT because of their small size. Adsorption of CNBr fragments to the histone matrix, followed by elution with a salt solution of stepwise increasing ionic strength permits an estimation of the relative binding strength of polypeptides to the histone matrix. Electrophoretic separation of CNBr fragments of pADPRT is shown in Fig. 6, lane 1. In lane 2, the nonadsorbed (flow-through) fragments, eluted by 50 mM Tris-HCl (pH 8.0) containing 10 mM 2-mercaptoethanol, are demonstrated. Lanes 3-7 show fragments emerging at 50 (lane 3), 100 (lane 4), 200 (lane 5), 400 (lane 6), and 1000 mM NaCl (lane 7) as components of the elution buffer. The polypeptide with an apparent mass of 14 kDa eluted between 50 and 200 mM NaCl, accompanied (lanes 3 and 4) by a broader immunopositive band which most probably consists of proteolytic breakdown products of this fragment.


Figure 6: Electrophoretic separation of CNBr fragments of pADPRT eluted from a histone-Sepharose affinity column. Lane 1 shows CNBr-generated peptides prior to passing through the affinity matrix; lane 2, the flow-through CNBr fragments; lanes 3-7, the eluates with 50, 100, 200, 400, and 1000 mM NaCl, respectively. For technical details, see ``Materials and Methods.''



The 14-kDa polypeptide was identified by sequencing and corresponds to residues 186-290 on the sequence of pADPRT (105 residues, M(r) = 11,826), its NH(2)-terminal being GFSVL. . . . This polypeptide is located between the 29- and 36-kDa domains of pADPRT obtained by digestion with plasmin(6) . It is only 22 residues downstream of the second zinc finger as apparent from the domain diagram (Fig. 7). We also sequenced the fragments which elute at 0.4 M NaCl (apparent molecular mass of 10 and 18 kDa, shown in Fig. 6, lane 6). The NH(2) terminus of the 18-kDa peptide was XXLTLGXLSQ. . . . This sequence places the peptide on the bovine enzyme at 396-525 (130 residues, M(r) = 14,002). Sequencing of the 10-kDa fragment yielded an NH(2)-terminal sequence of XEVKEANIRV. . . . The position of this peptide is at 446-525 (80 residues, M(r) = 8,603). These two polypeptides thus partly overlap, the larger includes the smaller one which is adjacent to the 56-kDa catalytic domain. These two polypeptides are part of the automodifiable region (45) of pADPRT and only the shorter one is shown as histone binder in Fig. 7.


Figure 7: Self- and histone-binding sites of pADPRT. In the upper diagram shaded rectangles indicate histone-binding CNBr peptides and open rectangles are peptides participating in self-binding. The lower diagram shows cutting sites by proteases. Small ellipses in the upper diagram mark the position of zinc fingers. N, the NH(2)-terminal; C, the COOH-terminal; pl, plasmin cutting sites; chy, chymotryptic cutting sites. The sign ∨ identifies the NH(2)-terminals of polypeptides obtained by direct sequencing. Established polypeptide domains of pADPRT are also indicated with double-ended arrows.



Our results are consistent with the existence of two histone-binding regions, one between residues 186 and 290, which is close to the second zinc finger, and a second histone-binding sequence that is between residues 446 and 525 that is part of the automodifiable portion of pADPRT (Fig. 7).

Since both direct chemical (12) and kinetic (13) evidence supports the significance of self-association of pADPRT molecules, we attempted to identify polypeptide domains of pADPRT that are participatory in the self-binding reaction. Chemical cross-linking experiments (12) earlier identified the 36- and 29-kDa polypeptides as binding to each other, implying self-association. When the pADPRT protein was digested with plasmin (6) a degradation pattern of polypeptides occurs as visualized after electrophoresis on 10% SDS-PAGE and transblot to nitrocellulose membranes followed by Western blot with anti-pADPRT antiserum(28) , as shown in Fig. 8(lanes 1 and 2). The native enzyme protein (Fig. 8, lane 1) contains traces of polypeptide degradation products of pADPRT having masses of 98, 80, 67, and 56 kDa(6) . As shown in Fig. 8, lane 2, digestion of pADPRT with plasmin (see legend to Fig. 8) yielded 64-, 56-, 36-, and 29-kDa polypeptides (6) and some minor peptides. When these transblotted polypeptides were probed with I-labeled pADPRT for self-binding, the intact pADPRT, and the 64-, 36-, and the 29-kDa polypeptides exhibited strongest binding (Fig. 8, lanes 3 and 4). The same polypeptides also bound DNA (Fig. 8, lanes 5 and 6), and the 29- and 64-kDa peptides incorporated Zn (Fig. 8, lane 7). Since it is known that zinc finger polypeptides of the 29-kDa fragment are required for the binding of DNA termini(27) , the role of Zn in self-binding of pADPRT had to be clarified. As illustrated in Fig. 8, lanes 8 and 9, zinc-depleted (lane 8) or zinc-replenished (lane 9) polypeptides of 64-, 36-, and 29-kDa mass equally bound radioiodinated pADPRT, suggesting that the basic (64 kDa) polypeptide fragment of pADPRT participates in self-binding and Zn may not be critical in this self-association reaction. Densitometric tracings of lanes 8 and 9 confirm the conclusions deduced from visual inspection of the bands and it is apparent that self-association of pADPRT at the 64- and 29-kDa peptide domains is Zn-independent. As shown in Fig. 7, the two discrete polypeptide domains specific for histone binding are also part of the 64-kDa proteolytic enzyme fragment.


Figure 8: Identification of the basic polypeptide domain (64 kDa) as the self-binding portion of pADPRT. Lanes 1 and 2 are immunostained blots of pADPRT (lane 1) and its plasmin-digested (lane 2) polypeptides. Lanes 3 and 4 are autoradiograms of transblotted pADPRT and its polypeptide fragments, both probed with I-labeled pADPRT. Lanes 5 and 6 are autoradiograms of transblotted pADPRT and its polypeptide fragments, both probed with [P]DNA. Lane 7 shows two polypeptide fragments (64 and 29 kDa) of pADPRT labeled with Zn. Lanes 8 and 9 are polypeptide fragments of pADPRT obtained by plasmin digestion of pADPRT which on nitrocellulose membrane transblots bind I-labeled pADPRT (64-, 39-, and 29-kDa peptides). The polypeptides shown in lane 8 contain no Zn, which was lost during electrophoresis and renatured in the presence of EDTA (see ``Materials and Methods''). In lane 9, Zn had been reincorporated into polypeptides on nitrocellulose transblots. Both Zn-free and Zn-replenished polypeptides bound pADPRT equally, as shown in the densitometric tracing made from lanes 8 and 9.



In further experiments, CNBr-generated peptides were separated by electrophoresis followed by transblot to PVDF membranes and incubation with radioiodinated pADPRT to detect self-association. This technique identified two discrete pADPRT-binding polypeptide fragments (separation not shown) and their NH(2)-terminal amino acid sequence was VKTQT. . . for the 13-kDa fragment (326-445, 120 residues, M(r) 13056) and KKTLK. . . for the 9-kDa fragment (526-606, 81 residues, M(r) 9070). The same 13-kDa polypeptide coeluted with the pADPRT molecule on a Sephadex G75 column as identified by SDS-PAGE (not shown), indicating self-binding in solution. Positioning of the 13- and 9-kDa CNBr fragments together with the histone-binding polypeptides (Fig. 7) shows the existence of contiguous and intermittent histone- and self-binding regions in the pADPRT molecule. The 29-kDa NH(2)-terminal polypeptide, which was not further fragmented, comprises the third domain involved in self-association. As stated above, CNBr fragmentation of the 29-kDa polypeptides yielded fragments too small for binding assays in our test system.

The Effect of Histone H3 on the Enzymatic Activity of pADPRT

Both the binding of histones to pADPRT (25) and self-association of the pADPRT protein (12, 13) exert significant stimulation of poly(ADP)-ribosylation. We therefore devised experiments which can demonstrate a correlation between protein-protein binding, as described above, and the enzymatic activity of pADPRT. Although all types of histones may behave similarly (24) we studied histone H3 in detail, because this histone was specifically poly(ADP)-ribosylated in activated thymocytes(26) . As illustrated in Fig. 9A, a protein concentration-dependent association of pADPRT molecules induces an increase of enzymatic rates between 0 and 30 nM pADPRT, and at higher enzyme concentrations the specific activity decreases, probably due to inhibitory higher order self-association of the monomers(12) . In the presence of histone H3, a dilute, by itself inactive, pADPRT solution exceeds its maximal polymerase activity assayed in the absence of histone H3. However, above 30 nM pADPRT the specific activity diminishes parallel with the increase of pADPRT concentration, similar to the behavior of the enzyme in the absence of histone.


Figure 9: The effect of histone H3 on the catalytic activity of pADPRT. A, effect of pADPRT concentration on the specific activity of the enzyme in the absence and presence of histone H3, determined in the presence of 200 µM [P]NAD and 200 µg/ml coenzymic DNA. Enzymatic activity of pADPRT at varying enzyme concentrations was assayed (6, 54) either in the absence (open circles) or in the presence (closed circles) of 200 µg/ml of histone H3 for 1 min at 23 °C. The reactions were started by adding 1 µl of pADPRT to reaction mixtures of varying volumes. The specific activity of the enzyme (ordinate) is plotted against the log of enzyme concentration (abscissa). Fig. 9B shows the effect of varying histone concentration on the specific activity of pADPRT. The pADPRT concentration in Fig. 9B was 5.5 nM and the histone concentration varied between 0.03 and 200 µg/ml.



The Effect of Histones on the Chain Length Distribution of Poly(ADP-ribose)

Since direct counting of P-labeled (ADPR)(n) polymer yields only an indication of enzymatic rates, expressed here as specific activity, we also analyzed the polymer distribution of the product by gel electrophoresis as illustrated in Fig. 10. At low (2 nM) enzyme concentration in the absence of histones mainly branched polymers, as seen at the origin, and long polymers were formed. Addition of histones tend to produce shorter oligomers (lane 2). Similar observations were made at 20 nM pADPRT concentration (lane 3 and 4) and at 200 nM pADPRT concentration the formation of short oligomers is readily observable (lanes 5 and 6).


Figure 10: The influence of variation in pADPRT concentration, in the absence and presence of a constant concentration of mixed histones, on the chain length distribution of poly(ADP-ribose). The concentrations of pADPRT in the reaction solution assayed were 2 nM (Lanes 1 and 2), 20 nM (lanes 3 and 4) and 200 nM (lanes 5 and 6). Odd numbered solutions contained no histones, even numbered ones contained mixed histone (200 µg/ml). The volumes of the reaction solutions were each 100 µl when the pADPRT concentrations were 20 and 200 nM, and the volume was 1000 µl when the pADPRT concentration was 2 nM. The assays were carried out under conditions described in legend to Fig. 9. At the end of incubation for 5 min at 23 °C the reaction was stopped with 20% trichloroacetic acid (4 °C), the precipitates were sedimented by centrifugation, washed four times with 10% trichloroacetic acid (4 °C), twice with absolute ethanol (200 µl/wash), and then air-dried and dissolved in 2% SDS containing TE buffer (50 µl) and 100 µg of proteinase K. Aliquots of the P-labeled poly(ADP-ribose) representing 30,000 cpm were withdrawn and adjusted to 20 µl with TE buffer, then loaded onto 20% PAGE with TBE as the running buffer. Xylene cyanol and bromphenol blue were used as polymer size markers.



Nucleosomal Structure-dependent Poly(ADP)-ribosylation of Histones

The activation of pADPRT by histone H3 (26) was dependent on histone concentration (Fig. 9B). The most obvious explanation for the activation of pADPRT by histones would suggest that histone H3 or other histones merely serves as an ADP-ribose acceptor. However, as tested by gel electrophoresis (Fig. 11, lane 2), only 1-5% of poly(ADP)-ribose was detectable on histone H3 or other histone species and the rest of ADP-ribose oligomers were covalently bound to pADPRT protein itself, comprising ``self-ADP-ribosylation.''


Figure 11: Poly(ADP)-ribosylation of pADPRT and histones in reconstructed nucleosome. Nuceosome reconstruction and assay of pADPRT activity was done as described under ``Materials and Methods.'' Proteins were separated by acidic SDS-PAGE(55) . Lane 1, pADPRT (1.2 µg) + coDNA (10 µg); lane 2, pADPRT (1.2 µg) + coDNA (10 µg) + histone H3 (10 µg); lane 3, pADPRT (1.2 µg) + reconstructed nucleosome (equal to 10 µg of DNA).



The apparent contradiction between these enzymological results and the known poly(ADP)-ribosylation of histones in nuclei was resolved by demonstrating that artificially reconstructed nucleosomes (40) indeed provided favorable conditions for extensive poly(ADP)-ribosylation of histones, as illustrated in Fig. 11. In Fig. 11, lane 1, auto-ADP-ribosylation of pADPRT is shown in the presence of DNA. Addition of mixed histones (lane 2) increased auto-ADPR-ribosylation of pADPRT without detectable histone ADP-ribosylation. However, in reconstructed nucleosomes extensive poly(ADP)-ribosylation of histones was apparent (Fig. 11, lane 3).


DISCUSSION

The previously outlined domain structure of pADPRT, which consists of DNA binding, self ADP-ribosylation and catalytic domains(31) , has now been expanded to include protein- and self-association participitating polypeptide regions. Because histones completely block the association of pADPRT to all proteins (Fig. 1), that in the absence of histones bind pADPRT, it seems reasonable to postulate that a more detailed analysis of histone binding sites of pADPRT may be a first approximation to the identification of general protein binding domain of pADPRT. Our technique of protein-protein binding assays has a size limitation (approximately 6-10 kDa) thus its accuracy is relatively limited. Furthermore binding strength is estimated only by salt gradients sufficient to disrupt association. Despite these shortcomings, our results clearly show that protein-protein binding regions coincide with DNA binding and automodification domains (Fig. 7). Since the DNA-binding zinc-finger region regulates, by as yet unknown mechanisms, the enzymatic as well as DNA binding activities of pADPRT, the proximity of protein binding sites implies that the two types of macromolecular associations, i.e. protein/DNA and protein/protein interactions, may have mutual regulatory functions, which are clearly of highly complex nature. Since auto-poly(ADP)-ribosylation by electrostatic repulsion inhibits pADPRT-DNA binding (9, 29) and an oscillating poly(ADP)-ribosylation) of histones has been recognized(46) , the inhibition of poly(ADP)-ribose synthesis, without altering the catabolism of polymers, would be expected to have profound effects on the above outlined macromolecular interactions. Consistent with this prediction we have observed large cellular phenotypic changes upon treatment of intact cells with specific pADPRT ligands, reflecting pleiotropic responses(57) . A more specific understanding of macromolecular association of pADPRT requires spectroscopic and fluorometric techniques, implementation of which is now made more feasible by our outline of protein binding domains of this complex protein.

Our results suggests that the presence of Zn in the basic polypeptide fragments of pADPRT appears to be unnecessary for binding to intact pADPRT. However the presence of Zn in the pADPRT molecule which is used as a binding reagent may weaken this argument since the Zn in the intact pADPRT may not be required for the binding to zinc-free polypeptides. A direct test of this assumption is technically not possible, because removal of Zn from pADPRT by specific reagents (47) results in large changes in the physical properties of pADPRT that make it experimentally unsuited for binding assays. (^2)

Both histone- and self-association have significant influence on the specific polymerase activity of this enzyme (Fig. 9), and it appears meaningful that both protein-binding domains reside within the 64-kDa basic part of the pADPRT molecule, which also contains DNA binding domains(31) , the 29-kDa part with its zinc fingers binding to DNA termini(27) , and the 36-kDa part (6) that binds to internal regions of certain double-stranded DNAs(48, 49) . The effect of histones on the enzymatic activity of pADPRT is not confined to an action on apparent specific activity, as illustrated at various protein concentrations (Fig. 9). Histones also induce the synthesis of many short chain oligomers (Fig. 10), an observation that agrees with previously obtained results with the carboxyl-terminal domain of human pADPRT (50) . The apparent nonselectivity of histones as pADPRT-binding proteins is in contrast to the well known specific biological function of histones in the nucleus(43) . It has been shown (51) that in isolated polynucleosomes the concentration of NAD determines the rate of poly(ADP)-ribosylation of various histone species, indicating that the concentration of NAD and the nucleosomal structure, or both, are both regulators of histone-specific poly(ADP)-ribosylation. The absence of poly-ADP-ribosylation of histones in solution by purified pADPRT has been previously reported (52) , but the changes in the polymer-accepting function of histones, as a consequence of supramolecular structural modifications, as we have shown here (Fig. 11), have not so far been considered. The apparent nonspecificity of histones to bind pADPRT in our model system appears to be explained by the commonality of lysine-rich polypeptide domains present in all histones (42, 43) as experimentally demonstrated here with proteolytic fragments of histone H1 (Fig. 3). It has been shown that the free poly(ADP-ribose) polymer binds to lysine-rich histone tails(41) , the same domain which associates with specific basic polypeptides of pADPRT, as documented here for H1. The binding of the acidic poly(ADP-ribose) to lysine-rich polypeptides appears to be explainable by electrostatic forces, but the association of basic polypeptide domains of histones to lysine-rich polypeptides is not consistent with ionic binding and may require hydrophobic mechanisms.

Our results, identifying the 29-kDa and two smaller domains (9 and 13 kDa) of pADPRT to be participatory in self-association, may be correlated with predictions made from the cDNA structure of pADPRT isolated from Drosophila(53) . A leucine zipper motif has been identified in the automodification domain of the Drosophila enzyme, and it is noteworthy that the amphipathic helix at 395-417 residues of the bovine pADPRT includes a degenerate ``bZip'' domain identified in Drosophila(53) . The functional importance of this domain in the self-association of pADPRT appears to be supported by our results.


FOOTNOTES

*
This research was supported in part by Octamer, Inc., and by the United States Air Force Office of Scientific Research F49620-92-J-0232-DEF. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
The first two authors contributed equally to this work.

To whom correspondence should be addressed: Laboratory for Environmental Toxicology and Chemistry and the Octamer Research Foundation, Romberg Tiburon Centers, San Francisco State University, 3150 Paradise Dr., P.O. Box 855, Tiburon, CA 94920. Tel.: 415-435-8851; Fax: 415-435-8853.

(^1)
The abbreviations used are: pADPRT, ADP-ribosyltransferase, polymerizing; ADPR, ADP-ribose; PMSF, phenylmethylsulfonyl fluoride; PBS, Dulbecco's phosphate-buffered saline without Ca and Mg; PVDF, poly(vinylidine fluoride); CHO, Chinese hamster ovary; PAGE, polyacrylamide gel electrophoresis; CAPS, 3-[cyclohexylamino]-1-propanesulfonic acid.

(^2)
K. G. Buki, P. I. Bauer, A. Hakam, and E. Kun, unpublished results.


ACKNOWLEDGEMENTS

We acknowledge with thanks Dr. Jerome Mendeleyev's contribution to the writing of this paper.


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